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A TAXONOMIC RE-EVALUATION OF
66Propionibacterium coccoides"
by
JANINE VAN NIEUWHOL TZ
Submitted in fulfilment of the requirements
for the degree of
MASTER OF SCIENCE
in the
Department of Microbiology and Biochemistry, Faculty of Natural Science
University of the Orange Free State
Bloemfontein, South Africa
Promotor: Dr. K-H.J. Riedel
Co-promotor: Prof. Dr. B.D. Wingfield
November 1998
To my parents, especially my mother
OTE K
"It is, however, important to realise that species are entities created by man to group
organisms into, and that these groupings are based on hypotheses
which are subject to continual re-evaluation"
K-H.J. Riedel (1996)
Acknowledgements
I wish to express my sincere gratitude and appreciation to the following persons for
their contributions to the successful completion of this study:
Or. K-I-I.J. Riedel, Department of Microbiology and Biochemistry, University of the
Orange Free State, for his adequate guidance, continual patience and for his
valuable and constructive criticism of the manuscript;
Prof. B.D. Wingfield, Department of Genetics, University of Pretoria, for her advice
and for granting a post-graduate bursary through the Foundation of Research
Development;
Prof. L.I. Vorobjeva, Department of Microbiology, Moscow State University, Russia,
for the two "Propionibacterium coccoides" strains and sincere interest
throughout this study;
Prof. LP. van der Walt, Department of Microbiology and Biochemistry, University of
the Orange Free State, for his invaluable assistance in the nomenclature of the
two "P. coccoides" strains;
The staff of the Department of Microbiology and Biochemistry, University of the
Orange Free State, and numerous people not mentioned by name who in some
way have contributed to this study;
To my parents, for their love, continuous encouragement, moral support as well as
financial support throughout my entire university career;
To my friend, Jakkie van Eden, for his love, patience, interest, encouragement and
support throughout the study; and
Above all, to Him who made it all possible.
Table of Contents
Page
Chapter 1: Introduction
Chapter 2: Literature review 7
Chapter 3: A numerical taxonomic study of "Propionibacterium coccoides",
other classical Propionibacterium species and Luteococcus
japonicus 61
Chapter 4: A molecular and phylogenetic analysis of the relationship between
the classical propionibacteria, "P. coccoides" and Luteococcus
japonicus 84
Chapter 5: General discussion and conclusions 119
Chapter 6: Summary 131
Language and style used in this dissertation are in accordance with the
requirements of the journal Systematic and Applied Microbiology)
Chapter 1
Introduction
The genus Propionibacterium consist of two principal groups from different habitats,
namely the "classical" or dairy and the "cutaneous" or clinical propionibacteria. The
classical group is isolated primarily from cheese and dairy products. One species of
the classical group namely Popionibacteriumfreudenreichii is used as a starter culture
during the manufacture of Swiss-type cheese. It has been reported that the propionic
acid bacteria contribute to the sweet nutty, as well as the buttery flavour of these
cheese (Glatz, 1992; Hettinga and Reinbold, 1972; Langsrud and Reinbold, 1973).
Propionibacteria are also responsible for the characteristic "eye" formation in Swiss-
type cheese through the production of carbon dioxide (Langsrud and Re inbold, 1974;
Chaia et al., 1990). The classical propionibacteria may also contribute to the natural
fermentation of silage and olives (Woolford, 1975), although spoilage in the olive
industry by members of the genus Propionibacterium has also been reported (Vaughn,
1981). The propionibacteria have, however, also been reported to be the causative
agents of some defects, including split and late blowing of Swiss-type cheese, due to
the excessive production of CO2 (Langsrud and Reinbold, 1974; Hettinga and
Reinbold, 1975; Massa et al., 1992). The presence ofpigmented propionibacteria can
also be responsible for a spoilage condition in the food industry which is often
referred to as red or brown spotting (Britz and Jordaan, 1976; Baer and Ryba, 1992;
Baer et al., 1993). The production of propionic and other minor organic acids, CO2,
bacteriocins, vitamins (Mantere-Alhonen, 1995) and the antimutagenic properties
displayed by propionibacteria (Vorobjeva et al., 1991:1996) may be of importance in
the application of these organisms as probiotic agents in foods. The propionibacteria
are also receiving increasing attention due to the fact that they rank amongst the most
potent immunomodulatory stimulating cell populations involved in non-specific
resistance (Roszkowski et al., 1990).
The genus Propionibacterium was first described in 1906 by Von
Freudenreich and Orla-Jensen. In 1957 Breed et al. described 11 classical species
within this genus. These 11 species were later consolidated in 4 species namely:
2
P. acidipropionici; P. freudenreichii; P. jensenii; and P. thoenii (Moore and
Holdeman, 1974). Recently a fifth classical species namely P. cyclohexanicum
(Kusano et aI., 1997) was also described which was isolated from spoiled orange
JUice.
Members of the cutaneous group have primarily been isolated from the
intestine and upper respiratory tract of humans (Cummins and Johnson, 1986).
Species was, however, also isolated from dogs (Goodacre et aI., 1994), pigs (Benno
and Mitsuoka, 1982) and paddy soils (Hayashi and Furusaka, 1979). Although these
organisms are generally considered opportunistic pathogens, recent reports published
by Brooke and Frazier (1991), Homer et al. (1992) and Ramos et al. (1995) indicate
the possibility that the species of the cutaneous group could be primary pathogens.
The cutaneous group was initially included in the genus Corynebacterium. In 1974,
Moore and Holdeman transferred four Corynebacterium to form the cutaneous
members of the genus Propionibacterium. These species include: P. acnes;
P. avidum; P. granulosum; and P. lymphophilum (Moore and Holdeman, 1974). In
1988, Charfreitag et al. proposed that Arachnia propionic us be transferred to the
genus Propionibacterium as P. propionieus. This was proposed based on sequence
analysis done on long regions of the 16S rRNA that indicates a more reliable
relationship with P. freudenreichii and P. acnes than with members of the genus
Arachnia.
In 1983, Vorobjeva et al. described a propionic acid producing coccus, which was
isolated from Soviet hard cheese in the early stage of ageing. When compared to the
other propionic acid bacteria, this propionic acid producing bacterium was shown to
be similar in fatty acid composition to the genus Propionibacterium, especially
P. jensenii (the old "P. technicum" strain). This bacterium also contained the anteiso
isomer of the Cwsaturated fatty acid (12-methyltetradecanoic) as the major type of
cellular lipids, which is characteristic of the other members of the genus
Propionibacterium. During lactate fermentation, propionic acid was also produced as
the major metabolic end-product, which is characteristic of the genus
Propionibacterium. Furthermore, considerable quantities of vitamin B!2, catalase,
3
superoxide dismutase and peroxidase are synthesised by both the propionibacteria and
this propionic acid coccus (Vorobjeva et al., 1983; Cummins and Johnson, 1992).
Based on the high degree of similarity in phenotypic characteristics, data on fatty acid
composition, DNA nucleotide composition and the high DNA:DNA homology values
observed between this propionic acid producing coccus and members of the genus
Propionibacterium, Vorobjeva et al. (1983) subsequently proposed that this propionic
acid coccus be included in the genus Propionibacterium as an independent species
under the name "Propionibacterium coccoides". Despite the fact that various
attempts have been made at solving the taxonomic dilemma in the genus
Propionibacterium, no further comparative evaluations of "P. coccoides" and the
propionic acid bacteria have ever been undertaken. Subsequently "P. coccoides" has
never been officially recognised as a new species within the genus
Propionibacterium. No mention of this strain or the proposal of Vorobjeva et al.
(1983) was made in Bergey's Manual of Systematic Bacteriology (Cummins and
Johnson, 1986). Furthermore, "P. coccoides" was also not included in Bergey's
Manual of Determinative Bacteriology (Holt et al., 1994) despite the fact that
Cummins and Johnson (1992) stated that there was sufficient evidence to include this
species in the genus Propionibacterium.
The objectives of this study were therefore, to firstly characterise "P. coccoides" and
the various classical species within the genus Propionibacterium phenotypically, in
order to determine to what extent the "P. coccoides" strains could be equated with the .
existing classical species. The next objective was to determine the relationship of the
"P. coccoides" strains to known Propionibacterium marker strains using various
molecular techniques, based on the 16S ribosomal RNA region, as well as DNA:DNA
hybridisation. Using the information gained from these studies, as well as that of
various other studies (Britz and Riedel, 1994; Riedel and Britz, 1993: 1996 and Riedel
et al., 1994), the systematic position of "P. coccoides" relative to the existing
Propionibacterium species would be evaluated.
4
References
Baer, A., Ryba, I.: Serological identification of propionibacteria in milk and cheese
samples. Int. Dairy J. 2,299-310 (1992).
Baer, A., Ryba, 1., Grand, M.: Study on the appearance of brown-spotting in cheese.
Schweiz. Milchw. Forschung 22, 3-7 (1993).
Benno, Y., Mitsuoka, T.: Anaerobic bacteria isolated from abscesses in pigs. Jap. J.
Vet. Sci. 44, 309-315 (1982).
Breed, R.S., Murray, E.G.D., Smith, N.R.: Genus l. Propionibacterium Orla-Jensen
1909, pp. 569-577. In: Bergey's Manual of Determinative Bacteriology (R.S.
Breed, E.G.D. Murray, N.R. Smith, eds.) 7th ed., Bailliére, London, Tindall
and Cox 1957.
Britz, T.J., Jordaan, H.F.: The rapid presumptive detection of propionibacteria as the
causative organisms of defects in Gouda cheese. S. Mr. J. Dairy Technol. 8,
79-83 (1976).
Britz, T.J., Riedel, K-H.J.: Propionibacterium species diversity lil Leerdammer
cheese. Int. J. Food Microbiol. 22, 257-267 (1994).
Brooke, 1., Frazier, E.H.: Infections caused by Propionibacterium species, Rev.
Infect. Dis. 13, 819-822 (1991).
Chaia, A.P., De Ruiz Holgado, A.P., Oliver, G.: Peptide hydrolyses of
propionibacteria: Effect of pH and temperature. J. Food Protect. 53, 237-240
(1990).
Charfreitag, 0., Collins, M.D., Stackebrandt, E.: Reclassification of Arachnia
propionica as Propionibacterium propionicus comb. nov. Int. J. Syst.
Bacteriol. 38,354-357 (1988).
Cummins, C.S., Johnson, J.L.: Genus 1. Propionibacterium Orla-Jensen 1909, pp.
1346-13 53. In: Bergey's Manual of systematic Bacteriolgy (P .H.A. Sneath,
N.S. Mair, M.E. Sharpe, J.G. Holt, eds.) vol. 2. Baltimore, Williams and
Wilkins 1986.
Cummins , C.S., Johnson, J.L.: The Genus Propionibacterium, pp. 834-849. In: The
Prokaryotes (A. Balows, H.G. Truper, M. Dworkin, W. Harder and K-H.
Schleifer, eds.) vol. l. New York, Springer-Verlag 1992.
5
Glatz, B.A.: The classical propionibacteria: their past, present and future as industrial
organisms. ASM News 58, 197-201 (1992).
Goodacre, R., Neal, M.l, KeIl, D.B., Greenham, L.W., Noble, W.C., Harvey, R.G.:
Rapid identification using pyrolysis mass speetrometry and artificial neural
networks of Propionibacterium acnes isolated from dogs. J. Appl. Bacteriol.
76, 124-134 (1994).
Hayashi, S., Furusaka, C.: Studies of Propionibacterium isolated from paddy soils.
Antonie van Leeuwenhoek 45,565-574 (1979).
Hettinga, D.H., Reinbold, G.W.: The propionic acid bacteria - a review. I. Growth. J.
Milk Food Technol. 35, 295-301 (1972).
Hettinga, D.H., Reinbold, G.W.: Split defect of Swiss cheese. II. Effect of low
temperatures on the metabolic activities of Propionibacterium. J. Milk Food
Technol. 38, 31-35 (1975).
Holt, lG., Krieg, N.R., Sneath, P.H., Staley, r.r., Williams, S.T.: Genus
Propionibacterium, p. 580. In: Bergey's Manual of Determinative
Bacteriology (lG. Holt, N.R. Krieg, P.R. Sneath, r.r. Staley, S.T. Williams,
eds.) 9th ed., Baltimore, Williams and Wilkins 1994.
Homer, S.M., Sturridge, M.F., Swanton, R.H.: Propionibacterium acnes causing an
aortic root abscess. Br. Heart l 68,218-220 (1992).
Kusano, K., Yamada, H., Niwa, M., Yarnasato, K.: Propionibacterium
cyclohexanicum sp. nov., a new acid-tolerant o-cyclohexyl fatty acid-
containing Propionibacterium isolated from spoiled orange juice. Int. J. Syst.
Bacteriol. 47, 825-831 (1997).
Langsrud, T., Reinbold, G.W.: Flavour development and microbiology of Swiss
Cheese. A review. II. Starters, manufacturing processes and procedures. J.
Milk Food Technol. 36, 531-542 (1973).
Langsrud, T., Reinbold, G.W.: Flavour development and microbiology of Swiss
Cheese. A review. IV. Defects. l Milk Food Technol. 37, 26-41 (1974).
Mantere-Alhonen, S.: Propionibacteria used as probiotics - A review. Lait 75, 447-
452 (1995).
Massa, S., Gardini, F., Sinigaglia, M., Guerzoni, M.E.: Klebsiella pneumonia as a
spoilage organism in Mozzarella cheese. J. Dairy Sci. 75, 1411-1414 (1992).
6
Moore, W.E.C., Holdeman, L.v.: Genus I. Propionibacterium OrIa-Jensen 1909, pp.
633-64l. In: Bergey's Manual of Determinative Bacteriology (R.E.
Buchanan, N.E. Gibbons, eds.) 8th ed., Baltimore, Williams and Wilkins 1974.
Ramos, J.M., Esteban, J., Soriano, F.: Isolation of Propionibacterium acnes from
central nervous system infections. Anaerobe 1,17-20 (1995).
Riedel, K-H.J., Britz, T.J.: Propionibacterium species diversity ill anaerobic
digesters. Biodiv. Conserv. 2, 400-411 (1993).
Riedel, K-H.J., Britz, Tl: Justification of the "classical" Propionibacterium species
concept by ribotyping. System. Appl. Microbiol. 19, 370-380 (1996).
Riedel, K-H.J., Wingfield, B.D., Britz, T.J.: Justification of the "classical"
Propionibacterium species concept by restriction analysis of the 16S
ribosomal RNA genes. Syst. Appl. Microbiol. 17, 536-542 (1994).
Roszkowski, W., Roszkowski, K, Ko, H.L., Beuth, J., Jeljaszewicz, J.:
Immunomodulation by propionibacteria. Zbl. Bakt. 274, 289-298 (1990).
VanNiel, C.B.: The propionic acid bacteria. Thesis. Haarlem, Delft, J.W.
Boissevain & Co 1928.
Vaughn, R.H.: Lactic acid fermentation of cabbage, cucumbers, olives and other
products, pp. 220-224. In: Prescott & Dunn's Industrial Microbiology. (G.
Reed, ed.) Connecticut, Saybrook Press 1981.
Von Freudenreich, E., OrIa-Jensen, S.: Ober die in Emmentalerkase stattfindende
Propionisauregarung. Zentralbl. Bakteriol. Parasitenk. Infekt. Hyg. Abt. II 17,
529-546 (1906). (As cited by Van Niel, 1928).
Vorobjeva, L.I., Turova, TP., Kraeva, N.l., Alekseeva, M.A: Propionic acid cocci
and their systematic position. Mikrobiol. 52, 368-373 (1983).
Vorobjeva, L.I., Cherdinceva, TA, Abilev, S.K, Vorobjeva, N.V.: Antimutagenicity
of propionic acid bacteria, Mutat. Res. 251, 233-239 (1991).
Vorobjeva, L.I., Khodjeav, E.Yu., Cherdinceva, T.A: The study of induced
antimutagenesis of propionic acid bacteria. J. Microbiol. Methods 24, 249-258
(1996).
Woolford, M.K: The significance of Propionibacterium species and Micrococcus
lactolyticus to the ensiling process. J. Appl. Bacteriol. 39,301-306 (1975).
7
Chapter 2
Literature Review
(Note: All the taxonomic and spelling styles used in this chapter are as reported in the
original scientific papers)
Characteristics of the genus Propionibacterium
Two principal groups of orgarusms from different habitats are currently
described within the genus Propionibacterium. The first group namely the "classical"
or dairy propionibacteria, was originally described by Von Freudenreich and Orla-
Jensen in 1906. The classical propionibacteria are isolated primarily from cheese and
dairy products, although they have also been isolated from soil (Van Niel, 1928;
Hayashi and Furusaka, 1979), silage (Beerens et al., 1986), natural fermentations
IJ
(Gonzalez-Cancho et al., 1970), anaerobic digesters (Dubourgier et al., 1988; Riedel
and Britz, 1993; Sarada and Joseph, 1994) and nematodes recovered from the hind gut
of zebra (Krecek et aI., 1992). In contrast to the classical group, the "cutaneous" or
clinical group are most frequently isolated from the human skin, especially areas rich in
sebaceous glands such as the alae nasi, as well as moist rather than oily regions, such
as the axilla, perineum and anterior nares (Cummins and Johnson, 1981). Members of
the cutaneous group have also been isolated from the intestine and the upper
respiratory tract of humans (Cummins and Johnson, 1986), as well as from dogs
(Goodacre et al., 1994), pigs (Benno and Mitsuoka, 1982) and paddy soils (Hayashi
and Furusaka, 1979). Although these organisms are generally considered
opportunistic pathogens, recent reports indicate that species of the cutaneous group
could be primary pathogens (Brooke and Frazier, 1991; Homer et al., 1992; Ramos et
al., 1995)
The genus Propionibacterium is characterised as pleomorphic rods, 0,5 - 0,8
urn in diameter x 1 - 5 urn in length. They are often diptheroid or club-shaped with
one end rounded and the other tapered or pointed. The cells may, however, also be
8
coccoid, bifid or even branched. The cells may occur singly, in pairs of short chains, in
V or Y configuration, or in clumps with "chinese character" arrangements (Cummins
and Johnson, 1986; Holt et al., 1994). Morphologically members of the genus
Propionibacterium are irregularly staining, Gram-positive, non-motile, nonsporing
rods. In general the classical propionibacteria tend to be shorter and rather thicker
than the cutaneous propionibacteria, although all strains may be very variable in
morphology, especially in early log phase cultures. Some strains may produce
extracellular slime consisting primarily of carbohydrates. The propionibacteria are
chemo-organotrophs and fermentation products include large amounts of propionic,
acetic, iso-valeric, formic, succinic or lactic acids and carbon dioxide (Cummins and
Johnson, 1986). These anaerobic mesophiles are generally catalase-positive, they
grow most rapid at 30 - 37°C and form small raised colonies that are cream, yellow,
orange, white, grey, pink or deep red (Cummins and Johnson, 1986; Glatz, 1992).
The mol% G + C content of their DNA varies from 53 - 67 mol% (Tm).
The cell walls of most of the strains m the genus typically contain
peptidoglycan in which L-DAP is the diamino acid and a polysaccharide consisting of
hexosamines and some combination of glucose, galactose and mannose.
Propionibacterium freudenreichii and certain strains of P. acnes, and P. avidum
contain meso-diarninopimelic acid, while P. granulosum has been reported to contain
lysine rather than glycine as the interpeptide bridge between DAP and the D-alanine of
the adjacent chain, as observed for the other species (Charfreitag and Stackebrandt,
1989). The various species of the genus Propionibacterium display rather uniform
menaquinone and fatty acid composition, with the cellular lipids of both the classical
and cutaneous species being characterised by large amounts of Cl5 branched-chain
fatty acids (principally iso- and anteiso-Cj, acids). Complex lipids that have
chemoattractant properties for phagocytes have also been extracted from strains of
P. acnes (Russel et al., 1976).
The nutritional requirements of both groups of propionibacteria are rather
similar, suggesting a close resemblance between the overall metabolism of the two
groups. Pantothenate is required by all strains and many also require biotin (Delwiche,
9
1949). Biotin plays a major role in the transcarboxylation reactions in the pathways to
propionic acid (Glatz, 1992). Thiamine and nicotinamide have been reported to
improve growth whilst some strains require p-aminobenzoic acid (Cummins and
Johnson, 1981). A number of other unknown factors in potato or yeast extract are
also stimulatory (Hettinga and Reinbold, 1972a). Although in general the nutritional
requirements of the two groups are very similar, the amino acid requirements of the
cutaneous group are more complex (Ferguson and Cummins, 1978). Many strains of
propionibacteria will, however, grow in a basal medium without the addition of amino
acids. Various organic and inorganic nitrogen sources can also be utilised. Some of
the classical propionibacteria can grow with ammonium sulphate as nitrogen source.
Due to the fact that the nutritional requirements of Propionibacterium strains may
differ significantly, defined media have generally included most amino acids, vitamins,
purines and pyrimidines to support the growth of all strains. The propionibacteria can
utilise a variety of sugars, including trioses, tetroses, pentoses, hexoses and their
corresponding polyols as well as lactic acid as carbon sources. To differentiate
phenotypically between species within the genus Propionibacterium, Holt et al., 1994
published an identification key consisting of nine characteristics (Table 1), where some
of these sugars are of significance.
Although initially considered strictly anaerobic, De Vries et al. (1972)
demonstrated that they were also able to grow under aerobic conditions. In terms of
metabolism, the propionibacteria have a fermentative metabolism and characteristically
produce large amounts of propionic acid as their fermentation end products via the
dicarboxylic acid pathway. Lesser amounts of acetic, iso-valeric, formic and succinic
acids as well as carbon dioxide are also generally formed (Cummins and Johnson,
1986). Generally the ratio of propionic acid to acetic acid is approximately 2: 1, but the
ratio may vary widely and be as high as 5:1 or more (Cummins and Johnson, 1981).
By using the Embden-Meyerhof-Parnas pathway, propionibacteria convert glucose to
pyruvate. Another carbon-source namely lactate, which are commonly found in milk,
can be used preferentially to glucose as a substrate by most propionic acid producing
bacteria. Propionibacterium freudenreichii and P. acidipropionici, however, do not
produce propionic acid from lactate under aerobic conditions but only acetic acid
(Pritchard et al., 1977). Lactate can be oxidised directly by a lactate dehydrogenase to
Table 1. Differential characteristics of species within the genus Propionibacterium (Halt et al., 1994)
Organism Acid from: Pigmentation 13- Hydrolysis of:
Sucrose Maltose L-Arabinose Cellobiose Glycerol of colonies Hemolysis Esculin Gelatin
P. aenes - - - - D White to grey d - +
P. avidum + + d - + White to cream + + +
P.granulosum + + - - + White to grey - - d
P. freudenreichii - - + - + May be tan or - + -
pink
P. jensenii + + - d + White to pink - + -
P. thoenii + + - - + Orange to red- + + -
brown
I
I P. acidipropionici + + + + + White - + -
P. lymphophilum d + - - - White - - d
Symbols: +, 90% or more of strains are positive; (+), 80-89% of strains are positive; (-), 11-20% of strains are positive; -, 90% or more of strains
are negative; d, 21-79% of strains are positive.
......
o
Il
pyruvate in a reaction requiring a flavoprotein as H-acceptor. The pyruvate is then
further transformed to form acetate and carbon dioxide but only in the presence of the
enzyme pyruvate dehydrogenase (a biochemical pathway that yields 1 mole of carbon
dioxide and 1 mole of ATP per mole of acetate). The reduction of lactate or pyruvate
to propionate follows the methylmalonyl-CoA pathway (Fig. 1) and occurs according
to the following overall equation (Schlegel, 1992):
3 CH3-CHOH-COOH
Oxaloacetate is also carboxylated from pyruvate by methylmalonyl CoA
carboxytransferase, in a transcarboxylation reaction with (S)-methylmalonyl-coenzyme
A (CoA) as the CO2-donor and biotin as the CO2-carrier. The reaction of malate
dehydrogenase and fumarase yields fumarate, which is reduced to succinate by
fumarate reductase. This reaction is coupled to an electron transport phosphorylation
with fumarate as the final electron acceptor and can thus be classified as anaerobic
respiration, called fumarate respiration. Thus, although succinate is generally an
intermediate, it can also be produced as a metabolic end product.
Succinyl-CoA is then formed in a CoA transferase (succinyl-CoA:propionate
CoA transferase) reaction and the rearrangement as catalysed by the coenzyme Bl2
(cyanocobalamin) containing methylmalonyl-CoA mutase, leads to (R)-methylmalonyl-
CoA. The (R)-methylmalonyl-CoA is, however, not a substrate for the
transcarboxylase. The (S)-enantiomer is subsequently formed by a specific racemerase.
It is this intermediate which loses carbon dioxide to yield propionyl-CoA by the CoA
transferase, whilst the methylmalonyl-CoA carboxytransferase mentioned above
acquires the CO2. Propionate is liberated from propionyl CoA by the CoA transferase,
which transfers CoA to succinate. One NADH is consumed during the oxidation of
lactate to acetate.
The enzyme (S)-methylmalonyl-CoA-pyruvate transcarboxylase is the key to
the cyclic nature of this pathway, since it enables a carboxyl group to be transferred
from (S)-methylmalonyl-CoA to pyruvate to form oxaloacetate and propionyl-CoA
(Hettinga and Reinbold, 1972b; Gottschalk, 1986; Schlegel, 1992). It should be noted
12
CH3-CO-COOH --------,------------- HOOC-CHTCO-COOH
Pyruvate Oxaloacetate
2 [HJ I 1 2 3 I 2[H]
CHrCHOH-COOH HOOC-CH2-CHOH-COOH
Lactate Malate
4 I H20
HOOC-CH=CH-COOH
Fumarate
Pi + ADP
Biotin 5 2[H]
ATP
HOOC-CHTCHz-COOH
Succinate
I
6 11-------,
CH3-CHTCOOH HOOC-CH2-CH2-CO~SCoA
Propionate Succinyl-CoA
7 B12-
6 Coenzyme
2
CH3-CHrCO-SCoA ____ .:...__ ---'- HOOC-CH-CO-SCoA
Propionyl-CoA I
CH3
Methylmalonyl-CoA
Fig 1. The Methylmalonyl-CoA pathway of propionate formation (Schlegel, 1992)
Enzymes: 1: Lactate dehydrogenase
2: Methylmalonyl-CoA carboxytransferase
3: Malate dehydrogenase
4: Fumarase
5: Fumarate reductase
6: CoA transferase
7: Methylmalonyl-CoA mutase
13
that in this process of propionate formation, two groups namely, C02 and CoA, are
transferred back from product to precursor without occurring in the free form.
Noteworthy feature of the metabolic pathway is the participation of three cofactors
(biotin, eoenzyme A and coenzyme BI2). Whilst biotin must be added to the growth
medium, most of the classical Propionibacterium strains produce vitamin Bl2 and some,
especially P. freudenreichii strains, produce such excessive quantities that these strains
can be used to produce this vitamin commercially (Glatz, 1992). The production of
vitamin B 12 by the cutaneous propionibacteria has, however, never been reported
iCummins and Johnson, 1986).
The propionibacteria can also utilise aspartate, which is also available in cheese,
as an energy source. Propionibacteria in the presence of both lactate and aspartate,
ferment lactate to propionate combined with the fermentation of aspartate to succinate
(Crow, 1986a:1986b). Due to the fact that aspartate is rapidly metabolised during the
ripening of Emmentaler cheese (Crow, 1986b), the theoretical propionate / acetate ratio
established in the propionic acid fermentation is modified to the disadvantage of
propionate. During this coupled fermentation reaction the production of acetate, carbon
dioxide and succinate is, however, enhanced.
Although the genus Propionibacterium is generally considered to have low
proteolytic activity, two types of activity have been found in all species of
propionibacteria, one acting on ~-casein and the other on Ust-casein as substrate. The
typical nutty flavour of Emmentaler cheese is also an important contribution made by
Propionibacterium freudenreichii, through the production of a minimum amount of
proline (Hettinga and Reinbold, 1972a; Langsrud and Reinbold, 1973).
Propionibacteria can also degrade various amino acids, especially aspartate, alanine,
serine and glycine, depending on the species. This results in additional CO2 production
in cheese (Langsrud et al., 1995; Tobiassen et al., 1996). Although lipases and esterases
may play an important role in the hydrolysis of fat and in the flavour composition of
Emmentaler cheese, knowledge of these activities is rather limited. In 1967, Oterholm
compared the lipolytic activity of P. freudenreichii subsp. shermanii with that of lactic
acid bacteria. He reported that P. freudenreichii subsp. shermanii displayed a hundred
14
fold higher lipolytic activity than lactic acid bacteria. Between three to six different
esterase activities were detected, two of them common to various strains of
P. freudenreichii subsp. freudenreichii.
The cutaneous Propionibacterium species were formerly, described in the
genus Corynebacterium. Based essentially on the work of Johnson and Cummins,
1972, they were transferred to the genus Propionibacterium primarily due to the fact
that they were: anaerobic; produced propionic acid as a major end product of
metabolism; contain mostly L-diaminopimelic acid (L-DAP) as the diamino acid of
peptidoglycan; contained CIS iso- and anteiso-acids as the principal fatty acids of cell
lipids; and they lacked mycolic acids and the arabingalactan which was characteristic of
Corynebacterium sensu stricto. This transfer was, however, not without controversy.
Prévot (1976) proposed that this cutaneous group of organisms should not be
transferred to the genus Propionibacterium but should instead be accommodated in a
separate subgenus Coryneformis in the family Corynebacteriaceae.
The designation of Propionibacterium freudenreichii as the type species of the
genus by Van Niel in 1928 is today perhaps rather anomalous in the light of more
recent information. Propionibacterium freudenreichii is rather atypical of the genus in
that: a) strains of P. freudenreichii contain meso-DAP instead of the LL-isomer found
in all other strains; b) it has a cell wall polysaccharide containing rhamnose; and c) it is
more distantly related to other Propionibacterium species by DNA:DNA homology
(Johnson and Cummins, 1972).
In 1993, Sutcliffe and Shaw reported on proportions and types of phospholipids
III Propionibacterium freudenreichii. The major phospholipids identified were
bis(phosphatidyl)glycerol, phosphatidylglycerol and phosphatidylinositol, with minor
components identified as phosphatidylethanolamine and lysophosphatidylinositol. In
contrast to the results of Prottey and Ballou (1968), no phosphatidylinositol
mannosides could be detected. The presence of this mannose containing phospholipid
in P. freudenreichii as reported by Prottey and Ballou (1968), was considered as an
additional factor indicating a close relationship between the genera Propionibacterium
and Mycobacterium (Hettinga and Reinbold, 1972c). The presence of
15
phosphatidylinositol, minor amounts of phosphatidylethanolamine and high
bis(phosphatidyl)glycerol and phosphatidyl glycerol contents in P. freudenreichii is,
however, consistent with the relative taxonomic position of the propionibacteria
between the other actinomycetes and the Clostridium-Bacillus-Streptococcus
subdivision of the Gram-positive eubacteria (Charfreitag and Stackebrandt, 1989;
Goodfellow, 1989; Suteliffe and Shaw, 1993). Members of this genus are readily
confused with some species of Corynebacterium or Clostridium (Holt et al., 1994).
Genera, which are phylogenetically related to the genus Propionibacterium, include
the members of the genera Corynebacterium, Clostridium, Arachnia (Cummins and
Johnson, 1986), Luteococcus, Aeromicrobium, Nocardioides (Yokota et al., 1994) and
Terrabacter (Kusano et al., 1997).
Industrial applications
The classical propionibacteria, especially Propionibacterium freudenreichii are
used extensively as starter cultures in the manufacture of Swiss-type cheeses. The
French cheese industry, for example, produced more than 240 000 tonnes of Swiss-
type cheese during 1994 (SIGF, 1994). The propionibacteria can use both the lactose
present in the milk and the lactic acid produced by the lactic acid starter cultures as
fermentation substrates. Lactic acid is, however, the preferred substrate. Through the
production of propionic and acetic acid, diacetyl and various amino acids, especially
leucine and proline, these organisms add to the characteristic sweet nutty flavour of
Swiss-type cheese (Langsrud and Reinbold, 1973; Chaia et al., 1990). In 1992, Glatz
reported that the sweet flavour of the cheese could be partly attributed to proline
production by these organisms, whereas the buttery flavour is caused by the production
of diacetyl. Propionibacteria also occur in some other cheese-types such as Appenzell,
Tilsit and Sbrinz (Baer et al., 1993), Parrniggiano, Reggiano and Grana Padano
(Thompson and Marth, 1985; Carcano et al., 1995) as well as Mozzarella
(Champagne and Lange, 1990). In these latter cheeses the conditions for their
proliferation are, however, not favoured, and the propionibacteria subsequently do not
contribute significantly to the ripening of these cheeses.
16
Propionibacteria are also responsible for the characteristic "eye" formation in
Swiss-type cheese through the production of carbon dioxide. Excessive carbon
dioxide production by propionibacteria, from both lactose and lactic acid fermentation
as well as amino acid metabolism can, however, result in split and late blowing defects
in Swiss cheese (Langsrud and Reinbold, 1974; Hettinga and Reinbold, 1975). This
phenomenon has also been observed in other types of cheese, such as Gouda (Britz and
Jordaan, 1976) and an American-type Mozzarella (Massa et al., 1992). Similarly,
insufficient CO2 production, due to the inhibition of propionibacterial growth, may also
result in inadequate "eye" formation in Swiss-type cheese (Langsrud and Reinbold,
1974).
With the technical improvement of cheese manufacturing today, the role of the
natural propionic acid bacteria in raw milk seems to have become less important
(Merildinen and Antila, 1976). The presence of pigmented Propionibacterium strains
in foods may also cause a spoilage condition referred to as "red" or "brown spot"
(Baer and Ryba, 1992; Baer et al., 1993). The development of "brown spots" caused
by P. freudenreichii, in Emmentaler cheese can be suppressed by adding increased
amounts of propionibacteria to the milk. Complete inhibition, however requires salt
concentrations of IS-20g/kg cheese (Sollberger, 1996), which greatly diminishes the
sensorial properties of the cheese. This defect is visible only after three months of
ripening, initially in the peripheral area and then in the whole cheese. Although very
rare, "red spots" can be attributed to the presence of red-pigmented "P. rubrum"
colonies in Swiss-cheese (Baer and Ryba, 1992; Baer et al., 1993). The classical
propionibacteria also contribute to the natural fermentation of silage and olives
(Woolford, 1975), although spoilage in the olive industry by members of the genus
Propionibacterium has also been reported (Vaughn, 1981),
The propionibacteria are further also important in the food industry, where they
are responsible for the production of organic acids, biomass, vitamin B 12 and other
specific metabolites (Marcoux et al., 1992). Although the classical propionibacteria
were extensively used for the commercial production of vitamin B 12, this vitamin is
currently produced using a faster growing Pseudomonas strain which has a higher yield
(Glatz, 1992). Vitamin B12 is an important co-factor for the metabolism of
17
carbohydrates, lipids, amino acids, nucleic acids and it is also reported to have been
used in chemotherapy (Quesda-Chanto et al., 1994).
Propionic acid is an important compound used in the production of thermo-
and cellulose-plastics, herbicides, flavours, perfumes, antiarthritic drugs and solvents.
The calcium, sodium and potassium salts of this acid are widely used as food and feed
preservatives (Lewis and Yang, 1992; Paik and Glatz, 1994). Although propionic acid
is currently produced mainly by chemical synthesis from petroleum feedstock, interest
in an alternative route for production of this acid using microbiological processes and
cheap renewable material such as whey, waste molasses or corn steep liquor is
increasing (Boyaval and Corre, 1995).
The production and application of bacteriocins may also have great potential as
natural food preservatives. Klaenhammer, in 1993 defined bacteriocins as proteins or
peptides, which are bactericidal to other, usually closely related bacteria. Further
characteristics are that bacteriocins have a narrow spectrum of activity and are mostly
plasmid-borne. Both classical and cutaneous propionibacteria have been reported to
produce bacteriocins.
Two bacteriocin-like substances were partially purified from a
Propionbacterium acnes strain isolated from plaque. The activity of these two
bacteriostatic substances namely: acneein CN-8 (Fujimura and Nakamura, 1978); and
bacteriocin-like substance RTT 108 were both inhibited by lysozyme at low ionic
strength. The bacteriocin-like substance RTT 108 had a rather large activity spectrum
in that it was active against both Gram-negative and Gram-positive anaerobes, while
acneein had a relatively narrow host range, inhibitory only to other strains of P. acnes
and Corynebacterium parvum.
The first bacteriocin purified and characterised within the classical propionic
acid bacteria group was PLG-1 from P. thoenii (Lyon and Glatz, 1991).
Characteristics of this propionicin included heat lability, sensitivity to different
proteolytic enzymes and its stability between pH 3 and 9. This bacteriocin was also
not inhibitory to the strain of P. thoenii that produced propionicin PLG-1. In 1993,
18
Lyon and Glatz further characterised PLG-l and they reported that this bacteriocin was
produced during the stationary growth phase of P. thoenii and that it was not plasmid-
coded. Propionicin Pl.GvI's spectrum of activity ranges from several Gram-positive
bacteria, including lactic acid bacteria, some Gram-negative bacteria as well as yeasts
and moulds. Inhibitory activity within the genus Propionibacterium was detected
against P. thoenii and P. jensenii, but no inhibitory effect against either three
subspecies of P. freudenreichii was observed.
Another bacteriocin similar to PLG-l, which was also coded for on the
chromosome rather than plasmid-borne, was isolated and described in 1992 by
Grinstead and Barefoot. This bacteriocin known as jenseniin G due to the fact that it
was isolated from a P. jensenii strain, was active at pH 7, sensitive to proteolytic
enzymes, heat stable and resistant to freezing and cold storage. Only three species
were, however, included in its activity spectrum namely: P. acidipropionici;
P. jensenii; and Lactobacillus delbrueckii subsp. lactis. (Paul and Booth, 1988).
A product such as "Microgard" (Wesman Foods, Inc., Beaverton, Oregon
USA), which has been approved by the Food and Drug Administration, contains a
small heat stable bacteriocin, produced by fermenting skimmed milk with
Propionibacterium freudenreichii subsp. shermanii. The product IS used as a
preservative in about 30% of the cottage cheese produced in the United States
(Grinstead and Barefoot, 1992). Microgard has an antagonistic effect against Gram-
negative bacteria and some yeasts and moulds, but not against Gram-positive bacteria
(Lyon and Glatz, 1991).
Another application of the genus Propionibacterium is their use as probiotics.
Probiotics are defined as a live microbial feed supplement which beneficially affects the
host animal by improving its intestinal microbial balance (Fuller, 1989). In order to
exert a probiotic impact in the intestine, bacteria have to survive the passage through
the stomach and duodenum and adhere to the intestinal mucosae. The probiotic effect
is based on the production of beneficial metabolites and antimicrobial compounds for
their host. As a consequence, higher resistance to harmful bacteria responsible for
19
intestinal disorders, stabilisation of the intestinal microflora, anticarcinogenic and
anticholesterolemic influences have been postulated (Fessler, 1997).
In 1995, Mantere-Alhonen proposed propionibacteria as probiotics for humans
and as probiotic growth promotors for pigs. As probiotic agents, propionibacteria in
combination with other bacteria such as lactobacilli, lactococci and enterococci can be
used to increase the weight gain of calves (Cerna et al., 1991) and curing intestinal
disorders in calves, piglets and hens (Vladimirov et aI., 1977; Antipov and Subbotin,
1980; Tuikov et al., 1980). According to Mantere-Alhonen (1982) P. freudenreichii
subsp. shermanii was observed to stimulate the growth of piglets. It has, however,
been suggested (Mantere-Alhonen, 1982; Fessler, 1997) that the high mineral content
(Mn, Zn, Cu, Fe) of propionibacteria could be partly responsible for the reported
probiotical effect, since no colonisation or adhesion of propionibacteria to the intestinal
mucosae of the piglets could be found.
Perez Chaia et al. (1994) reported that propionibacteria useful in the dairy
industry, could also favourably effect the lipid metabolism in the gut and the immune
system of mice. Pro biotic food products in which propionibacteria were combined
with bifidobacteria or lactic acid bacteria, have also been described (Fessler, 1997).
An example is a sour milk product which was developed by Mantere-Alhonen (1987)
containing P. freudenreichii subsp. shermanii and Lactobacillus acidophilus. The
product was reported to exhibit favourable sensory quality, mild flavour, good
consistency and storage properties.
During the manufacturing process of Swiss-type cheese, interactions between
the lactic acid bacteria and the propionibacteria have both favourable and unfavourable
impacts on each other (Perez Chaia et al., 1982: 1994; Beide et al., 1977; Jimeno et al.,
1995). Perez Chaia et al. (1994) reported that the rapid pH reduction by the growth
of L. helveticus and consequently lactate accumulation in the cheese due to the slow
lactate utilisation by P. acidipropionici was the major reason observed for the
inhibition of propionibacteria in mixed cultures. When propagating L. helveticus and
another species of the genus Propionibacterium namely P. jreudenreichii subsp.
shermanii together, a lower biomass yield of lactobacilli, and an increase in the
20
fermentation activities of both micro-organisms with an increase in propionic acid
production, was observed (Perez Chaia et al., 1982). A mixed liquid cultivation of
L. acidophilus and P. freudenreichii subsp. shermanii also seems to be beneficial for
the proliferation of both bacteria (Liu and Moon, 1982). Beide et al. (1977), however,
reported that the addition of increasing amounts of L. bulgaricus to cheese resulted in
a lower pH, lower levels of acetyl production and proteolysis and a decrease in the
number of propionibacteria. Jimeno et al. (1995) suggested that the relative amount of
copper and citrate in cheese could play an important role in L. rhamnosus and L. casei
both having an inhibitory effect on P. freudenreichii. This suggestion was verified
when they found that the inhibition of P. freudenreichii growth could not be
reproduced in cultures with the usual media (Jimeno et al., 1995).
The production of propionic and other minor organic acids, CO2, bacteriocins,
vitamins (Mantere-Alhonen, 1995) and the antimutagenic properties displayed by.
propionibacteria (Vorobjeva et aI., 1991: 1996) may be of importance in the application
of these organisms as probiotic agents in foods. The propionibacteria are also
receiving increasing attention due to the fact that they rank amongst the most potent
immunomodulatory stimulating cell populations involved in non-specific resistance
(Roszkowski et al., 1990).
Early isolations
Despite the initial reports by Fitz in 1878 and 1879 that organisms from cheese
could ferment lactate to propionic and acetic acids and liberate carbon dioxide in the
process, the first pure cultures of propionic acid producing bacteria were isolated and
described in 1906 by Von Freudenreich and Orla-Jensen. The organisms isolated by
Von Freudenreich and Orla-Jensen in 1906 from Emmentaler cheese were named
"Bacterium acidi propionici a", "Bacterium acidi propionici b" and "Bacillus acidi
propionici" based on Ferdinand Cohn's taxonomic system of classification. This
classification system concentrated primarily upon structural characteristics and in
particular the shape of the bacterial cells. The short rods were classified in the genus
Bacterium and the longer rods in the genus Bacillus.
21
In 1908, Thoni and Alleman isolated two more species from the cut surface of
Emmentaler cheese. Using the same nomenclatural system, they named their isolates
"Bacterium acidi propionici var. rubrum" and "Bacterium acidi propionici var.
fuscum", The variants were differentiated on the basis of red and brown pigmentation,
respectively. A year later Troili-Petersson (1909) while studying a Swedish type of
Emmentaler cheese discovered yet another propionic acid producing bacterium. Due
to it's fermentative behaviour and clumping characteristics displayed in liquid medium
which differed from the already reported propionic acid producing bacteria, she named
this organism "Bacterium acidi propionici c",
Orla-Jensen in 1909 was the first researcher to indicate a relationship between
the propionic acid bacteria and other groups of microbes. The propionic acid bacteria
were considered as a separate genus under the order Peritrichinae. Orla-Jensen
(1909) proposed a new genus Propionibacterium with "Bacterium acidi propionici
a", "Bacterium acidi propionici b" and "Bacillus acidi propionici" being the only
species within this genus.
This genus, was, however, not recognised by the committee on the
characterisation and classification of bacterial types of the Society of American
Bacteriologists (1917). Criticising their lack of recognition of the genus
Propionibacterium, Orla-Jensen (1921) stated that if morphological characteristics
were of such significance then a division in the genus requires that besides the genus
Propionibacterium, a genus "Propionicoccus" should also be created for the spherical
forms. In the same year, Sherman (1921) reported the isolation of another type of
propionic acid bacterium which he named "Bacterium acidi propionici d". The
various propionic acid producing bacteria which had been described before 1921 are
presented in Fig. 2.
No mention of the genus Propionibacterium or the genus "Propionicoccus"
was made in the first and second edition's of Bergey's Manual of Determinative
Bacteriology (Bergey, 1923: 1925). Buchanan (1925) in his "General Systematic
Bacteriology" merely stated that a genus Propionibacterium as well as
"Propionicoccus " had been proposed by Orla-Jensen in 1909 and 1921, respectively,
22
"Bacterium acidi propionici var. rubrum"
(Thoni and Alleman, 1908)
"Bacterium acidi propioniel var. rubrum"
(Thoni and Alleman, 1908)
"Bacillus acidi propionici"
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici b"
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici c"
(Troili-Petersson. 1909)
"Bacterium acidi propionici var.fuscum"
(Thoni and Alleman, 1908)
"Bacterium acidi propionici a"
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici d"
(Sherman, 1921)
Fig. 2. A list of the species of propionic acid bacteria described before 1921 and the literature
citations of the descriptions.
23
but emphasised that the status of the genus was doubtful since no species had been
described or referred to.
Primarily due to the work of Orla-Jensen (1909) on the metabolic processes of
the propionic acid bacteria, Kluyver and Donker (1925), were able to place the
propionic acid bacteria between the lactic acid bacteria and the butyl alcohol and
butyric acid bacteria. In 1927 Rippel, however, placed the propionic acid bacteria in a
subgroup of the heterofermentative lactic acid bacteria. This placement was
undertaken based primarily on the results of Virtanen (1923) and his own findings on
the intermediate fermentation products, such as lactic acid. This was the start of the
problems concerning the taxonomic placement of the propionic acid bacteria.
The Van Niel era
The propionic acid producing bacteria were grouped together in a systematic
unity for the first time under the generic name Propionibacterium in the family
Lactobacillaceae when Van Niel (1928) published a comprehensive treatise on the
propionibacteria. Orla-Jensen's (1921) proposal of a second genus "Propionicoccus JJ
was, however, rejected as Van Niel (1928) was convinced that different cultural
conditions which could change the morphology of an organism, could result in the
same species being classified under different genera. Despite this fact, Van Niel (1928)
used cell morphology successfully in differentiating the various groups. Other
characteristics which played a significant role in the placement of the groups in the
genus Propionibacterium were the fact that they were all nonmotile, Gram-positive,
formed no spores, grew into short rods under anaerobic conditions in a neutral medium
and into irregular long rod shaped cells under aerobic conditions, catalase positive,
producing propionic acid, acetic acid and carbon dioxide during the fermentation of
lactic acid, carbohydrates and polyalcohols. Complex nitrogen compounds were also
required for growth and development.
Van Niel (1928) designated Propionibacterium Freudenreichii as the type
species and recognised eight species (Fig. 3). Van Niel (1928) also included a concise
description of each species, with the properties summarised in the form of a key. This
24
"Bacterium acidi propionici var. rubrum" - 1) "P. rubrum"
(Thoni and Alleman, 1908)
"Bacterium acidi propionici var. rubrum" - 2) "P. Thënii'
(Thoni and Alleman, 1908)
"Bacillus acidi propionici" - 3) "P. pentosaceum"
(Von Freudenreich and Orla-Jensen, 1906)
- 4i) "P. Jensenii var. raffinosaceum"
"Bacterium acidi propionici b" - 4ii) "P. Jensenii"
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici c" - 5) "P. Peterssonii"
(Troili-Petersson, 1909)
- 6) "P. technicum"
"Bacterium acidi propionici var.fuscum" - 7ii) P. Freudenreichii
(Thoni and Alleman, 1908) (Synonym)
"Bacterium acidi propionici a" - 7i) P. Freudenreichii
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici d" - 8) "P. Shermanii"
(Sherman, 1921)
(1-8 = Van Niel, 1928)
Fig. 3. A list of the Propionibacterium species as proposed by Van Niel (1928).
25
key was based on morphological characteristics, fermentation patterns, pigment
production and growth in liquid or stab culture. Orla-Jensen (1909) proposal to use
differences in acid formation as a distinguishing characteristic was rejected.
"Bacterium acidi propionici var. rubrum" isolated by Thoni and Alleman
(1908), was represented by four strains, two distinguishable from the other two. Van
Niel (1928) named the one group "Propionibacterium rubrum" and the other
"Propionibacterium Thorui ". "Propionibacterium acidi propionici var. fuscum"
(Thoni and Alleman, 1908) was, however, considered to be a synonym of
Propionibacterium Freudenreichii. Van Niel (1928) further suggested that the genus
Propionibacterium should rather be placed between the bacteria of the colon group
and the lactic acid bacteria, rather than between the latter and the butyric acid and
butyl alcohol bacteria due to the characteristic presence of the catalase enzyme in the
propionic acid bacteria, despite their strictly anaerobic nature.
The genus Propionibacterium was subsequently officially recognised for the
first time, as a legitimate genus in the third publication of Bergey's Manual of
Determinative Bacteriology (Bergey, 1930). The genus was placed in the family
Bacteriaceae, as one of the genera of the tribe Propionibacteriaceae, using Van Niel's
(1928) method for differentiation.
Further isolations: 1931 - 1944
Werkman and Kendell (1931) raised "P. Jensenii var. raffinosaceum" to
specific rank as "P. raffinosaceum" (Fig. 4). They criticised Van Niel (1928) for using
pigmentation to differentiate between the species of the propionic acid bacteria. In
1934, Hitchner described two new species isolated while studying the physiological
characteristics of the propionic acid producing bacteria. These two isolates were
named "P. zeae" and "P. arabinosum" (Fig. 4). In 1933, the validity of the eleven
Propionibacterium species was re-evaluated by Werkman and Brown. They reported
that without exception it was possible to recognised the various species individually,
whatever criterion for classification was used. Three subgeneric groups were formed
based on serological differentiation (Fig. 4). "Propionibacterium zeae" and
26
"Bacterium acidi propioniel var. rubrum" - 1) "P. rubrum"
(Thont and Alleman, 1908)
"Bacterium acidi propionici var. rubrum" - 2) "e ThOnil~~
iThoni and Alleman, 1908)
"Bacillus acidi propionicï" - 3) "P. pentosaceum'~
(Von Freudenreich and Oria-Jensen, 1906)
"P. arabinosum'~
- 4i) "P. Jensenii var. raffinosaceum"
---t "P. raffinosaceum"
"Bacterium acidi propionici b" - 4ii) "e .lensenii'~ (Werkman and KendelI, 1931)
(Von Freudenreich and Oria-Jensen, 1906)
"Bacterium acidi propionici c" - 5) "P. Peterssonii':
(Troili-Petersson, 1909)
"P. zeae"
"Bacterium acidi propionlei var.fuscum" - 7ii) P.:..f..~~~U!.~!!.~~i~h#
(Thoni and Alleman, 1908) (Synonym)
"Bacterium acidi propionici a" - 7i) P.:..f.r.~~!!~!!r.~~t;,hi!.
(Von Freudenreich and Oria-Jensen, 1906)
"Bacterium acidi propionici d" - 8) ~~P..$: .h~r.'1!~!!!.~f~
(Sherman, 1921)
(1-8 = Van Niel, 1928)
(_ /= / = Werkman and Brown, 1933)
Fig.4. The three subgeneric groups formed within the genus Propionibacterium (Werkman and Brown,
1933).
27
"P. rubrum" were found to show relatively little relationship to any of the three
groups. Werkman and Brown (1933) also proposed an identification key based
primarily on carbohydrate fermentation. Pigment production, catalase and nitrate
reduction were also mentioned, but to a lesser degree.
Controversies experienced during the placement of strains III specific
species
The first indication that the Il published and accepted species did not include
all forms of the propionic acid bacteria arose when Rodenkirchen (1938) found that
three of his 25 isolates could not be identified using the Werkman and Brown (1933)
classification system.
In 1941, Sakaguchi and eo-workers described five new species and one new
variant which they had isolated from Japanese cheese. They named their species
"P. globosurn", "P. amylaceum", "P. japonicum Jl, "P. orientum" and "P. coloratum"
(Fig. 5). The new variant was named "P. amylaceum var. auranticum" (Fig. 5).
Using the classification systems of both Van Niel (1928) and Werkman and Brown
(1933), Sakaguchi et aI., (1941) reported that a revision of the classification systems
would be required to establish the taxonomic position of their newly isolated species.
Furthermore, .Janoschek (1944) reported that of his 200 isolates, mostly
isolated from milk and cheese, 53 could not be identified further than the generic
level. .Janoschek (1944) subsequently described three more species, namely: "P.
casei"; "P. pituitosum"; and "P. sanguineum " (Fig. 5). The new species described by
both Sakaguchi et al. (1941) and Janoschek (1944) were clearly separable from the Il
previously described species in the 5th edition of Bergey's Manual (Bergey et aI.,
1939). These species subsequently constituted valid descriptions, since in both cases,
the newly proposed species were clearly described and correctly named (Buchanan et
aI., 1966). In the next edition of Bergey's Manual (Bergey, 1948), the newly
described species were, however, considered as variants of existing species and were
only mentioned in the appendix.
28
"Bacterium acidi propionici var. rubrum' - 1) "P. rubrum'
(Thoni and Alleman, 1908)
"Bacterium acidi propionlei var. rubrum' - 2) "e ThOn;;"
(Thoni and Alleman, 1908)
"Bacillus acidi propionici" - 3) "P. pentosaceum"
(Von Freudenreich and Orla-Jensen, 1906)
"P. arabinosum"
- 4i) "P. Jensenii var. raffinosaceum"
~ "P. raffinosaceum"
"Bacterium acidi propionici b" - 4ii) "e .lensenii" (Werkman and KendelI, 1931)
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici c" - 5) "P. Peterssonii"
(Troili-Petersson, 1909)
"P. zeae"
"P. amylaceum"
"P. amylaceum var.
auranticum"
"P. japonicum"
"P. pituitosum"
"Bacterium acidi propionlei var.fuscum" - 7ii) f.:..!:t~ff!k!!t~!f~#
(Thoni and Alleman, 1908) (Synonym)
"Bacterium acidi propionici a" - 7i) f.:..f.t~ff4~!!!.!!.if~#
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici d" - 8) ~~f...$:.~~r.1JJ;!mi.e
(Sherman, 1921)
"P. case;"
(1-8 = Van Niel, 1928)
"P. globosum"
(_ /_ / . = Werkman and Brown, 1933)
(Hitchner, 1934) "P. orientum"
(Sakaguchi et al., 1941) "P. coloratum"
(Janoschek, 1944)
"P. sanguineum"
Fig. 5. The eight five new species and one new variant isolated by Sakaguchi et al. (1941) and
Janoschek, 1944.
29
The family Propionibacteriaceae
In the 7th edition of Bergey's Manual (Breed et al., 1957), the propionic acid
producing bacteria were placed in the order Eubacteriales, in a new family
Propionibacteriaceae as proposed by Delwiche (1954). The family consisted of the
genera Propionibacterium, Butyribacterium and Zymobacterium. This family was
placed between the Lactobacillaceae and the Corynebacteriaceae because of its
relationships to these families. The existing eleven species (Van Niel, 1928) were
retained with only minor changes to the identification key. The species described by
Sakaguchi et al., (1941) and Janoschek (1944) were, however, once again not
mentioned by Breed et al. (1957).
In 1963, Antila and Gyllenberg while doing a study on the propionic acid
producing bacteria of dairy origin found that the species "P. casei" of Janoschek
(1944) should be given specific rank. Using the identification key of Janoschek (1944)
they were, however, not able to separate "P. Peterssonii" and P. Jensenii, but divided
"P. arabinosum" into two subgroups. They further differentiated the genus
Propionibacterium into four main groups (Fig. 6), namely: "P. arabinosum",
P. Freudenreichii, "P. Shermanii" and "P. casei"; "P. pentosaceum" and
"P. technicum"; P. Jensenii together with "P. raffinosaceum" and "P. Peterssonii";
and finally "P. Thonii''. Antila and Gyllenberg (1963) did, however, not find
"P. arabinosum JJ and "P. pentosaceum JJ to be closely related. They also concluded
that species consolidation was required.
Seyfried again studied the position of the family Propionibacteriaceae relative
to that of Lactobacillaceae in 1968 while doing a computer aided classification of the
lactobacilli. She found that the propionibacteria were clearly separated from the lactic
acid bacteria. Furthermore, she reported that the propionibacteria clustered together in
one group, with two unnamed cultures clearly distinguishable from the existing species.
Other cultures that could not be associated with known species of propionibacteria
were also reported by Malik et al. (1968). Whilst in studying the spoilage of pickled-
olives, Gonzalez-Cancho et al. (1970) also reported that certain cultures identified in
their study, using the identification key of Breed et al. (1957), could not be grouped
30
"Bacterium acidi propionici var. rubrum" - 1) "P. rubrum"
(Thoni and Alleman, 1908)
"Bacterium acidi propionici var. rubrum" - 2) "P. Thanii" (d)
(Thoni and Alleman, 1908)
"Bacillus acidi propionici" - 3) "P. pentosaceum" (b)
(Von Freudenreich and Oria-Jensen, 1906)
"P. arabinosum" (a)
- 4i) "P. Jensenii var. raffinosaceum" (c)
~ "P. raffinosaceum"
"Bacterium acidi propionici b" - 4ii) "e .lensenii" (c) (Werkman and KendeIl, 1931)
(Von Freudenreich and Oria-Jensen, 1906)
"Bacterium acidi propionici c" - 5) "P. Peterssonii" (c)
(Troili-Petersson, 1909)
"P. zeae"
"P. amylaceum"
"P. amylaceum var.
auranticum"
"P.japonicum"
"P.pituitosum"
"Bacterium acidi propionici var.fuscum" - 7ii) f..t..!:t~~4.l!!!t~ifh(#a)
tThoni and Alleman, 1908) (Synonym)
"Bacterium acidi propionici a" - 7i) f:.f.r.~'l:4~!!r.~~fM(aj)
(Von Freudenreich and Oria-Jensen, 1906)
"Bacterium acidi propionici d"
(Sherman, 1921)
"P. casei" (a)
(1-8 = Van Niel, 1928)
"P. globosum"
( _ /= / _= Werkman and Brown, 1933)
(Hitchner, 1934) "P. orientum"
(Sakaguchi et al., 1941) "P. coloratum"
(Janoschek, 1944)
"P. sanguineum"
(a-d) =Antila and Gyllenberg, 1963)
Fig.5. The four main groups as described by Antiia and Gyllenberg (1963)
31
within the existing eleven species of the genus Propionibacterium. It was thus evident
that not all propionibacteria had been studied and described, and that the existing
identification system of Breed et al. (1957) would have to be amended to incorporate
these unnamed strains.
The anaerobic coryneforms
Unna (1916) made the first observation of the organism originally referred to in
medical literature as the "acne bacillus". In 1900, Gilchrist named this bacterium
Bacillus acnes. Bacillus acnes was subsequently placed within the genus
Corynebacterium by Bergey (1923) primarily due to similarity in morphological
relationships between these two groups. Since the inclusion of this organism in the
genus Corynebacterium seemed questionable, Douglas and Gunter (1946) considered
it worth while to make a comparative study of the series of isolates.
They found that on a morphological basis C. acnes might be placed in either the
genus Corynebacterium or the genus Propionibacterium, for these two groups have
certain morphological features in common. Two characteristics, however, indicated
that the C. acnes strains were more related to the propionic acid bacteria than to the
corynebacteria. These characteristics were the nature of the catabolic process and the
effect of oxygen which either completely or strongly inhibited the growth of C. acnes.
This characteristic distinguished C. acnes from the typically aerobic corynebacteria.
Douglas and Gunter (1946) subsequently suggested that C. acnes should be
transferred to the genus Propionibacterium as Propionibacterium acnes inspite of the
fact that their data indicated that the organism did not ferment lactate to propionate.
Furthermore, Douglas and Gunter (1946) proposed that the definition of the
genus Propionibacterium should be modified to include non-lactate-fermenting
species. In addition, they found that the strains of C. acnes formed a homogeneous
group based on the following characteristics: optimum temperature; gelatin
liquefaction; action on milk; nitrate reduction; lactate fermentation; and habitat.
32
The nomenclatural changes proposed by Douglas and Gunter (1946) were,
however, not accepted, since the description of C. acnes under the genus
Corynebacterium was maintained in the 7th edition of Bergey's Manual (Breed et al.,
1957). In 1980, Skerman accepted the fermentation of lactate as a diagnostic criterion
for the genus Propionibacterium. Lactate fermenting cultures, which were apparently
otherwise similar to C. acnes, were isolated from the rumen of cattle by Gutierrez
(1953). Gutierrez (1953) subsequently assigned these isolates to C. acnes.
The validity of Corynebacterium acnes was tested in 1963 byMoore and Cato.
They demonstrated that the strains of C. acnes consistently fermented lactate with the
production of propionic acid under anaerobic conditions, thereby fulfilling the
diagnostic criteria as prescribed for members of the genus Propionibacterium. Their
results subsequently supported the proposal of Douglas and Gunter (1946) to
reclassify C. acnes to the genus Propionibacterium. Furthermore, their transfer would
not require a redefinition of the genus to include non-lactate-fermenting species as had
originally been suggested.
It wasn't until 1968 that any investigation was again initiated to evaluate this
observation. In this year Zierdt et al., proposed that C. acnes should be retained in the
genus Corynebacterium. This was based on differences they observed between these
two genera. Their motivation was founded on the morphological differences observed
between C. acnes and members of the genus Propionibacterium. Corynebacterium
acnes was delicate, of quite uniform bacillary morphology, occurred in pairs and single
and stained unevenly during Gram-staining. In contrast, species of the genus
Propionibacterium were robust, almost coarse, pleomorphic, occurred singly and
stained uniformly.
They further stated that it should be noted that C. acnes requires anaerobic
conditions for production of propionic acid from glucose or lactate, while
Propionibacterium produce it aerobically as well as anaerobically. To confirm their
results, they also named three other organisms which produced similar products during
their fermentation. These bacteria included Corynebacterium diptheriae, which
produced propionic acid from glucose (Tasman and Brandwijk, 1938); Veillonella
33
gazogenes (Johns, 1951); and an even more closely resembled example, a unidentified
Gram-positive coccus which was isolated from the rumen of sheep which was found to
produce propionic acid, acetic acid and CO2 from lactate (Elsden, 1945).
In 1972, Johnson and Cummins observed that several of the species classified
in the genus Corynebacterium were similar in their cell wall composition and the mol%
G+C content of their DNA to members of the genus Propionibacterium. Moore and
Holdeman (1973) subsequently reclassified several of the anaerobic to aerotolerant
members of the genus Corynebacterium as members of the genus Propionibacterium.
These bacteria were anaerobic, produced propionic acid as a major end-product of
their metabolism, contained mostly L-DAP as the diamino acid of peptidoglycan,
produced CIS iso-and anteiso-acids as the principle fatty acids of celllipids and lacked
mycolic acids and the arabinogalactan which was characteristic of the genus
Corynebacterium. These anaerobic coryneforms were subsequently reclassified as
Propionibacterium acnes, P. avidum, P. granulosum and P. lymphophilum. This was
partially in agreement with the proposal of Douglas and Gunther (1946), that C. acnes
should be transferred to the genus Propionibacterium.
The transfer of these anaerobic coryneforms to the genus Propionibacterium
was, however, very controversial. Some workers were in favour of the transfer
(Johnson and Cummins, 1972), while others considered the dissimilarities between the
two groups sufficient enough to support their separation (Langsrud and Reinbold,
1974). Prévot (1976) proposed that the P. acnes group of organisms should not be
transferred to the genus Propionibacterium, but should instead be accommodated in a
separate subgenus Coryneformis in the family Corynebacteriaceae, primarily due to
their pathogenic and reticulostimulatory properties.
These four species currently form the cutaneous members of the genus
Propionibacterium (Holt et al., 1994). The proposal of a new species
Propionibacterium propionicus (Charfreitag et al., 1988), which was closely related
to these four cutaneous members of the genus Propionibacterium, was only mentioned
by Holt et al. (1994) in the ninth edition of Bergey's Manual of Determinative
34
Bacteriology, although it was retained In the genus Arachnia for determinative
purposes.
Changes in the current taxonomy
A major change in the taxonomic status and the species arrangement of the
genus Propionibacterium occurred in the 8th edition of Bergey's Manual (Moore and
Holdeman, 1974). According to this system the propionibacteria were part of the
Actinomycetales and related organisms (Part 17) of Class 1, the Bacteria, in the
Kingdom Procaryotae. The family Propionibacteriaceae was retained with two
genera, the Propionibacterium and the Eubacterium.
Based essentially on cell wall composition and deoxyribonucleic acid
similarities as reported by Johnson and Cummins (1972), Moore and Holdeman (1974)
consolidated and reduced the 21 species within the genus Propionibacterium to eight
species. The species proposed by Moore and Holdeman (1974) were:
P. acidipropionici; P. freudenreichii; P. jensenii; and P. thoenii, which formed the
classical propionic acid bacteria group as well as P. acnes; P. avidum; P. granulosum;
and P. lymphophilum, which formed the cutaneous group. The species
"P. pentosaceum", "P. arabinosum" and "P. rubrum", "P. Thonii" were consolidated
to form the species P. acidipropionici and P. thoenii respectively (Fig. 7).
Furthermore, rune species namely: "P. raffinosaceum''; P. Jensenii;
"P. Peterssonii"; "P. technicum"; "P. zeae''; "P. amylaceum"; "P. amylaceum var.
auranticum"; "P. japonicum "; and "P. pituitosum" were consolidated to form the
species P. jensenii (Fig. 7). Propionibacterium Freudenreichii; "P. Shermanii";
"P. casei"; "P. globosum"; "P. orientum"; "P. coloratum"; and "P. sanguineum "
were consolidated to form the fourth species of the classical propionic acid bacteria
group, namely P. freudenreichii (Fig. 7). Propionibacterium freudenreichii was
further differentiated into three subspecies primarily due to their significance in the
cheese industry, namely: P. freudenreichii subsp. freudenreichii; P. freudenreichii
subsp. shermanii; and P. freudenreichii subsp. globosum. Propionibacterium acnes
was divided into two subgroups on the basis of cell wall composition and cell wall
35
"Bacterium acidi propionici var. rubrum" - 1) "P. rubrum'
(Thani and Alleman, 1908) P.thoenii
"Bacterium acidi propionici var. rubrum" - 2) "P. ThOnii"
(Thoni and Alleman, 1908)
"Bacillus acidi propionici" - 3) "P. pentosaceum" (b) _____
(Von Freudenreich and Orla-Jensen, 1906) ______ P. acidipropionici
"P. arabinosum" (a)
- 4i) "P. Jensenii var. raffinosaceum" (c)
~ "P. raffinosaceum"
"Bacterium acidi propionici b" - 4ii) "P. .lensenii" (c) \werkman and KendeII, 1931)
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici c" - 5) "P. Peterssonii"
(Troili-Petersson, 1909)
- 6) \
"P. amylaceum"
auranticum"
"P. japonicum"
"P. pituitosum"
"Bacterium acidi propionici var.fuscum" - 7ii) f.: ..f.):~~4l!!!.n~ifh(fai)
(Thoni and Alleman, 1908) (Synonym)
"Bacterium acidi propionici a" - 7i) f.:..f.r.~/:Y!~!!r.~if!t:(#a.)
(Von Freudenreich and Orla-Jensen, 1906)
"Bacterium acidi propionici d"
(Sherman, 1921)
(1-8 = Van Niel, 1928) "P. casei" (a)
P. freudenreichii
(_ /_ / = Werkman and Brown, 1933)
(Hitchner, 1934) "P. globosum"
(Sakaguchi et al., 1941) "P. orientum"
(Janoschek, 1944)
"P. coloratum"
(a-d) = Antila and Gyllenberg, 1963
"P. sanguineum"
(Moore and Holdeman, 1974)
Fig.7. The four species of the classical Propionibacterium group (Moore and Holdeman, 1974)
36
antigens. Propionibacterium avidum, P. granulosum and P. lymphophilum were
classified as individual species.
Moore and Holdeman (1974) subsequently also proposed a identification key
where differentiation between the species was based on the fermentation of certain
carbohydrates, the hydrolysis of both gelatine and esculin, and their reduction of
nitrate. According to Reinbold (1978) the change in the species arrangement within
the genus Propionibacterium, improved the taxonomy of the cutaneous
propionibacteria, but worsened the situation with respect to the identification of the
classical species and other propionic acid producing isolates. Despite using several
alternative techniques in an attempt to confirm the species groupings of Moore and
Holdeman (1974), Britz and Steyn (1979a: 1979b: 1979c) were largely unsuccesfull.
Techniques evaluated by them included pyrolysis-gas-liquid-chromatography, cellular
fatty acid profiles and deoxyribonucleic acid base composition (mol% G + C). Despite
the fact that Britz and Steyn (1979a) could divide the genus Propionibacterium into its
two major groups corresponding their ecological differences, they were not able to
differentiate the various species of the genus. Using cellular fatty acid profiles (Britz
and Steyn, 1979b), would also not differentiate between the various species of the
genus Propionibacterium. During an evaluation of the deoxyribonucleic acid base
composition (mol% G + C), Britz and Steyn (1979c) reported that the mol% G + C of
the classical group varied from 64-68 mol% whereas that of the cutaneous group
varied from 57-63 mol%. Using this technique, Britz and Steyn (1979c) were,
however, again not successful at differentiating the various species.
Even after this major change in the taxonomic status, several reports are to be
found in the literature concerning isolates which could not be identified using the
classification key of Moore and Holdeman (1974). In 1975 and 1978, Britz reported
the isolation of various propionibacteria from South African cheese, which were
difficult to identify using the key of Holdeman et al. (1973; 1977). In 1983, Bushel
mentioned a Propionibacterium denitrificans, which is not a recognised species since
no description of this strain appeared in Bergey's Manual of Systematic Bacteriology
(Cummins and Johnson, 1986). According to Beerens et al. (1986), Samain (1983)
reported the isolation of a Propionibacterium sp. from a methanogenic fermentation,
37
which could not be identified further than the generic level. Unfortunately no further
references or literature are available concerning this bacterium. Furthermore,
Dubourgier et al. (1988) considered Propionibacterium as one of the main genera of
the microbial conglomerate present in granular sludge, from an anaerobic
methanogenic reactor.
Reclassification of "Arachnia propioniea"
In 1959, Pine and Hardin isolated an organism morphologically resembling
Actinomyces israelii, but which produced propionic and acetic acids as major products
of glucose fermentation. Based primarily on the major metabolic products produced
by this strain, Buchanan and Pine (1962) classified this organism as Actinomyces
propionicus. Seven years later, however, Pine and Georg (1969) discovered the
presence of diaminopimelic acid in the cell wall of Actinomyces propionicus. Based on
these observations, they subsequently reclassified A. propionicus by creating a new
genus Arachnia, with Arachnia propionica as the only species.
Arachnia propionica differed from the other Actinomyces species in that it
produced propionic acid as one of the major end-products of glucose fermentation, it
possessed a murein based on the L-DAP and contained major amounts of iso- and
ante iso-methyl branched fatty acids. Most of these characteristics were similar to that
of the genus Propionibacterium. Based on cell wall composition and deoxyribonucleic
acid similarities, Johnson and Cummins (1972) could, however, not find any homology
between Arachnia propionica, the classical Propionibacterium and the anaerobic
coryneforms. The bacterium was subsequently retained as the only species within the
genus Arachnia by both Moore and Holdeman (1974) and Cummins and Johnson
(1986).
Using reverse transcriptase sequencing of long regions of the 16S ribosomal
ribonucleic acid, Charfreitag et al., (1988) found that A. propionica was unrelated to
members of the genus Actinomyces, but formed a distinct group with P. freudenreichii
and P. acnes. Charfreitag et al. (1988) subsequently stated that the retention of
Arachnia as a separate genus was unjustified and proposed that Arachnia propionica
38
be reclassified as Propionibacterium propionicus. The placement of A. propionica
with the propionibacteria on the basis of comparative analysis of 16S rRNA sequences
is in accordance with the physiological and chemical similarities of these organisms.
Additional data which supported the transfer of Arachnia propionica to the
genus Propionibacterium were the cellular fatty acid composition as determined by
O'Donnell et al., (1985) and Cummins and Moss in 1990. They reported that the cell
walls of Arachnia propionica contained large amounts of 13-methyltetradecanoic acid
(isoC15:0) and 12-methyltetradecanoic acid (antisoC15:0), resembling the pattern
found in propionibacteria. Cummins and Moss (1990), however, further argued that
the name should rather be Propionibacterium propionicum, since Propionibacterium
is a neuter noun.
Systematic position of "P. coccoides"
"Propionibacterium coccoides" was first described in 1983 by Vorobjeva et al.
This bacterium was isolated from "Soviet" cheese where it plays a significant role in
the aging process of hard cheeses. Vorobjeva et al., (1983) designated "P. coccoides"
as a member of the genus Propionibacterium based on the following characteristics:
the same phenotypic characteristics as for the propionic acid bacteria;
propionic acid was formed as the major product of lactate fermentation. This
propionic acid was produced from methylmalonyl-CoA, formed as a coenzyme
B12-dependent isomerization reaction, which is a key systematic characteristic
of the propionic acid bacteria;
the electrophoretic mobility of the enzymes (catalase, superoxide dismutase and
peroxidase) were the same as those observed for the propionic acid bacteria;
the mol% G + C content of their DNA was determined to be 63.4 mol %. That
of the other propionic acid bacteria was found to be 65 - 67 mol %; and
their genome structure (DNA homology) showed closest similarity (49%) to
the propionic acid bacterium P. jensenii.
39
According to the definition of the genus Propionibacterium as given by Moore and
Holdeman (1974), this propionic acid producing coccus should subsequently be
classified in this genus. No mention of this strain or the proposal of Vorobjeva et al.
(1983) was made in Bergey's Manual of Systematic Bacteriology (Cummins and
Johnson, 1986). Furthermore, "P. coccoides" was also not included in Bergey's
Manual of Determinative Bacteriology (Holt et aI., 1994) despite the fact that
Cummins and Johnson (1992) stated that there was sufficient evidence to include this
species in the genus Propionibacterium.
Reclassification of Propionibacterium innocuum
In 1991, Pitcher and Collins proposed Propionibacterium innocuum as a new
species within the genus Propionibacterium to accommodate strains of coryneform
bacteria from human skin with phenotypic characters similar to those of the classical
Propionibacterium group. The only differences between these strains and the classical
Propionibacterium group were that they exhibited aerobic respiration and possessed a
unique cell wall composition in which LL- diaminopimelic acid and arabinose occurred
together. Based on partial 16S rRNA sequence data, Pitcher and Collins (1991)
observed that these strains represented a distinct line within the genus
Propionibacterium.
In 1994, Yokota et al., based on 16S ribosomal DNA analysis, reported that the
phylogenetic neighbour of Propionibacterium innocuum was Luteococcus japonicus
and that this pair of organisms branched intermediately between the genus
Propionibacterium on the one side and both genera Aeromicrobium and Nocardioides
on the other side. The P. innocuum strains differed from the species of
Aeromicrobium and Nocardioides by the formation of propionic acid and from the
species of Luteococcus in their morphology. Yokota et al., (1994) subsequently
proposed the transfer of P. innocuum (Pitcher and Collins, 1991) to a new genus,
Propioniferax, as Propioniferax innocua.
40
Reclassification of "Propionibacterium rubrum"
Moore and Holdeman (1974) consolidated "P. Thonii IJ, P. rubrum, and
"P. sanguineum" to form the species P. thoenii. This consolidation was considered
valid due to the high DNA homology values observed between these old species
(Johnson and Cummins, 1972) as well as many characteristics in common, including
the production of an intense red or reddish brown pigment and being p-hemolytic on
blood agar. Malik et al. (1968) as wel! as Britz and Riedel, (1991: 1995), however,
reported a low level of phenotypic similarity between the "P. rubrum" and P. thoenii
species. These results were subsequently also confirmed by Riedel and Britz (1992)
who observed a high degree of similarity between the P. jesenii and "P. rubrum"
strains based on electrophoretic protein profiles.
In 1995 De Carvalho et al. investigated the application of 16S rRNA sequence
analysis in determining the taxonomic relationship of strains previously designated as
"P. rubrum" to the species P. thoenii and P. jensenii. They reported that in their
comparison of the complete 16S rRNA gene sequence, only twelve bases differed
between the old "P. rubrum" species and the P. jensenii species. De Carvalho et al.,
(1995) considered these two species to be almost identical. Confirming the results of
Malik et al., (1968), Britz and Riedel (1991) as well as Riedel and Britz (1992). De
Carvalho et al., (1995) thus subsequently proposed that "P. rubrum" be reclassified as
a p-hemolytic biovar of P. jensenii. They further differentiated the genomic species
P. jensenii and P. thoenii by biochemical characteristics such as production of acid
from myo-inositol and starch.
The description of Propionibacterium cyclohexanicum
The latest addition to the genus Propionibacterium was the description of a
new species, namely P. cyclohexanicum, which was isolated from spoiled orange juice
by Kusano et al. (1997). The results obtained of a phylogenetic analysis of the 16S
rRNA gene indicated that its highest level of homology is with the representative of the
classical propionibacteria, P. freudenreichii (97.1%). Although this strain was
41
phenotypically also similar to P. jreudenreichii, it produced a large amount of lactic
acid and had a distinct fatty acid composition, acid tolerance and heat resistance, which
differentiated it from P. jreudenreichii and the other propionic acid-producing
bacteria. In their unrooted phylogenetic tree, Kusano et al. (1997) reported that the
most closely related genera to the genus Propionibacterium were Luteococcus,
Nocardioides (Yokota et al., 1994) and Terrabacter.
The genus Luteococcus was first described in 1997 by Kusano et al. for a new
Gram-positive, non-motile coccus. Isolated from water and soil, Luteococcus
japonicus has the following chemotaxonomie characteristics: Menaquinone .MJ(-9(~);
67mol% G + C content; and L-diaminopimelic acid, alanine, glycine and glutamic acid
being produced in a molar ratio of ea. 1:2:1:1. The partial 16S rRNA sequence data
further indicated the genus represented a distinct line of descent among the Gram-
positive bacteria with a high G + C content.
Characteristics of the genus Nocardioides, include the formation of
pleomorphic elements to branched vegetative mycelium, which break up into short to
elongated rodlike fragments. Members of the genus Nocardioides are susceptible to
specific phages and contain diphosphatidylglycerol, phosphatidylglycerol and two
incompletely characterised lipids. Tetrahydrogenated menaquinones with either eight
or nine isoprene units (MK-8(~), 9(~)) predominate (Holt et al., 1994).
Characteristics of the genus Terrabacter include a rod-coccus growth cycle
that occurs during growth on complex media. Although no aerial mycelium are
produced, the long rods show pnmary branching. They contain
diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and some
unknown amino-containing phosphoglycolipids. Tetrahydrogenated menaquinone with
eight isoprene units saturated at sites II and III (MK-8(I1, III-~)) are the predominant
isoprenolog (Holt et al., 1994).
42
Present work done on propionibacteria
The taxonomic re-evaluation of the groupings amongst the propionibacteria are
currently dominated by the application of molecular techniques. In 1989, Charfreitag
and Stackebrandt determined the phylogenetic structure of the genus
Propionibacterium based on the comparative analysis of the 16S rRNA sequence data.
Their results confirmed the relationships within the genus Propionibacterium as
reported by Johnson and Cummins (1972), based on total DNA similarities.
Propionibacterium jensenii and P. thoenii were observed to be closely related and
formed a tight cluster with P. acidipropionici. NI three these species contain the LL-
isomer of diaminopimelic acid in their peptidoglycan and can ferment sucrose and
maltose (Charfreitag and Stackebrandt, 1989). This phylogenetic cluster was
observed to be well separated from the fourth classical species P. freudenreichii, which
contains meso-diaminopimelic acid in their peptidoglycan. Propionibacterium
freudenreichii was, however, found to be equidistantly related to both P. propionicus
and P. acnes representing the cutaneous Propionibacterium group. The data of
Charfreitag and Stackebrandt (1989) subsequently did not support the proposal of
Prévot (1976) to accommodate P. acnes and related taxa into a separate subgenus
Coryneformis in the family Corynebacteriaceae.
Using two-dimensional gel electrophoresis of ribosomal proteins Dekio et al.
(1989) could differentiate between the two serological types of P. acnes.
Differentiation between P. acnes and P. granulosum was also possible using this
technique. The technique is, however, not suitable for the rapid and routine
identification of propionibacteria, since it is cumbersome and time consuming.
In 1991, Britz and Riedel undertook a numerical taxonomic study on
propionibacteria obtained from dairy sources. NI four classical Propionibacterium
species could be differentiated. Two distinct groups within the P. jensenii species
could, however, be identified if pigmentation was used as differential characteristic. In
a numerical taxonomic study of propionibacteria isolated from anaerobic digesters,
Riedel and Britz (1993) reported that some isolates could be identified as a specific
species using the identification system of Cummins and Johnson (1986), but based on
43
overall similarity, many of the strains clustered among members of another species.
This was especially obvious in species identified as P. jensenii and P. acidipropionici.
Their explanation for this observation was that the strains were identified as a specific
species based on only five biochemical characteristics, and that the major differential
characteristic between the species P. jensenii and P. acidipropionici was the reduction
of nitrate, which was observed to be a highly variable phenotypic characteristic.
Riedel and Britz (1992) could differentiate all four the classical species based
on numerical analysis of electrophoretic protein profiles. Certain clusters were,
however, electrophoretically heterogeneous and P. jensenii and P. thoenii were
observed to cluster together. Delineation between the various P. freudenreichii
subspecies was also not achieved.
Baer and Ryba in 1992 evaluated serological methods for the identification of
the classical propionibacteria and compared their results with that of Werkman and
Brown (1933). Werkman and Brown could only distinguish three subgeneric groups
containing the 11 species described at that stage. Bacr and Ryba (1992), were,
however, able to serologically differentiate the four classical species within the genus
Propionibacterium. Werkman and Brown (1933) also succeeded in serologically
differentiating P. freudenreichii subsp. freudenreichii from P. freudenreichii subsp.
shermanii. These results could, however, not be confirmed by Baer and Ryba (1992).
By obtaining these close relationship between the various P. freudenreichii subspecies,
Baer and Ryba (1992) also confirmed the DNA-hybridisation results of Johnson and
Cummins (1972) and the SDS-PAGE profiles obtained by both Baer and Ryba (1988)
and Riedel and Britz (1992).
While studying the complexity of the esterase system of the propionic acid
bacteria using PAGE separation, Dupuis and Boyaval (1993) only observed three
different patterns for the five species examined. Strains of P. freudenreichii could be
differentiated from P. acidipropionici, and the P. jensenii / P. thoenii group, which
displayed an identical profile. Propionibacterium freudenreichii subsp. freudenreichii
and P. freudenreichii subsp. shermanii could not be differentiated, again confirming
44
the results obtained by Baer (1987), Baer and Ryba (1988) as well as Riedel and Britz
(1992) who evaluated electrophoretic protein profiles.
Plasmid profiling is a simple and expressrve technique and the presence of
plasmids in propionibacteria has been described by numerous researchers (Panon,
1988; Perez Chaia et al., 1988; Rehberger and Glatz, 1990). The occurrence of
plasmids in the genus Propionibacterium varies between 25 and 38% of all strains.
Subsequent studies report that approximately 70% of all propionic acid bacteria either
have no plasmids (Rehberger and Glatz, 1990). The plasmid size is also an arguable
issue since it varies from 3kb to over 150kb. The only drawback of this technique is
the instability of the profiles obtained primarily due to the acquisition, loss or transfer
of plasmids.
With the exception of the ability to ferment lactose, associated with plasmid
pRG03 in a strain of P. freudenreichii subsp. globosum (Rehberger and Glatz, 1990)
and a clumping phenomenon associated with plasmid pRG05 in a strain of P. jensenii
(Rehberger and Glatz, 1990), no evidence has been found to indicate plasmid
association for bacteriocin production, pigment production, fermentation
characteristics, growth characteristics or antibiotic resistance.
In the differentiation of species within the genus Propionibacterium, phage
typing is generally considered an unsuccessful technique primarily due to the fact that
the technique is cumbersome and lacks the precision required for routine strain
differentiation. Presently, only bacteriophages active against the cutaneous P. acnes
species (Jong et al., 1975) and the classical P. freudenreichii species (Gautier et al.,
1992b; 1995) have been detected. The application of phagetyping (Webster and
Cummins, 1978) and a combination of phage typing with biotyping (Jong et al., 1975)
resulted in the differentiation of the two serotypes of P. acnes. Propionibacterium
acnes I strains were observed to be more susceptible to phage lysis than serotype II
strains. Although only 18% of P. freudenreichii strains analysed by Gautier et al.
(1995) were sensitive to phages, bacteriophages that were active against
propionibacteria were found in at least 50% of the Swiss cheese examined by them.
45
In 1992, Gautier et al. (1992a) determined that the genome size of the four
classical propionibacteria ranged from 2300 to 3200 kb depending on the specific
species. A year later, however, Rehberger (1993) undertook a genomic analysis of
P. freudenreichii and determined that the estimates for the genome size ranged from
1600 - 2300 kb.
In 1994 and 1998, Riedel et al. reported an analysis of restriction fragment
length polymorphisms within the 16S rDNA genes of the classical propionibacteria.
Using the restriction endonucleases HaelII, AluI and HpaII, all four classical species
could be differentiated. A higher degree of similarity was observed between both the
old "P. rubrum" and "P. sanguineum" species and the P. jensenii species than between
these two species and the P. thoenii and P. freudenreichii species, respectively.
Later in the same year, De Carvalho et al., reported a study where the
classical species were evaluated using ribotyping. Using the restriction endonucleases
BamHI and ClaI, De Carvalho et al. (1994) could differentiate between all four the
classical species and it was also possible to distinguish P. freudenreichii subsp.
freudenreichii from P. freudenreichii subsp. shermanii. This was, however, in
contrast to the results reported by Riedel et al., in 1994, where no differentiation was
obtained between the three subspecies of P. freudenreichii. De Carvalho et al. (1994)
reported ribotyping to be an excellent tool for species determination, since different
patterns with species specific fragments could be obtained for the four classical
Propionibacterium species. The ribotype patterns obtained also differed markedly from
those obtained for phylogenetically closely related genera (Bifidobacterium,
Brevibacterium, Corynebacterium and Mycobacterium) as well as for other dairy
bacteria (Lactococcus, Lactobacillus and Leuconostoc).
In 1996, Riedel and Britz also reported the evaluation of ribotyping as a
technique for the differentiation of the classical propionibacteria. In contrast to De
Carvalho et al. (1994), Riedel and Britz (1996) used the restriction endonucleases
HaelI and Smal. Sixteen distinct profiles could be observed within the classical
propionibacteria. Two profiles were observed within the P. acidipropionici species,
one profile for P. freudenreichii, nine profiles were observed in all strains representing
46
the P. jensenii species and four profiles for the P. thoenii species. The ribotyping
profiles observed for "P. rubrum" and "P. sanguineum" were similar to that of the
P. jensenii species, confirming the RFLP results of Riedel et al., (1994).
Gautier et al. (1996) differentiated between various Propionibacterium strains
by applying DNA fingerprinting using pulsed-field gel electrophoresis. Poor pattern
variability was, however, observed amongst the P. acidipropionici, P. jensenii and
P. thoenii species and the application of this technique was subsequently limited. A
high degree of restriction fragment length polymorphism was observed which
confirmed the results obtained by Rehberger (1993). The high degree of heterogeneity
in the restriction fragment patterns among the majority of P. freudenreichii strains was
rather unexpected, especially due to the high DNA relatedness among
P. freudenreichii strains, that has been reported to be 90% (Johnson and Cummins,
1972).
Despite the application of various conventional and molecular techniques, it
is, however, still evident from the literature that many discrepancies are still apparent
concerning the isolation, identification and differentiation of the various classical
members of the propionic acid producing bacteria. Inspite of the fact that
"P. coccoides" was originally proposed as a new Propionibacterium species by
Vorobjeva et al. in 1983, no systematic evaluation of "P. coccoides" and the propionic
acid bacteria has ever been undertaken. Subsequently "P. coccoides" has never been
officially recognised as a new species within the genus Propionibacterium. The exact
systematic position of "P. coccoides" relative to that of the other Propionibacterium
species is subsequently still unresolved.
The objectives of this study were thus to phenotypically characterise
"P. coccoides" and the varIOUS classical Propionibacterium species in order to
determine the relationship between these orgarusms. The next objective was to
determine the relationship of the "P. coccoides" strains to known Propionibacterium
marker strains using various molecular techniques, based on the 16S ribosomal RNA
region, as well as DNA:DNA hybridisation.
47
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59
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60
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Bacteriol. 1,33-47 (1968).
61
Chapter 3
A numerical taxonomic study of" Propionibacterium coccoides", other
classical Propionibacterium species and Luteococcus japonicus
Summary
Despite the fact that "Propionibacterium coccoides" was originally proposed
as a new species within the genus Propionibacterium in 1983, the exact systematic
position of this bacterium still remains unresolved. The systematic position of
"P. coccoides" relative to that of the classical Propionibacterium species and
Luteococcus japonicus was subsequently evaluated using numerical taxonomic
techniques. Four type, 13 reference and 15 classical Propionibacterium strains as well
as two "P. coccoides" strains were included in this study. The type strain of
Luteococcus japonicus, which is a phylogenetic neighbour of the genus
Propionibacterium, was also included as an outgroup. A data set of 75 phenotypic
characters, obtained using standardised AP! systems and conventional methods was
analysed. To delineate the clusters, use was made of the Sokal and Michener (SSM)
coefficient and the average linkage clustering algorithm (UPGMA). All the classical
Propionibacterium strains were observed to group into four major clusters, with a final
linkage at the 81% S-level. These clusters could be equated with the
P. acidipropionici, P. freudenreichii, P. jensenii and P. thoenii species using the
current classification system. The two "P. coccoides" strains, however, grouped in a
separate cluster with the Luteococcus japonicus type strain. The L. japonicus type
strain and the two "P. coccoides" strains clustered together at the 80% S-level. This
cluster was clearly delineated from the various classical Propionibacterium species and
linked with these clusters at a 72% similarity level. Based on these results it is evident
that a higher phenotypic similarity exists between the L. japonicus and the
"P. coccoides" strains than between the latter and the various classical
Propionibacterium species.
62
Introduction
The genus Propionibacterium consists of two principal groups, the classical or
dairy and the cutaneous or clinical strains. During the last 20 years, the taxonomy of
the genus Propionibacterium has undergone extensive changes. Twenty three classical
forms of the genus Propionibacterium were isolated and described during the period
1906 to 1944 (Britz and Steyn, 1980). Of these only 11 were regarded as species by
Breed et al. (1957). In 1974, Moore and Holdeman consolidated these 11 species into
four classical species, namely: Propionibacterium acidipropionici, P. freudenreichii,
P. jensenii, and P. thoenii. Recently Kusano et al. (1997) described a new classical
species namely P. cyclohexanicum. Although isolated from spoiled orange juice,
results obtained from a phylogenetic analysis of the 16S rRNA genes indicated that its
highest level of 16S rRNA similarity was with a representative of the classical
propionibacteria, namely P. freudenreichii.
Based primarily on the research of Johnson and Cummins (1972), Moore and
Holdeman (1974) transferred four species from the genus Corynebacterium to the
genus Propionibacterium. These species subsequently constituted the cutaneous
members of the genus Propionibacterium. These species included: Propionibacterium
acnes; P. avidum, P. granulosum; and P. lymphophilum. In 1988 Charfreitag et al.
proposed that Arachnia propionica be reclassified as Propionibacterium propionicum,
confirming the research of 0 'Donnell et al. (1985).
In 1983, Vorobjeva et al., described a bacterium which plays a significant role
in the aging process of Soviet hard cheeses. Due to the high degree of similarity
observed between this bacterium and members of the classical Propionibacterium,
Vorobjeva et al. (1983) proposed that this bacterium be named "Propionibacterium
coccoides". When compared to the other propionic acid bacteria, this bacterium also
produced propionic acid as the major metabolic product of lactate fermentation.
Furthermore "P. coccoides" also contained the anteiso isomer of the Cwsaturated
fatty acid (12-methyltetradecanoic) as the major type of cellular lipids, which is
characteristic of members of the genus Propionibacterium. The cellular fatty acid
composition of "P. coccoides" was also observed to be very similar to that of
P. jensenii (the old "P. technicum" strain). "Propionibacterium coccoides" was also
63
observed to produce considerable quantities of vitamin BI2, catalase, superoxide
dismutase and peroxide, which are characteristic of the propionic acid bacteria. In
addition, the high mol% G + C content of the "P. coccoides" strains were similar to
that of members of the genus Propionibacterium. Furthermore, the highest
DNA:DNA homology (49%) was observed between "P. coccoides" and P. jensenii
(Vorobjeva et al., 1983).
In spite of this very thorough investigation and the detailed report concerning
the systematic position of "P. coccoides" (Vorobjeva et al., 1983), no further
comparative evaluations between "P. coccoides" and the classical propionibacteria
have ever been undertaken. Although numerous taxonomic studies have attempted to
solve the taxonomic dilemma within the classical propionibacteria, no studies have
included "P. coccoides", Subsequently the correct systematic grouping of this
organism still remains unresolved. Furthermore, although this proposal was
mentioned by Cummins and Johnson (1992), no mention of this has ever been made in
either the Bergey's Manual of Systematic Bacteriology (Cummins and Johnson, 1986)
or Bergey's Manual of Determinative Bacteriology (Holt et al., 1994).
The aim of this study was thus to further characterise "P. coccoides"
phenotypically and to determine it's systematic position relative to that of the classical
Propionibacterium species and the phylogenetically related Luteococcus japonicus.
Materials and Methods
Bacterial strains, growth conditions and preservation. The various bacterial
strains used during this study are presented in Table 1. The 32 Propionibacterium
strains used in this study comprised of the four type and 13 reference strains from the
ATCC (Anon. 1985), NCFB (Anon. 1986) and DSM as well as strains from the dairy
industry and anaerobic digesters. The two "P. coccoides" strains were kindly supplied
by Prof. L.I. Vorobjeva of the Moscow State University, Russia. The type strain of
Luteococcus japonicus (ATCC(R) 51527) was included as an outgroup during the
numerical analysis.
64
Table 1. List of bacterial strains used In this study. (The names are listed as
received).
No. Lab. Name Sourc Strain Isolated from Identification key#
No. e
I 78 "P. arabinosum" 5 P 50 P. acidipropionici
2 93 P. acidipropionici 7 Gouda P. acidipropionici
3 120 P. acidipropionici 7 Gruyere P. acidipropionici
4 263 P. acidipropionici 7 Anaerobic digester P. acidipropionici
5 349 P. acidipropionici 7 Leerdanuner P. acidipropionici
6 421 "P. pentosaceum" 4 NCFB 570 Enunentaler P. acidipropionici
7 424 P. acidipropionici I ATCC 25562* P. acidipropionici
8 456 "P. arabinosum" 5 P78 P. acidipropionici
9 73 P. freudenreichii 5 P 57 P.freud. ss.freudenreichii
10 131 P. freudenreichii 8 Enunentaler P. freud. ss. globosum
Il 308 P. freudenreichii 7 Anaerobic digester P. freud. ss. shermanii
12 348 P. freudenreichii 7 Leerdammer P. freud. ss. freudenreichii
13 423 P. freudenreichii I ATCC 6207* Swiss cheese P. freud. ss. freudenreichii
14 435 P. freudenreichii 6 B 3523 P.freud. ss.freudenreichii
15 453 "P. shermaniï 5 P 67 P. freud. ss. shermanii
16 454 P. freudenreichii 5 P 73 P. freud. ss. freudenreichii
17 75 P. jensenii 7 75 Enunentaler P. jensenii
18 80 P.jensenii 2 DSM 20535* Buttermilk P.jensenii
19 89 P. jensenii 7 Gouda P. jensenii
20 122 P.jensenii 7 Enunentaler P.jensenii
21 357 P. jensenii 7 Leerdammer P. jensenii
22 425 "P. sanguineum" 4 NCFB 1080 P.jensenii
23 427 "P. pituitosum" 4 NCFB 1077 P. jensenii
24 431 "P. peterssonii" 4 NCFB 565 Enunentaler P. jensenii
25 97 P. thoenii 7 Enunentaler P. thoenii
26 260 P. thoenii 7 Anaerobic digester P. thoenii
27 294 P. thoenii 7 Anaerobic digester P. thoenii
28 297 P. thoenii 7 Anaerobic digester P. thoenii
29 307 P. thoenii 7 Anaerobic digester P. thoenii
30 419 P. thoenii 4 NCFB 568* Enunentaler P. thoenii
31 426 "P. wentii" 4 NCFB 1083 Anaerobic digester P. thoenii
32 447 P. thoenii 5 P 15 P. thoenii
33 362 "P. coccoides'' 3 KM252 Soviet cheese
34 363 "P. coccoides" 3 KM375 Soviet cheese
35 364 L. japonicus ATCC(R) Ground and water
51527*
*. Type strain
#: Identification key of Cummins and Johnson (1986).
I: ATCC, American Type Culture Collection, Rockville, Maryland, USA.
2: DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany.
3: KM, Katedra Mikrobiologii, Department of Microbiology, Moscow State University, Russia.
4: NCFB, National Collection of Food Bacteria, Shinfield, Reading, UK.
5: G.W. Reinbold, Iowa State University, Iowa, USA.
6: U.S. department of Agriculture, USA.
7: Environmental Microbiology Culture Collection, University of the Orange Free State,
Bloemfontein, South Africa.
65
The bacterial strains were streaked onto yeast extract lactate (YEL) medium,
and incubated anaerobically for four days at 30°C in a Forma Scientific (Mallinckrodt,
Inc., Ohio, USA) anaerobic cabinet, using an oxygen-free nitrogen gas phase (10%
R21l0% COibalance N2) as recommended by Holdeman et al., (1977). The YEL-
medium consisted of (gil): yeast extract 6.0; sodium lactate (70% v/v) 20.0; peptone
2.0; KlI:zP04 10.0; hemin 10.0 ml and Tween 80 l.0 ml (Holdeman et al., 1977). The
pH was adjusted to 7.2 before sterilisation. Culture purity was regularly checked by
means of microscopical examination of Gram-stained preparations and by gas
chromatographical determination of the major metabolites produced. All the cultures
were preserved according to the lyophilisation technique as described by Joubert and
Britz, (1987). Lyophilised cultures were stored at -20°C. Lyophilized discs were used
to inoculate YEL-broth to obtain working cultures.
AP! and conventional tests. Seventy four substrate utilisation and biochemical
tests, as previously described (Britz and Riedel, 1991: 1994; Riedel and Britz, 1993),
were determined using the API 20E, API 20NE and API SOCR systems (API System
S.A., La Balme le Grottes, 38390 Montalieu Vercieu, France) as well as numerous
conventional tests. The conventional tests were undertaken to confirm phenotypic
characteristics, which are important in the identification of propionibacteria using the
identification key described by Moore and Holdeman (1974), Cummins and Johnson
(1986) and Holt et al. (1994). Phenotypic characteristics of all the Propionibacterium
strains, the two "Propionibacterium coccoides" strains as well as Luteococcus
japonicus were determined in a Forma Scientific anaerobic cabinet, using oxygen-free
nitrogen as gas phase (10% ~/10% COibalance N2). Each test was done in duplicate
and when different results were obtained the test was repeated. The majority result
was taken as being representative.
To determine the optimum growth temperature, growth was determined at
2SoC, 30°C, 3SoC, 37°C and 40°C by comparison of the optical density every 4 h.
Morphological characteristics of the various cultures were determined by bright-field
microscopy of Gram-stained preparations. Motility was observed after 48 h in MRS-
medium and colony pigmentation after 10 days on YEL plates. Extracellular
polysaccharide production was determined using the Indian ink wet method after 10
66
days incubation in YEL-medium and the formation of endospores was observed using
standard methods (Gerhardt et al., 1981).
Metabolic end-products were determined using a Hewlett Packard (Avondale,
PA, USA) gas chromatograph equipped with a flame ionisation detector and a 30 m x
0.53 mm i.d. Nukol (Supelco, Inc., Avondale, PA, USA) capillary column. The
chromatograph was programmed at an initial temperature of 120°C, then increased at a
rate of 6°C/min and finally held at 165°C. The detector and inlet temperatures were
250°C and 135°C, respectively. Nitrogen, at a flow rate of 5 rnlImin was used as the
earner gas.
A Varian 3300 gas chromatograph, equipped with a thermal conductivity
detector and column (2.0 m x 3.4 mm i.d.) packed with Porapak Q (Waters
Association, Inc., Milford, MA. USA), 80-100 mesh, was used for the determination
of CO2 in the gas phase. The oven temperature was set at 50°C and hydrogen, at a
flow rate of 40 ml/min, was used as the carrier gas. A Varian 3300 gas
chromatograph, equipped with a thermal conductivity detector and a stainless steel
column (1.2 m x 3.4 mm i.d.) packed with Molsieve 5A (Supelco, Inc., Avondale, PA,
USA) was used for the determination of H2 in the gas phase. The oven temperature
was set at 50°C and the detector temperature at 130°C. Nitrogen, at a flow rate of 30
rnlImin, was used as carrier gas.
Numerical analyses. Phenotypic and biochemical tests which gave uniform
results as well as duplicates of the different API systems, were excluded from the
computational analyses. The remaining 75 phenotypic characters were included in a
data set and analysed using the simple matching (SSM) coefficient of Sokal and
Michener (1958). The unsorted similarity matrix was rearranged into groups using the
average linkage cluster a.i.gorithm (upGMA) (Lockhart and Liston, 1970). Several
strains, randomly chosen, were examined in duplicate to estimate the average
probability of error (P) using the formula of Sneath and Johnson (1972).
67
Results
Strain characteristics. The phenotypic characteristics as determine for the 32
Propionibacterium strains, both "P. coccoides" strains and those for Luteococcus
japonicus are presented in Table 2. All 35 bacterial strains examined during this study
were Gram-positive, non-motile, non-sporing, anaerobic to aerotolerant cocci to
pleomorphic rods which produced propionic acid as major metabolic end-product with
lesser amounts of acetic acid and CO2.
During this study, the phenotypic data obtained for the vanous
Propionibacterium strains confirmed the data previously reported by Britz and Riedel
(1994: 1995) and Riedel and Britz (1993). Only two characteristics (lactose and nitrate
reduction) were found to differ from those results as reported for "P. coccoides" (KM
252) by Vorobjeva et al. (1983). With the exception of lactose fermentation which
was observed to be negative, the phenotypic data obtained during this study for
"P. coccoides" (KM 375) was identical to that as reported by Vorobjeva et al. (1983).
Gelatin liquefaction which was observed to be positive during this study, was found to
be the only alteration when the phenotypic data as obtained for .Luteococcus japonicus
(ATCC(R) 51527) during this study was compared with that as reported by Tamura et
al. (1994). All strains, including the two "P. coccoides" and L. japonicus strains were
lipase positive, produced acid from galactose, D-fructose and were D-xylose and 5
keto-gluconate negative.
Clustering of strains. The average probability of error (P) (Sneath and
Johnson, 1972) as calculated for several randomly chosen duplicate strains, was found
to be 2.8%. No serious distortion of the taxonomic structure would thus be expected.
A dendogram based on the overall similarity of the 32 Propionibacterium, the two
"P. coccoides" strains and the Luteococcus type strain based on the SSM similarity
coefficient and the UPGMA clustering algorithm is presented in Fig. 1. All thirty-five
strains were recovered in five major clusters, which linked at, or above the 72%
similarity level.
The first major cluster (cluster A) consisted of the P. freudenreichii type, four
reference and three P. freudenreichii strains with a overall similarity of 86%. Cluster
B consisted of the P. thoenii type, two reference and five P. thoenii strains. The
Table 2: Comparison of the various phenotypic characteristics obtained for the 35 strains evaluated during this study.
Organisms (as listed in Table 1)
Tests 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Acid from:
Glycerol + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + +Erythritol
D-Arabinose +-+++++-+ + + - + - + + +
L-Arabinose + + + + + + + + + +++++++++++++ - ++ --+
Ribose + + + + + + + + + + -------+
D-Xylose + + - +
L-Xylose - +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - +Adonitol + + + + + + + + + + + + + + + + + + + +
p-Methyl xyloside + + + + + + + + + +
Galactose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + - + + + + + + + + + + + + + + + + + + + +D-Glucose
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +D-Fructose + + +
D-Mannose +
L-Sorbose + - - + + + +
Rhamnose + + + + + + + + + + + +
Dulcitol + + + + + + + + - + + + + + + + + + + + +
Inositol + + + + + + + + + + + + + + + + + +
Mannitol + + + + + + + + + + + +
Sorbitol + + + + + + +
a.-Methyl-D-mannose + + + + + + + +
a.-Methyl-D-glucoside + +
+ + - - +
+
N-Acetyl-glucosamine +++-+++-+
Amygdalin + + + - + + + + +
+----++
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - +Arbutin
Esculin + + + - + + + + + + +
+ + + + + +
Salicin + + + + - + + + + + +
+
Cellobiose + + + + + + + + + + + +
+ - + + + + + + + + - - +
+ + + + + + + + + + - + + + + + + +- -++++ + + + + +Maltose
+ + + + - + + + +Lactose + - - + - + 0\00
+ + + + + + + + + + - + + + + + + + + + + + + + + +Melibiose
Table 2. Continued
+ + + + + + + + + + + + + + + + + + + + + + + + + + + +Trehalose + - +
Inulin + + + +
Melezitose + + + + + + + + + + +
D-Raffinose + - - + + + + + + + + + + - +
Amidon + + + + + +
Glycogene + + + +
+ - +
+ + + - + + + + + - + + + + + + + + + + +Xylitol
~-Gentiobiose + + + + + + + + - + +
+ + + + + + + + + - + + + + + + + + + + + + +D-Turanose
D-Lyxose
D-Tagatose + +
+ + + + + + + + + ++++++++ + + + + + + + + + +D-Arabitol
+ + + + + + + + + + + + + + + + + + - + + + + + + - +L-Arabitol
Gluconate + - + + + + + + + + + +
2 keto-gluconate + -
5 keto-gluconate
ONPG + + + + + + - + - - + + +
+ + + + + +
ADH + + + + +
+
+
LDe + + + + + +
ODe + + + +
+
eIT + + - + + +
+ - + +URE +
TDA + + + + + + + + + + + - + + + + + + + + +
IND - +
+ + + + + - - + + + + + - + + + + + + + +VP + +
GEL + +
Extracellular Polysacch:
L-DAP + + + - + - + +-+++-+----+
+ + - - +
+ + + N N + + + NN- - - -++++N+ + + + N N N N + + + N N Nmeso-DAP
Litmus milk: _ NN __ - + + NN + + + + - - - - N - - - - NNNN - - - NNN
acid
reduction +++ - - ++ - - - -+- - -+++ - + - - - - - - - ++ 0\
stormy + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
\.0
Table 2. Continued
fermentation + +
clot + + + ++----+---+++-+ + +
Catalase + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Pigment:
cream/white + + + + + + + + + + + + + + + + + + + - + - + +
yellow/orange + + +
brown/red +-+- -++++++++
Lipase
Opt. temp.:
30C + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + +
37C - ++ - - - - - + - - - - - - - - - - - - - - +
Morphology:
cocci +----+- + + + + - + - + + +
rod + + + + + + + + + - + + + + - + + + + + + + + + + + - + - + - +
pleomorphic + - + + ++--+---------+
Nitrate reduction + + + + + + ++-+-+++-+ +
+ Positive
Negative
N: Not determined
ONPG: Ortho-nitro-phenyl-galactoside
ADH: Arginine dihydrolase
LDC: Lysine dihydrolase
ODC: Ornithine utilisation
CIT: Citrate utilisation
URE: Urease
TDA: Tryrophane desaminase
IND: Indole
VP: Acetoin production
GEL: Gelatin liquefaction
L-DAP: L-diaminopimelic acid
Meso-DAP: Meso -diaminopimelic acid o-..l
71
Fig. 1. Dendrogram, based on the SSMcoefficient and the average linkage clustering
algorithm, showing the relationship between the classical Propionibacterium, the
"P. coccoides" and the L. japonicus strains. * = Type strain. ATCC, American Type
Culture Collection, Rockville, Maryland, USA; DSM, Deutsche Sammlung von
Mikroorganismen und Zellkulturen, Braunschweig, Germany; NCFB, National
Collection of Food Bacteria, Shinfield, Reading, UK; K.t\1, Katedra Mikrobiologii,
Department of Microbiology, Moscow State University, Russia.
72
Percenlage similarity Cluster (No. of strains) Type and Reference strains
so 60 70 80 90 100
P·frc:udC:/lreichii P 57
J'. freudcnreichii ATCC 6207'
P. [reudcnreichii P 73
A (8)
P.frel/Jel/reichii B 3523
"I'. shermanii" P 67
B(8)
P. theonil NCFB 568'
P. thoenii P 15
"I'. wentii" NCFB :083
"I'. peterssonii" NCFB 565
"I'. sanguineum" NCFB 1080
C (8)
P. species 75
"I'. pituitosum" NCFB 1077
Pi jensenii DSM 20535'
"I'. arabinosum" P 50
P. acidipropionici ATCC 25562'
0(8)
"I'. arabinosum" P 78
"I'. pentosaceum" NCFB 570
"I'. coccoides" KM 252
E (3) "I'. coccoides" KM 3 7 5
Luteococcus japonicus ATCC(R) 51527'
so 60 70 80 90 100
73
overall similarity of this cluster was 88%. The third cluster (Cluster C) contained the
eight strains of P. jensenii, namely the type, three reference and four other P. jensenii
strains. These strains clustered together at an overall similarity level of 83%. The last
cluster representing the genus Propionibacterium (Cluster D) was comprised of the
P. acidipropionici type, a single reference as well as six P. acidipropionici strains,
with a overall similarity of 87%. The two "P. coccoides" strains clustered together
(84%) with the L. japonicus type strain at an overall similarity level of 80% (Cluster
E). This cluster could clearly be differentiated from the four clusters representing the
various classical Propionibacterium species. Cluster E linked with the major clusters
representing the various classical Propionibacterium species at a 72 % S-level.
Differential phenotypic characteristics of the five major clusters observed
during this study are given in Table 3. Nine phenotypic characteristics which could be
used to differentiate clusters A, B, C, D and E from each other were identified during
this study (Table 4). Differentiation between cluster A and C, representing the
P. freudenreichii and P. jensenii species, respectively was only possible on
P. jensenii' s inability to ferment both dulcitol and inositol. It was also possible to
differentiate both clusters A and C from clusters B, D and E on the basis of the
fermentation of trehalose (cluster B), L-arabinose or melezitose (cluster D and E) and
D-mannose, sorbitol or yellow / orange pigmentation (cluster E). Based on the results
of this study, cluster B representing the P. thoenii species could be differentiated from
all the other cluster due to the inability of the P. thoenii strains to ferment trehalose
and D-arabitol. The ability of the "P. coccoides" and L. japonicus strains within
cluster E to ferment D-mannose and sorbitol as well as their characteristic yellow
pigmentation facilitated differentiation of this cluster from the other clusters
representing the classical Propionibacterium species. Bacterial strains within both
clusters D and E representing the P. acidipropionici and "P. coccoides" / L. japonicus
species, respectively were observed to ferment L-arabinose as well as melezitose,
which enabled differentiation of these clusters from the other major clusters.
Although the "P. coccoides" strains clustered with the L. japonicus strain in
cluster E, the "P. coccoides" strains could still be differentiated phenotypically from
the L. japonicus type strain. Differential characteristics included: acid production
from ribose; adonitol; L-sorbose; inositol; a.-methyl-D-glucoside; Neacetyl-glucosamin;
74
Table 3. Differential phenotypic characteristics of the various clusters as obtained
during this study
Tests Cluster (No. of strains)
A (8) B (8) C (8) D (8) E (3)
Acid from:
Erythritol + d+ + + +
D-Arabinose d- d+
L-Arabinose + +
Ribose + + + d-
D-Xylose d-
Adonitol + + + + d-
~-Methyl xyloside + d+ + +
D-Glucose + d- + + +
D-Mannose +
L-Sorbose ± d-
Dulcitol d+ + +
Inositol d- + + d-
Mannitol d+ + +
Sorbitol +
a-Methyl-D-mannose d+ +
a-Methyl-D-glucoside d- d-
N-Acetyl-glucosamine d+ d-
Amygdalin d- +
Arbutin + + + + d-
Esculin d+ +
Salicin d- + d-
Cellobiose + ± + d-
Maltose d+ d- d+ + +
Lactose d- d+ d-
Melibiose + d- + d-
Trehalose + + + +
Inulin d+
Melezitose + +
D-Raffinose d- d- +
Amidon d- + d+
Glycogene ± d+
Xylitol + d- d- + d-
~-Gentiobiose d- d- d+
D-Turanose + d- + +
D-Arabitol + + + +
L-Arabitol d+ d- + + d+
Gluconate d+ d- ±
ONPG d+ d- ± d+
ADH ± d-
LDC d+ d-
ODC ± d-
CIT d-
75
Table 3. Continued
URE d- d-
TDA ± d+ d+ d+
VP + ± d+ d-
GEL d-
Extracellular Polysacch:
L-DAP d- d+ d+
meso-DAP d+ d- d+ d+ N
Litmus milk:
acid d- d+ d- N
reduction d- d- ± d-
stormy + + + +
fermentation d-
clot d- d- ± d-
Catalase + + + d- +
Pigment:
cream/white + d+ +
yellow/orange +
brown/red + d-
Morphology:
COCCl d+ d- +
rod d+ d+ + +
pleomorphic d- d+
Nitrate reduction d+ d-
+: > 80% positive;
< 20% positive;
d-: 20-50% positive;
d+: 50-80% positive;
±: 50% positive and 50% negative reactions in strains respectively.
N: Not determined
ONPG: Ortho-nitro-phenyl-galactoside
ADH: Arginine dihydrolase
LDe: Lysine dihydrolase
ODe: Ornithine utilisation
eIT: Citrate utilisation
URE: Urease
IDA: Tryrophane desaminase
VP: Acetoin production
GEL: Gelatin liquefaction
L-DAP: L-diaminopimelic acid
meso-DAP: meso-diaminopimelic acid
76
Table 4. Differentiation of the major clusters based on nme differential
phenotypic characteristics as obtained during this study
Tests Cluster (No. of strains)
A (8) B (8) C (8) D (8) E (3)
Acid from:
L-Arabinose + +
D-Mannose +
Dulcitol d+ + +
Inositol d- + + d-
Sorbitol +
Trehalose + + + +
Melezitose + +
D-Arabitol + + + +
Pigment:
cream/white + d+ +
yellow/orange +
brown/red + d-
+: > 80% positive;
< 20% positive;
d-: 20-50% positive;
d+: 50-80% positive;
±: 50% positive and 50% negative reactions in strains respectively.
N: Not determined
77
arbutin; salicin; cellobiose; melibiose; inulin; amidon; glycogene; xylitol; L-arabitol;
arginine dihydrolase; lysine dihydrolase; ornithine utilisation; urease; and nitrate
reduction (Table 5).
Discussion
Five major clusters, four representing the various classical Propionibacterium
species included in this study, as well as a single cluster containing the L. japonicus
type strain and the two "P. coccoides" strains could be delineated during this study.
All the P. acidipropionici strains included in this study and which grouped
together in the major cluster D could be characterised and differentiated from the other
major clusters in their ability to ferment L-arabinose or melezitose (cluster A, B and
C), trehalose or D-arabitol (cluster B) as well as their inability to ferment D-mannose
and sorbitol (cluster E). The P. acidipropionici strains were further characterised by
their ability to hydrolyse esculin, their inability to hydrolyse gelatin as well as their
white to creamish and orange yellow pigment. These results obtained during this study
subsequently confirm the reports of Cummins and Johnson (1986), Britz and Riedel
(1995) and Holt et al. (1994).
According to Cummins and Johnson (1986), nitrate reduction can be used as
the major differential characteristic between the P. jensenii and P. acidipropionici
species. All the P. acidipropionici strains evaluated during this study could not reduce
nitrate, confirming the results of Britz and Riedel (1995). Subsequently, if the
identification key of Cummins and Johnson (1986) was used, these strains would
phenotypically have been incorrectly identified as P. jensenii.
Although numerous phenotypic characteristics were evaluated, it was,
however, not possible to differentiate the P. jreudenreichii strains which grouped
together in cluster A from all the other major clusters, on the basis of one unique
characteristic. Furthermore, all the P. freudenreichii strains included in this study
could ferment glycerol and hydrolyse esculin, confirming the data of Cummins and
Johnson (1986) and Holt et al. (1994), as reported in their identification keys. None of
the P. jreudenreichii strains could, however, utilise L-arabinose which is in accordance
to the results obtained by Britz and Riedel (1995), but in contrast to the results as
reported by Holt et al. (1994).
78
Table 5. Differential phenotypic characteristics of the two "P. coccoides" strains
and the L. japonicus type strain as obtained during this study.
Tests Bacterial strain
"P. coccoides" "P. coccoides" L. japonicus
(KM 252) (KM 375) (ATCC(R) 51527)
Acid from:
Ribose +
Adonitol +
L-Sorbose +
Inositol +
a-Methyl-D-glucoside +
N-acetyl-glucosamine +
Arbutin +
Salicin +
Cellobiose +
Melibiose +
Inulin + +
Amidon + +
Glycogene + +
Xylitol +
L-Arabitol + +
ADH +
LDC +
ODC +
URE +
Nitrate reduction +
+ Positive
Negative
ADH: Arginine dihydrolase
LDC: Lysine dihydrolase
ODC: Ornithine utilisation
URE: Urease
79
According to Cummins and Johnson (1986) as well as Holt et al. (1994), the
only differential characteristic between the P. jensenii (cluster C) and P. thoenii
(cluster B) species is p-hemolysis and their pigmentation characteristics. Since P-
hemolysis was not evaluated during this study, only the pigmentation of white to pink
and orange to red-brown as reported for P. jensenii and P. thoenii, respectively, could
be validated. Except for the hydrolysis of esculin which was observed to be negative
for the P. jensenii strains and not positive as expected, all the other differential
phenotypic characteristics as determined for both P. jensenii and P. thoenii were in
accordance to that as described by Holt et al. (1994) as well as Cummins and Johnson
(1986).
Based on the data obtained during this study it is possible to differentiate
cluster E (representing the two "P. coccoides" strains and the L. japonicus type strain)
from the P. acidipropionici, P. freudenreichii, P. jensenii and P. thoenii major clusters
(clusters ft., B, C and D) on their ability to ferment D-mannose and sorbitol, as well as
their characteristic bright yellow pigmentation.
On the basis of the phenotypic characteristics evaluated during this study, the
"P. coccoides" strains as described by Vorobjeva et al. (1983) could not phenotypically
be identified as any of the classical Propionibacterium species as currently described in
the genus Propionibacterium (Cummins and Johnson, 1986). Furthermore, the
"P. coccoides" strains grouped in a separate cluster together with the L. japonicus type
strain. This cluster was clearly delineated from the clusters representing the various
classical Propionibacterium species.
Based on the results of this study, it is evident that the two "P. coccoides"
strains could also be phenotypic differentiated from the Luteococcus japonicus type
strain. Furthermore the two "P. coccoldes strains could also be phenotypic
differentiated from each other. L. japonicus (ATCC(R) 51527), "P. coccaides (KM
252) and "P. coccaides (KM 375) could be differentiated on the basis of: ribose
fermentation; the ability to ferment L-arabitol; inulin; glycogen; nitrate reduction; and
melibiose fermentation, respectively. It is subsequently apparent that the proposal of
Vorobjeva et al. (1983) to include "P. coccoides" as a new species within the genus
Propionibacterium could possibly be incorrect. It is evident that a higher degree of
phenotypic similarity exists between Luteococcus japonicus and the "P. coccoides"
80
strains, than between the latter and members of the genus Propionibacterium. In spite
of the fact that "P. coccoides" was probably incorrectly classified by Vorobjeva et al.
(1983), it should, however, be taken in consideration that the genus Luteococcus was
only proposed in 1994 by Tamura et al. (1994). This is Il years after the description
of "P. coccoides" by Vorobjeva et al. (1983). Unfortunately Vorobjeva et al. (1983)
did not have the advantage of the inclusion of the phylogenetic neighbour of the genus
Luteococcus in their study. To confirm the relationships observed during this study,
alternative techniques such as ribotyping, restriction fragment length polymorphisms,
DNA:DNA hybridisation and sequencing of the 16S rRNA regions should, however,
also be evaluated. Furthermore, additional "P. coccoides" strains should also be
characterised phenotypically to confirm the results of this study.
81
References
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Vectors, pp. 128-129. (R. Gherna, W. Nierman, P. Pienta, eds.) 16th ed.,
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Anon.: National Collection of Food Bacteria Catalogue of Strains, p. 161. 3rd ed.,
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Breed, R.S., Murray, E.G.D., Smith, N.R.: Genus 1. Propionibacterium Orla-Jensen
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and Cox 1957.
Britz , T.J. Riedel, K-H.J.: Propionibacterium species diversity in Leerdammer
cheese. Int. J. Food. Microbiol. 22,257-267 (1994).
Britz, T.l, Riedel, K-H.J.: Numerical evaluation of the species grouping among the
"classical" propionibacteria. Lait 75, 309-314 (1995).
Britz, T.l, Riedel, K-H.J.: A numerical taxonomic study of Propionibacterium
strains from dairy sources. 1. Appl. Bacteriol. 71, 407-416 (1991).
Britz, T.l, Steyn, P.L.: Comparative studies on propionic acid bacteria.
Phytophylactica 12, 89-103 (1980).
Charfreitag, 0., Collins, M.D., Stackebrandt, E.: Reclassification of Arachnia
propionica as Propionibacterium propionicus comb. nov. Int. J. Syst.
Bacteriol. 38, 354-357 (1988).
Cummins, C.S., Johnson, J.L.: Genus Propionibacterium Orla-Jensen 1909, pp.
1346-1353. In: Bergey's Manual of Systematic Bacteriology (P.H.A. Sneath,
N.S. Mair, M.E. Sharpe, 1.G. Holt, eds.) vol. 2. Baltimore, Williams and
Wilkins 1986.
Cummins , C.S., Johnson, lL.: The Genus Propionibacterium, pp. 834-849. In: The
Prokaryotes (A. Balows, H.G. Truper, M. Dworkin, W. Harder and K-H.
Schleifer, eds.) vol. 1. New York, Springer-Verlag 1992.
Cummins, C.S., Moss, C.W.: Fatty acid composition of Propionibacterium
propionicum (Arachnia propionicay. Int. J. Syst. Bacteriol. 40, 307-308
(1990).
82
Gerhardt, P., Murray, RG.E., Castilow, R.N., Nester, E.W., Wood, W.A., Krieg,
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Holdeman, L. V., Cato, E.P., Moore, W.E.C.: Arachnia and Propionibacterium, pp.
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Holt, J.G., Krieg, N.R., Sneath, P.H., Staley, r.r., Williams, S.T.: Genus
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Johnson, lL., Cummins, C.S.: Cell wall composition and deoxyribonucleic acid
similarities among the anaerobic coryneforms, classical propionibacteria, and
strains ofArachniapropionica. J. Bacteriol. 109,1047-1066 (1972).
Joubert, W.A., Britz, T.J.: A simple and inexpensive method for the long-term
preservation of microbial cultures. J. Microbiol. Methods 7, 73-76 (1987).
Kusano, K., Yamada, H., Niwa, M., Yamasato, K.: Propionibacterium
cyclohexanicum sp. nov., a new acid-tolerant ro-cyclohexyi fatty acid-
containing Propionibacterium isolated from spoiled orange juice. Int. J. Syst.
Bacteriol. 47, 825-831 (1997).
Lockhart, W.R, Liston, J.: In: Methods for numerical taxonomy. AmericAN Society
for Microbiology, Washington DC 1970.
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633-641. In: Bergey's Manual of Determinative Bacteriology (R.E Buchanan,
N.E. Gibbons, eds.) 8th ed., Baltimore, Williams and Wilkins 1974.
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Schaal, K.P.: Lipid and wall amino acid composition in the classification and
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Hyg. Abt. 1260,300-310 (1985). (As cited by Curnmins and Moss, 1990).
Riedel, K-H.J., Britz, T.J.: Propionibacterium species diversity in anaerobic
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Sneath, P.H.A., Johnson, R.: The influence on numerical taxonomic similarities of
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84
Chapter 4
A molecular and phylogenetic analysis of the relationship between the
classical propionibacteria, "P. coccoides" and Luteococcus japonicus
Summary
Despite the fact that "P. coccoides" was originally proposed as a new species
within the genus Propionibacterium in 1983 it is, however, apparent that a higher
degree of phenotypic similarity exists between L. japonicus and "P. coccoides" than
between the latter and members of the genus Propionibacterium (Chapter 3). The
phylogenetic relationship between "P. coccoides", various classical Propionibacterium
species as well as L. japonicus were subsequently evaluated during this study using
various molecular techniques. Certain regions of the 16S ribosomal RNA genes of the
various bacterial strains were amplified using the polymerase chain reaction. Visual
differentiation between the various classical Propionibacterium type strains and the
two "P. coccoides" / L. japonicus strains was possible after restriction endonuclease
digestion of the PCR products using the restriction endonucleases HaeIII and AluI.
Differentiation between the L. japonicus strain and the two "P. coccoides" strains was,
however, not possible despite the evaluation of various other restriction endonucleases.
The two "P. coccoides" and L. japonicus strains could only be differentiated on the
basis of their EstEII rRNA gene restriction endonuclease patterns (ribotyping).
Phylogenetic analysis of the 16S ribosomal RNA gene sequence data (PAUP)
confirmed the high phenotypic similarity observed between the "P. coccoides" and
L. japonicus strains. Both the "P. coccoides" strains and the L. japonicus type strain
grouped together in a phylogenetically tight cluster which could clearly be
differentiated from the various clusters representing the genus Propionibacterium.
Furthermore, the DNA:DNA probe hybridisation technique resulted in a clear
differentiation of the classical propionibacteria from the two "P. coccoides" and the
L. japonicus strains which exhibited a high degree of DNA:DNA homology,
confirming the incorrect placement of "P. coccoides" within the genus
Propioni bacterium.
85
Introduction
In 1983, Vorobjeva et al. described a certain coccus isolated from Soviet hard
cheese with a high mol% G + C content. Since various characteristics were observe to
resemble the description of the genus Propionibacterium as described by Moore and
Holdeman (1974), Vorobjeva et al. (1983) proposed that this bacterium be included in
the genus Propionibacterium as an independent species under the name
"Propionibacterium coccoides". Despite the fact that this species constituted a valid
description, "P. coccoides" was never included in any studies concerning the taxonomy
of the genus Propionibacterium (Britz and Riedel, 1991: 1994; Riedel and Britz, 1993;
Riedel et al., 1992: 1994). Furthermore the proposal of Vorobjeva et al. (1983) was
never mentioned in either the Bergey's Manual of Systematic Bacteriology (Cummins
and Johnson, 1986) or Bergey's Manual of Determinative Bacteriology (Holt et al.,
1994), although it was mentioned by Cummins and Johnson in 1992. Subsequently the
exact systematic position of this bacterium still remains unresolved.
Based on results obtained during the numerical analysis of phenotypic
characteristics of the various classical propionibacteria, the two "P coccoides" strains
and L. japonicus, which was included as an outgroup (Chapter 3), it became evident
that a higher degree of phenotypic similarity existed between L. japonicus and the
"P. coccoides" strains than between the latter and members of the genus
Propionibacterium. It was subsequently evident that the proposal of Vorobjeva et al.
(1983) to include "P. coccoides" as a new species within the genus Propionibacterium
could possibly be incorrect.
In order to verify the results obtained during the numerical taxonomic study of
the phenetic characteristics, alternative molecular techniques such as ribotyping,
restriction fragment length polymorphisms, DNA:DNA hybridisation and 16S rRNA
gene sequencing data would have to be evaluated. The aim of the present study was
thus to characterise the various strains on a molecular level and to further determine
the phylogenetic relationship between "P. coccoides", Luteococcus japonicus and the
classical propionibacteria.
86
Materials and Methods
Bacterial strains and growth conditions. The various bacterial strains
evaluated during the analysis of the restriction fragment length polymorphisms section,
the ribotyping and the 16S rRNA sequence analysis section of this chapter are listed in
Table 1. The bacterial strains analysed during the DNA:DNA probe hybridisation
section of this chapter are listed in Table 2. These bacterial strains comprised of
various type, reference and other Propionibacterium strains, two "P. coccoides"
strains kindly supplied by Prof L.I. Vorobjeva of Moscow State University, Russia, as
well as the type strain of Luteococcus japonicus. The bacterial strains were incubated
anaerobically in a Forma Scientific (Mallinckrodt, Inc., Ohio, USA) anaerobic cabinet,
using oxygen free nitrogen gas phase, as recommended by Holdeman et al. (1977).
The strains was cultured at 30°C for 4 days in the medium of Charfreitag et al. (1988),
with the addition of 4% (v/v) mineral salts solution (Holdeman et al., 1977). Culture
purity was regularly checked by means of microscopical examination of Gram stains
and by comparison of morphological characteristics (Britz and Riedel, 1991: 1994;
Riedel and Britz, 1993).
DNA isolation and purification. The various bacterial strains were cultured
anaerobically for four days at 30°C in the medium of Charjreitag et al. (1988), with
the addition of 4% (v/v) mineral salts solution (Holdeman et al., 1977). Genomic
DNA was isolated according to the method described by Sambrook et al. (1989). The
methods used for agarose and polyacrylamide gel electrophoresis were as described by
Perbal (1988). DNA purity was confirmed using the ratio between the
spectrophotometric readings at 260 nm and 280 nm (OD 260/280).
Polymerase chain reaction. Primer design, reaction mixture composition and
the optimum conditions for amplification of the small subunit ribosomal RNA region
(16S rRNA) using primers 16sP1 (5'-GGGTGACCGGCCACA-3') and 16sP4 (5'-
TCGGGTGTTACCGAC-3') were as described by Riedel et al. (1992:1994). The
reactions were performed using an automated thermal cycler (Techne PHC 2, Techne
Incorporated, Brunswick, New Jersey, USA). Polymerase chain reaction fragments
87
Table 1. List of bacterial strains used in this study. (The names are listed as received).
No. Lab. Name Source Strain Isolated from Identification ke:y#
No.
1 424 P. acidipropionici ATCC 25562* P. acidipropionici
2 423 P. freudenreichii ATCC 6207* Swiss cheese P. freud. ss. freudenreichii
3 80 P. jensenii 2 DSM 20535* Buttermilk P.jensenii
4 419 P. thoenii 4 NCFB 568* Emmentaler P. thoenii
5 362 "P. coccoides" 3 KM252 Soviet cheese
6 363 "P. coccoides" 3 KM375 Soviet cheese
7 364 L. japonicus 1 ATCC(R) Ground and water
51527*
Type strain
#: Identification key ofCummins and Johnson (1986).
1: ATCC, American Type Culture Collection, Rockville, Maryland, USA.
2: DSM, Deutsche Sarnrnlung von Mikroorganismen und Zellkulturen, Braunschweig,
Germany.
3: KM, Katedra Mikrobiologii, Department of Microbiology, Moscow State University,
Russia.
4: NCFB, National Collection of Food Bacteria, Shinfield, Reading, UK.
88
Table 2. List of bacterial strains used during DNA:DNA Hybridisation. (The names are listed as
received).
No. Lab. Name Source Strain Isolated from Identification key#
No.
1 424 P. acidipropionici 1 ATCC 25562* - P. acidipropionici
2 421 "P. pentosaceum" 4 NCFB 570 Emmentaler P. acidipropionici
3 423 P. freudenreichii 1 ATCC 6207* Swiss cheese P. freud. ss.freudenreichii
4 453 "P. shermanii" 5 P67 P. freud. ss. shermanii
5 80 P.jensenii 2 DSM 20535* Buttermilk P. jensenii
6 130 "P. intermedium" 5 NCIB 8728 P. jensenii
7 427 "P. pituitosum" 4 NCFB 1077 P.jensenii
8 75 "P. technicum" 6 P74 Emmentaler P. jensenii
9 419 P. thoenii 4 NCFB 568* Emmentaler P. thoenii
10 294 P. isolate 7 Anaerobic digester P. thoenii
Il 297 P. isolate 7 Anaerobic digester P. theonii
12 362 "P. coccoides" 3 KM252 Soviet cheese
13 363 "P. coccoides" 3 KM375 Soviet cheese
14 364 L. japonicus 1 ATCC(R) Ground and water
51527
*. Type strain
#: Identification key of Cummins and Johnson (1986).
1: ATCC, American Type Culture Collection, Rockville, Maryland, USA.
2: DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig,
Germany.
3: KM, Katedra Mikrobiologii, Department of Microbiology, Moscow State University,
Russia.
4: NCFB, National Collection of Food Bacteria, Shinfield, Reading, UK.
5: NCIB, National Collection of Inustrial Bacteria, UK.
6: G.W. Reinbold, Iowa State University, Iowa, USA.
7: Environmental Bacteriology Culture Collection, University of the Orange Free State,
South Africa.
89
were electrophoresed in a 0.8% agarose gel in TBE buffer (0.089 M Tris-borate, 0.089
M Boric acid, 0.002 M EDTA) at a constant voltage of 45 V for 3 h. The fragments
were visualisated using Ethidium Bromide (0.5 Ill/ml final concentration prepared from
alO mg/ml stock solution).
Restriction fragment length polymorphisms. The resulting 16S rDNA peR
products were extracted from the peR reaction mixture using chloroform, purified
using Qiagen columns (Diagen, Dusseldorf), precipitated with iso-propanol and
redissolved in SABAX water. The peR products were subsequently digested using
the restriction endonucleases HaelII and Smal as described by Riedel et al.
(1994: 1998) and Riedel (1997). Other restriction endonucleases evaluated during this
study included AvaIl, Bcll, BstEII, CfoI, DdeI, DraI, HaelI, HpaIl, KspI, NotI, Sac!
and SspI (Boehringer Mannheim, GmbH, Germany) according to the manufacturing
instructions. Restriction fragments were electrophoresed on a 12% Sodium Dodecyl
Sulfate Polyacrylamide gel (SDS-PAGE) (Merck) in TBE buffer (0.089 M Tris-borate,
0.089 M Boric acid, 0.002 M EDTA) at a constant voltage of 240 V per 2 gels
(Hoefer PS 2500 De power supply), at lOoe in a water cooled tank (Hoefer SE 600
vertical slab gel unit), for 5 h. DNA molecular markers V and VIII (0.25 ug) were
used as reference standards (Boehringer Mannheim, GmbH, Germany). The gels were
stained using the silver staining technique as described by Perbal (1988). Restriction
endonuclease fragment sizes were determined by linear regression analysis, using the
molecular weight markers as standards.
Ribotyping. Restriction endonuclease digestion of the genomic DNA of the
four Propionibacterium type strains, the two "P. coccoides" strains and the
Luteococcus japonicus type strain were performed using the restriction endonucleases
HaelI, Smal, AvaIl, Bcll, BstEII, CfoI, DdeI, DraI, HaelII, HpaIl, KspI, NotI, Sac!,
and SspI (Boehringer Mannheim, GmbH, Germany): The method used was the same
as described by Riedel and Britz, (1996). The restriction fragments were separated by
electrophoresis through a 0.85% molecular grade agarose gel (promega, USA) at 50 V
for 3,5 h in 1 x TBE buffer (0,089 M Tris-base, 0,089 M Boric acid, 0,002 M EDTA,
pH 8,0). To determine the sizes of the DNA fragments, molecular weight standard
phage Lambda DNA digested with EcoRI-HindIII was used as standard reference.
90
After electrophoresis, the DNA was transferred to a positively charged nylon
membrane (Hybond Amersham International plc, Amersham Place,
Buckinghamshire, UK), using the Southern blotting technique (Southern, 1975). The
DNA was cross-linked to the nylon membrane by exposure to a UV transilluminator
for 3 min. The membranes were then prehybridised for 5 h at 68DC in hybridisation
solution containing 5 x SSC prepared from a 20 x SSC stock solution (0.3 M sodium
citrate, 3 M NaCI), 0.1% N-Iaurylsarcosine, 0.02% sodium dodecyl sulfate (SDS) and
1% blocking reagent (Boehringer Mannheim, GmbH, Germany). Following
prehybridisation the membranes were incubated overnight at 68DC in the presence of
the hybridisation buffer containing the heat-denatured (100DC for 10 min) DIG-
labelled probes (16sPI-16sP4 PCR fragment of Propionibacterium freudenreichii
subsp. freudenreichii (ATCC 6207) and phage Lambda DNA). After hybridisation,
the membranes were washed twice for 5 min at room temperature with 2 x SSC-O.l %
SDS and twice for 15 min at 68DC with 0.1 x SSC-O.l % SDS. The presence of the
DIG-labelled DNA probes were determined with an alkaline phosphatase-conjugated
antibody as described by the manufacturer of the DIG detection system (Boehringer
Mannheim, GmbH, Germany). A similarity matrix was constructed on the basis of the
presence or absence of ribotyping bands at a given position over the size range from
1.0 - 8.8 kilobase pairs (kb). Similarity coefficients were calculated using the simple
matching coefficient (SSM)(Sokal and Michener, 1958). The unsorted similarity
matrix was rearranged into clusters by single linkage cluster analysis (Lockhart and
Liston, 1970)
Sequencing of the 16S rDNA. In order to reduce the possibility of nucleotide
misincorporation errors during the amplification process, the PCR products of several
independent amplifications were combined for each sequencing reaction. The PCR
products were sequenced either directly using the fmol™ DNA sequencing system
(Promega, Madison, USA), according to the manufacturers protocol or via
autosequencing using an ABI PRISM™ 377 DNA sequencer. The autosequencing
reactions were carried out using a ABI PRISM™ Dye Terminator Cycle Sequencing
Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS (Perkin Elmer,
Warrington, U.K.) according to the protocol of the manufacturer. Synthetic
91
oligodeoxyribonucleotide primers 16sPl, 16sP3, 16sP4 (Riedel et al., 1992:1994) as
well as the primers: 16sP3R (5'-CCTCATCCCCACCTTCCTCC-3'); 16sP5 (5'-
TCGCTTCTCAGCGTCAGGAA-3'); and 16sP5R (5'-
TTCCTGACGCTGAGAAGCGA-3') (Riedel, 1997) enabled forward and reverse
sequence reactions of the amplified PCR products. The primer sequences were
selected from the conserved regions of the 16S rRNA genes (Charfreitag and
Stackebrandt, 1989).
The sequences were aligned manually with the published sequences of
representatives of the classical and cutaneous propionibacteria (Charfreitag and
Stackebrandt, 1989), the complete 16S rRNA sequence of P. acnes (EMBL Accession
No. M61903) as well as the partial 16S rRNA sequence data of Propionibacterium
cyclohexanicum (Kusano et al., 1997), Propioniferax innocua (EMBL Accession No.
S93388), Terrabacter tumescens (EMBL Accessions No. X53215), Nocardioides
jensenii (EMBL Accession No. X53214) (Collins et al., 1989), Nocardioides luteus
(EMBL Accession No. X53212), Nocardioides albus (EMBL Accession No.
X53211), Luteococcus japonicus (Tamura et al., 1994) and Aeromicrobium erythreus
(EMBL Accession No. M37200). The aligned sequence data was analysed using
PAUP version 3.1 (Phylogenetic Analysis Using Parsimony) (Swofford, 1993). With
PAUP, the heuristic and random sequence addition options were utilized to determine
the most parsimonious tree. Bootstrapping (1000 replicates) was performed on the
sequence data to determine the confidence intervals of the tree branching points
(Felsenstein, 1988).
DNA:DNA Probe Hybridisation. The DNA:DNA homology between 14
strains, including the four type strains of the four classical Propionibacterium species,
seven reference and other Propionibacterium strains, two "P. coccoides" strains as
well as the type strain of L. japonicus (Table 2) were evaluated during this study using
the DNA:DNA probe hybridisation technique as described by Viljoen (1996). The
DNA of each bacterial strain was quantified using a TKO 100 Fluorometer (Roefer
Scientific Instruments, San Francisco, California, USA) according to the manufacturers
specifications. Lambda DNA (Boehringer Mannheim, South Africa) was used as
concentration standard. Hoechst 33258 dye (Sigma-Aldrich, Dorset, UK) was used as
fluor at 0.1 ug/ml in 0.1 NaCl, 10 mM Tris-HCl and 1.0 mM EDTA (pH 7.4).
92
Reference DNA (50 ng) was radiolabelled with 32p using the Random Primed DNA
Labeling Kit (Boehringer Mannheim, Randburg, South Africa) according to the
manufacturers protocol. Unincorporated 32p was removed by gel-filtration
chromatography (Maniatis et al., 1989). Aliquots containing the radiolabelled DNA
were pooled for hybridisation reactions.
The DNA (300 ng) from the various cultures was denatured using 1.5 M
NaOH (final concentration). The denatured DNA was blotted onto a positive charged
nylon membrane (Hybond N, Amersham International plc, Amersham Place,
Buckinghamshire, UK). For each hybridisation reaction, DNA from the different
cultures was placed at 1.5 cm intervals from each other. A set of dot blots was
prepared in the same manner for each radiolabelled reference DNA used in a
hybridisation reaction. The DNA was covalently linked to the membrane using UV-
transillumination for three minutes. The individual membranes were pre-hybridised in
hybridisation buffer (5 x SSC (0.075 M NaCitrat, 0.75 M NaCl), pH 7.0), 0.1% N-
lauroylsarcosine (w/v), 0.02% SDS, and 1% blocking reagent (Boehringer Mannheim,
Randburg, South Africa) for 1 hr at 85°C. After addition of the heat denatured (100°C
for 10 min) radiolabelled probe, the membranes were hybridised for 16 hrs at 80°C.
Following hybridisation, the membranes were washed twice for 5 min in excess 2 x
SSC, 0.1% SDS and twice for 15 min in excess 0.1 SSC, 0.1% SDS at room
temperature. The membrane blocks were allowed to dry on paper towels at room
temperature.
The membrane blocks were cut into individual squares (1.5 ern"), each square
contained DNA from one culture and the reference radiolabelled DNA that had bound
to this DNA. The amount of radiolabelled probe bound to the membrane was
determined by Cerenkov counting (Wilson and Gou/ding, 1986) using a Beckman LS-
6000 scintillation counter (Beckman, South Africa). Hybridised membrane blocks
containing no DNA were used to determine the amount of background radiation.
The cpm (counts per minute) from individual hybridisations for each reference
DNA were expressed as a percentage, using the reference DNA hybridised to itself as a
measure of maximum hybridisation (100%). The degree of hybridisation of other
strains simultaneously hybridised by the same reference DNA were then determined as
a percentage of this value. The experiment was done in duplicate and repeated twice.
93
Results
DNA purity. All DNA used during this study was extensively purified and the
260/280 ratio gave values of approximately 1.8.
Polymerase chain reaction. The 16S rRNA genes from the genomic DNA of
the four classical Propionibacterium type strains, the two "P. coccoides" strains and
Luteococcus japonicus type strain were successfully amplified using peR. Products of
the predicted size (111 0 bp), using primers 16sP 1-16sP4 were obtained for all the
species included in this study (data not shown).
Restriction fragment length polymorphisms. The results obtained from the
restriction endonuclease analysis (HaelIl and AluI) of the peR products (16sP1-
16sP4) are shown in Fig. 1. Visual differentiation between the four classical
Propionibacterium type strains was possible after restriction endonuclease digestion of
the peR products, using the restriction endonucleases HaelIl and AluI confirming the
results of Riedel et al. (1994: 1998). Although the two "P. coccoides" and the
L. japonicus strains could be differentiated from the other classical propionibacteria
species on the basis of their restriction fragment length polymorphisms it was not
possible to differentiate them from each other (Fig. 2). Similar results were observed
when the restriction endonucleases HaelI, Smal, AvaIl, Bcn, BstEII, CioI, DdeI, DraI,
HpalI, KspI, NotI, Sad, Smal and SspI were also evaluated.
Differentiation between the P. acidipropionici strains and the "P. coccoides" /
L. japonicus strains was possible since the restriction endonuclease pattern obtained
with both the restriction endonucleases HaelIl and AluI differed. Fragment sizes
displayed by the P. acidipropionici type strain were: A/uI - 520, 363 and 220; HaelIl
- 470,215,203,95 and 85, whilst those for "P. coccoides" / L.japonicus were: AluI-
363, 320, 281 and 220; HaelII - 404, 320 and 203 bp. The restriction pattern
obtained for the P. freudenreichii, P. jensenii, P. thoenii and the "P. coccoides" /
L. japonicus strains using the restriction endonuclease AluI were identical and
differentiation was not possible. Similar results for the various classical
Propionibacterium species were reported by Riedel et al. (1994: 1998). Four
fragments were obtained with sizes determined to be 363, 320, 281 and 220 base pairs
94
1 2 3 4 5 6 7 8 9 10 11 12 13 14
504 -
504
404 -
320 - 404 -
242 - 320 -
184 - 242 -
124 - 184 -
89 -
Fig. l. Silver-stained 12% polyacrylamide gel displaying fragments obtained after
restriction endonuclease digestion (HaeIII and A/uI) of the amplified products (1110
bp) obtained using primers 16sPI-I6sP4. Lanes: 1, DNA molecular weight marker
VIII; 2, DNA molecular weight marker V; 3, P. acidipropionici ATCC 25562 _
HaeIII; 4, P. acidipropionici ATCC 25562 - A/uI; 5, P. freudenreichii subsp.
freudenreichii ATCC 6207 - HaeIII; 6, P. freudenreichii subsp. freudenreichii ATCC
6207 - A/uI; 7, P. jensenii DSM 20535 - HaelII; 8, P. jensenii DSM 20535 - A/uI;
9, P. thoenii NCFB 568 - HaelII; 10, P. thoenii NCFB 568 - A/uI; Il, DNA molecular
weight marker VIII; 12, DNA molecular weight marker V; 13, "P. coceetdes KM
252 - HaelII; 14, "P. coccoides" KM 252 - A/uI.
95
2 3 4 5 6
04
404 -
242 -
184 -
124
Fig. 2. Silver-stained 12% polyacrylamide gel displaying fragments obtained after
restriction endonuclease digestion (HaelII and A/uI) of the amplified products (1110
bp) obtained using primers 16sPl-16sP4. Lanes: 1, DNA molecular weight marker V;
2, DNA molecular weight marker VIII; 3, "P. coccoides" KM 375 - HaelII; 4,
"P. coccoides" KM 375 - A/uI; 5, L. japonicus ATCC(R) 51527* - Haelll; 6,
L. japonicus ATCC(R) 51527* - A/uI.
96
(bp). Differentiation between the P. freudenreichii, P. jensenii, and P. thoenii species
as well as the "P. coccoides" I L. japonicus group was, however, possible based on the
HaeIIl restriction endonuclease profiles. For the "P. coccoides" I L. japonicus profile,
three fragments were obtained with sizes of 404, 320 and 203 bp. The fragment sizes
observed for P. freudenreichii were 448, 255, 203, 85 and 76 bp, for P. jensenii, 470,
385,95 and 85 bp and for P. thoenii, 428, 215, 203, 95 and 85 bp, confirming the data
as reported by Riedel et al. (1994: 1998) .
Ribotyping. Both "P. coccoides" strains and the L. japonicus type strain
displayed an identical and unique ribotype profile (profile Q). The ribotype profile
obtained using the restriction endonucleases HaeIl and Smal (Fig. 3) was found to
differ significantly from the ribotyping data obtained for all four the classical
Propionibacterium species (Riedel and Britz, 1996). The fragment size obtained using
the restriction endonuclease HaeIl (Fig. 3, lane 2) was 4782 (bp). Three different
fragments were, however, obtained using the restriction endonuclease Smal (Fig. 3,
lane 3). These fragment sizes were: 4260; 3766; and 2227 bp. From the cluster
analysis performed on the basis of the ribotype profiles observed for 135
Propionibacterium strains as reported by Riedel and Britz (1996) and the ribotype
profile as observed for the two "P. coccoides" and L. japonicus strains (ribotype
profile Q), a dendogram was produced (Fig. 4). Four major clusters, above the 70%
similarity level could be delineated in the dendogram. Each of these major clusters
represented a classical Propionibacterium species. A 82% similarity was observed
between the ribotyping profiles obtained for the two "P. coccoides" and the
L. japonicus strains (profile Q) with the various ribotyping profiles obtained for the
p. jensenii species.
To confirm the differentiation of the two "P. coccoides" strains and that of the
L. japonicus type strain as observed during the numerical analysis of phenotypic
characteristics (Chapter 3), numerous other restriction endonucleases (AvaIl, Bell,
BstEIl, Cfol, Ddel, Dral, HaelIl, HpaIl, Kspl, Notl, Sac! and Sspl) were also
evaluated. The two "Propionibacterium coccoides" strains and the L. japonicus type
strain could only be differentiated from each other when their genomic DNA was
digested with restriction endonuclease BstEIl (Fig. 5). The fragment sizes obtained
after digestion of the genomic DNA with BstEIl of: "P. coccoides" (KM 252) were
97
1 2 3
21092074 --
1584 -
1330 -
Fig. 3. Representative patterns of the ribotyping profile obtained for the two
"P. coccoides" and the L. japonicus strains after digestion of the genomic DNA with
the restriction endonucleases HaeII and Smal, separation on an agarose gel and
hybridisation with the DlG-labeled 16S rDNA probe of P. jreudenreichii subspecies
jreudenreichii (ATCC 6207). Lanes: 1, Molecular mass standard (EcoRl/HindlII
digested Phage Lambda DNA); 2, Ribotype Q - HaelI; 3, Ribotype Q - Smal.
98
Fig. 4. Simplified dendrogram showing the grouping of the vanous ribotypes.
Similarity coefficients were calculated using the simple matching (SSM) coefficient.
The unsorted similarity matrix was rearranged into groups by single linkage cluster
analysis.
99
Percentage similarity Ribotype (No. of Strains) Type and Reference Strains
40 so 60 70 80 90 100
A(7)
"1'. pcntosaceum' NCFB 570
B (33)
P. jensenii NCFB 572
P. acidipropionici ATCC 25562'
"P. arabinosum" NCFB 563
"P. arabinosum" ATCC 4965
C(16)
P·frelldellreichii ATCC 6207'
"P. peterssontt" VPI 5169
P. jensemi DSM 20535'
D (23) ·'P. raffinosaceum" NCFB 1078
"P. rubrum" NCFB 571
"1'. peterssonii" NCFB 565
"1'. pituitosum: NCFB 1077
"1'. lntermedium" NCm 8728
"1'. sanguineum" NCFB 1080
l-uteococcustaponicus ATCC(R) 51527
"1'. wentii" NClB 8915
"P. wentii" NCFB 108:
P. thoenii NCFB 568'
"1'. technicum" NCFB 567
100
1 2 3 4
5148 -
4973 -
4277 -
2027 --
1904 -
1584 -
1330 -
Fig. 5. Representative patterns of the ribotyping profile obtained for the two
"P. coccoides" strains and the L. japonicus type strain after digestion of the genomic
DNA with the restriction endonuclease BstEII, separation on an agarose gel and
hybridisation with the DIG-labeled 16S rDNA probe of P. freudenreichii subspecies
freudenreichii (ATCC 6207). Lanes: 1, Molecular mass standard (EcoRl/Hindlll
digested Phage Lambda DNA); 2, "P. coccoides" KM 252 - BstEII; 3, "P. coccoides
KM 375 - BstEII; 4, L. japonicus ATCC(R) 51527* - BstEII.
101
3929, 3341 and 2972; (Fig. 5, lane 2), of "P. coccoides" (KM 375) were 3929 and
2972; (Fig. 5, lane 3) and of Li japonicus (ATCC(R) 51527) were 3413 and 3069 bp.
(Fig. 5, lane 4).
16S rDNA sequencing. An alignment of the 1137 bases of DNA sequence
obtained from the amplified 16S rRNA genes of the four classical Propionibacterium
type strains, as well as that of the two "P. coccoides" strains, P. acnes,
P. cyclohexanicum, Propioniferax innocua, Terrabacter tumescens, Nocardioides
jensenii, Nocardioides luteus, Nocardioides albus, Luteococcus japonicus (ATCC(R)
51527) and Aeromicrobium erythreus is presented in Fig. 6. Regions of DNA that
could not be sequenced, especially in the areas of priming were excluded from the
PAUF analysis. The phylogenetic analysis was thus subsequently based on a
comparative analysis of921 bases covering the regions 40-274,297-804,920-1100 bp
(Fig. 6).
The phylogenetic analysis of the PCR-amplified 16S rRNA gene sequence data
of the four Propionibacterium type species, P. acnes, the two "P. coccoides" strains,
Luteococcus japonicus and the phylogenetically related genera produced one most
parsimonious tree. The phylogenetic tree, rooted to Nocardioides luteus, is illustrated
in Fig. 7. The confidence intervals for the main branches of the tree obtained using
bootstrapping analysis (1000 replicates) are also indicated on the figure.
The various Propionibacterium species grouped together as a major
phylogenetic cluster which was observed to be well separated from the various
Terrabacter, Aeromicrobium and Nocardioides outgroups. Two major phylogenetic
groups, based on the branching pattern of the phylogram were, however, evident
within the classical propionibacteria, confirming the data obtained by Riedel (1997).
The larger of the two phylogenetic groups, which consisted of the P. acidipropionici,
P. jensenii and P. thoenii species, could clearly be separated with a confidence interval
of 100% from the smaller P. freudenreichii and P. cyclohexanicum group.
Comparison of the sequence data for the four type strains of the classical
Propionibacterium species as obtained during this study and the sequence data as
reported by Charfreitag and Stackebrandt (1989), revealed a 19 bp difference for the
P. thoenii species, a 13 bp difference for both the P. acidipropionici and P. jensenii
species and a 3 bp difference for the P. freudenreichii strains. The sequence data
Fig.6. Aligned DNA sequence (5'-3') of the 16S rDNA region from the two "P. coccoides" and the L. japonicus strains, the various
classical and a cutaneous Propionibacterium species, as well as the phylogenetically closely related Propioniferax innocua, Terrabacter
tumescens, Nocardioides jensenii, Nocardioides luteus, Nocardioides albus and Aeromicrobium erythreus. (Stack), indicates the
sequence data as determined by Charfreitag and Stackebrandt, (1989); (Tamu), indicates sequence data as determined by Tamura et
al. (1994); N, indicates a base composition that could not be determined; (-), indicates a gap enabling alignment of the sequence; (.),
indicates a base homologous and identical to the corresponding base in P. jensenii - (Stack)
......
o
N
P. jensenii (Stack) GGGTGACCGG CCACATT-GG GACTGAGATA CGGCTCAGAC TCCTACGGGA GGCAGCAGTG GGGAAT-ATT GCACAATGGG CGCAAGCCTG ATGCNGNNAC [lOOJ
P.jensenii - DSM 20535" NNNNNNNNNN NNNNNNN-NN NNNNNNNNNN NNNNNNNNN. ....A.CA ..
P. thoenii (Stack) .•..•.•..• .•.... . - N . N .•••• - .•. ·.A N.CN ..
P. thoenii - NCFB 568" NNNNNNNNNN NNNNNNN-NN NNNNNNNNNN NNNNNN . C ••••. - ... ·.A A.CA ..
P. acidipropionici (Stack) ..•....•.• •....• • - •.•.•.••..••...• N .••.• ·.G A.CA ..
P. acidipropionici - ATCC 25562" NNNNNNNNNN NNNNNNN-NN NNNNNNNNNN NNNNN T · T . ..G ..NNN .. . A .CA ..
P.freudenreichii (Stack) •.. . N .•... N....• - ••. ........ N ..... A.CN ..
P. freudenreichii - ATCC 6207" NNNNNNNNNN NNNNNNN-NN NNNNNNNNNN NNNNNNNNN. ....A.CA ..
P. cyc/ohexanicum .• . C •............ T ..•...•.••...... C . ·.G A.CA ..
Propioniferax. innocua .......... . C.- C . ·.A N N.CN ..
P. acnes • , .. C ••••• N ..... - . ·.G A.CA ..
"P. coccoides" - KM 252 NNNNNNNNNN NNNNNN.- C.NN.N ..N . .... .. T . ·.G N A.CA ..
"P. coccoides" - KM 375 NNNNNNNNNN NNNNNN.- CN C.NA T . ...... AT .. ·.G A.CA.G
Luteococcus japonicus - ATCC(R) 51527" NNNNNNNNNN NNNNNN.- N C ..A.N ..N . -G. ....... ..G. ...... ....A.CA ..
Luteococcusjaponicus (Tamu) ., .C . ....... .• C .. A .. ·.G A.CA ..
Nocardioides. jensenii .... .C.- C C . ·G. ....... ..A N ..C .A .CA ..
Nocardioides /uteus .....C. - C CT . ·G. ....... ..G N ..C .A .CA ..
Nocardioides a/bus .... .C.- C C . ·G. ....... ..G N ..C .A .CA ..
Aeromicrobium erythreus .... .C.- C C . .G A N ..C.A.CA ..
Terrabacter tumescens ...C C.- C C .. ·..G. ..... ..A N A .CG ..
P. jensenii (Stack) -GCCGCGTGC GGGATGACGG CCTTCGGGTT GTNAACTGCT TTCNCCAGGG ACGAAGT--- -GCCTNTCGG GGTGTNACGG TACCTG-AGA AGAAGCACCG [200J
P. jensenii - DSM 20535· TT ........ . NNNN .. ..A C .N . ...A. ..... . --- - T. ... . G. ... . G. .. . .
P. thoenii (Stack) - N A C A G--- - A A.N G ..
P. thoenii - NCFB 568· ..A C A G--- - T T.G G .
P. acidipropionici (Stack) ·.A C. .. ...A. ..... . G-CA TT. T .T .A.. .TGT .N. ... . G .
P. acidipropionic - ATCC 25562· -.NN . ·.A C. .. ...A. ..... . G-CA TT. T .T .A.. .TGT .G. ... . G .
P. freudenreichii (Stack) ..G C AT.CAT CGCA A.-------- ----.N GTN.G .
P. freudenreichii - ATCC 6207· ..A C AT.CAT CGCA A.-------- ----.G GTG.G .
P. cyclohexanicum ·.A C A ..CAT CGTG A.-------- ----.G GTG.G .
Propioniferax. innocua - •. N ...•.. ·.N C NG CTNA C.-------- ----.G C .
P. acnes ·.A C G.TGT- CGTG A.-------- ----.G ATG.GTA.
"P. coccoides" - KM 252 -N- . ·.A C NA.GCA .. GCG.ACGAA A.-------- ----.G TGC.T - .
"P. coccoides" - KM 375 -N . ·.A C. .. ...NA. GCA. . CGAA A. -------- ----. G. ... ..TGC. T. .. . .
Luteococcusjaponicus - ATCC(R) 51527· -N . ·.A C. -. ...AA. GCA. .GCG. ACGAA A. -------- ----. G. ... ..TGC. T .
Luteococcus japonicus (Tamu) ..A C AA.GCA CGAA A.-------- ----.G TGC.T .
Nocardioides jensenii - .•..•••. A ·.A CT AG.G CGAA A.-------- ----.G C.C .
Nocardioides luteus - ••••.•.• A ·.A CT.. ...AG ..CA. . CGCA A. -------- ----. G. ... ..TG ..C. .. . G .
Nocardioides a/bus - •••••.•. A ·.A CT.. ...AG ..CA. . CGAA A. -------- ----. G. ... ..TG ..C. .. . G .
Aeromicobium erythreus - •..•.••. A ·.A CT.. ...AG. .... . CGAG A. -------- ----. G. ... . C. .. . G . -o
Terrabacter tumescens - A ·.A CT AG A CGAA A.-------- ----.G C .
VJ
P.jensenii (Stack) GCTAACTACG TGCCAGCNGC CGCGGTNATA CGTAGGGTGC NAGCGTTGTC CGGAATTATT GGGCGTAAAG AGCTCGTAGG TGGTTGATCN CGTCGGAAGT [300]
P. jensenii - DSM 20535· •.•••••••....•.•• A ••••.••• G ••..••••••••• G ••••••••..... T ..•••.....•••.•....•.••........••• A ••.....•.•
P. thoenii (Stack) .•..••.•......... N .....•.• N .• N G ..•......................••••..........•........ G •.........
P. thoenii - NCFB 568· .•••.• • A •••.•••• G ••• G ••••••••••.•. T ..••• •.. NNN ..• G
P. aeidipropioniei (Stack) ...... . A .••••..• N .•• N .•................. G •.. N ••••• C ... N •••• G
P. aeidipropioniei - ATCC 25562· •••••• • A •••••••• G ••• G ••...••...... T ..... G .••. NNNNN NNNNN .. NNG
P. freudenreiehii (Stack) ..••••••.•..••••• A ••.•.••• G ••. G ...•••••• •••. N ••.•. C .••••••. A
P.freudenreiehii - ATCC 6207· ••..••.... ...••. . A ••.•.... G •.. G ••••••••• .•.. T •.... C •....... A
P. cyclohexanicum • .C .......•...... A .••••... G . G •••••.... • ••• T •••.• C •...•.•• G ••..• A .•..
Propioniferax. innocua · .C .....•........ A .•••••.. A . G •••••...•...•.• C ••• . ... T ..... C .... TG .. A
P. aenes ••.•..••.••••..•• A ••••••.• G .•• G .••.•......•• T ••••• G •...•••••......... G
"P. eoeeoides" - KM 252 · .C •..••.......•• A .•.....• G .•........... G .•..•.... A. . . . . . . .. • ... T. . . .. C TG . TG A .....
"P. eoeeoides" - KM 375 • .C •.•......••..• A ••••.... G ............• G . A. . . . . . . .. . .•• T. . . .. C TG . TG A .•...
Luteoeoecusjaponicus - ATCC(R) 51527- · . C. . . . . .• . ••.... A.. • ••..• G. •. NN........ G . A. . . . . • . •. .. N . T. . . .. C TG . TG A .....
Luteoeoecusjaponicus (Tamu) • .C •••••••••••••• A •••••••• G •••••.•.••••• G •..•••••. . T. . . .. C ..•. TG . TG A .•...
Noeardioides jensenii · .C ......•....... A A •••........ C. N •..... A .. G CC .. NTG .. A G .
Noeardioides /uteus · .C A A C. T C .. A .. G T. . . .. CN .. NTG .. G N ..
Nocardioides a/bus · .C .••.•........• A A •.•...•.... C. T ..• C .• A .. G T. . . .. CN .. NTG .. G G .
Aeromierobium erythreus · .N •••••......•.• A ••.•.... A .•.•.••.... C. N .•..••••. . ..... N ... G. • • . . . . .. CN TG .. G G ..•
Terrabaeter tumeseens .•.•.. . N .•.•••.. A .•• N N. .... N . N . .. . •... NG .. G T . CT ..
P. jensenii (Stack) CAAAACTTGG GGCTTAACTC TGGGCGTGCT TTCGATCCGG GTTNACTTGA GGAAGGTAGG GGAGAATGGA ATTCCCGGTG GAGCGGTGGA ATGCGCAGAT [400]
P.jensenii - DSM 20535· G .........•....... C ......••.•....... A ..••.. G ......•...••.....•.••.....•.••• T ••....•••.•......•••••.•
P. thoenii (Stack) .•.... A •••.•. G .••••••••. C ...•.•• N ....•••
P. thoenii - NCFB 568- G C. . •••.. A ••••.. G ..•.•. • •... T ....
P. aeidipropioniei (Stack) .............. N .•.•... A ..••......... A •..... G . • . N ...••••
P. acidipropionici - ATCC 25562· G ......••••.•..••• C ••. A •••••••..•..• A •.•... G ..••.••..•....•• • ..•. T .
P. freudenreiehii (Stack) G TTCCA . •......... . .... T. . .. . A. .. . .• G. . . . .. . T . .C .. T .
P.freudenreiehii - ATCC 6207· G TTCCA. .......•.. • •... T . . •. . A. .. . .. G. . . . .. . T . .C .. T .
P. cyclohexanicum G ... TTCCA. •••.• T •••....... A •••.. CG .
Propioniferax. innocua G CA. A......... . .A .. C. . .. . C A. .. . C. G A.. . C. .. . .•.. T .
P. acnes GT .. T. . . .. . C . .. A. . . . . .. . A. .. . .. G. . . . .. .. N. . . . . .. . T .
"P. coccoides" - KM 252 G TN . CA. T A. •• A. . . . . .. • CT ..• A. .. . CAG A.. . T. . .. . .•... C. .. . T .. - .
"P. coccoides" - KM 375 G TN . CA. T ......• A. .• A. . . . . .. . CT A. .. . CAG A.. . T. . .. . C. .. . T .. C . . .. N .
Luteoeoecus japonicus - ATCC(R) 51527· G TA.CA. T ....••• AN .• A ••.•.... CT A CAG A T C TN .
Luteococcusjaponicus (Tamu) G T •. CA. T A. .. A. . . . . .. . CTC .. A. .. . CAG . G . A.. . T. . .. . C. .. . T. . .. . . ....... . C.
Nocardioides jensenii G CA.. T ..•.... A. CTN .• C. . .. . C A. .. . CAG N.. .. T . N . CN.. . C. .. . TN . .. T A.
Noeardioides /uteus G CA .. T A. CTN .. C A CAG N.G T.TG .. G .. C .A.C.GGATT .GATA.CC TAGTCCAC. CC.TAA.CG.
Noeardioides albus G CA.. T A. CTN .. C . N.. . C A. .. . CAG N.. .. T . CTC. .. . TN. .. T A. . .
Aeromierobium erythreus .....G CA. . C .•. C . .• A . N. . . .• . C A. .. . CAG A.. .. T . T . C. .. . C. .. . T. . .. T......... . . o
Terrabacter tumescens G TCC . A C CT C .. A . TN .. NG. G . G . A. .. . CAG N.. . TGT. . . . .. . C. . .. . T. . .. T . .j:>.
P.jensenii (Stack) ATCGGGA-A- GAACACCAGT GGCGAAGGCG GTTCTCTGGA CCNTTCCTGA CGCTGAGAAG CGAAAGCGTG GGGAGCNAAC AGGCTTAGAT ACCCTGGTAG [5001
P.jensenii - DSM 20535· •. . A ... --G ......... N NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNN ...
P. thoenii (Stack) .•••..••.••• T ••••••..•...•.....•.••.••.......• A ..•
P. thoenii - NCFB 568· •. • A ... --G .NNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNN .
P. acidipropionici (Stack) •• • C •••••• ....•••••... T .....••....•.••..•••••••••....•.• N .
P. acidipropionici - ATCC 25562· .•• A ••. --G ......... N NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNN .
•• . C •..... .AT . •••••• A •.•P.freudenreichii (Stack)
P.freudenreichii - ATCC 6207" ... A ... --G ...NNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN
P. cyc/ohexanicum ....... --G . . T .
•.•••. A •••
Propioniferax. innocua .. . A --G G N T .
....T.A ...
P. acnes ...A --G N G ..T G .. .•.... G •..
"P. coccoides" - KM 252 ...A G.G G - T.T G A.T N A N A N N .
"P. coccoides" - KM 375 ·..N ..NG .G G.. ..N - T .T GA. T. ...... . A. .. . .
Luteococcusjaponicus - ATCC(R) 51527· ...A G.G G T.T G A.T A N .
Luteococcusjaponicus (Tamu) .,.A --G G G A.T A .
Nocardioidesjensenii ...A --G G G .AT.A N G T G A .
Nocardioides /uteus TGG.C.CT.G .TGTGGG.TC CATTCCACG. N CG ..CC G.AGCTAACG .AT.A ..CGC .CC.G.TACT CA.G.GAG.A T ..AA.TCC. GGTG.A.CG.
Nocardioides a/bus ...A --G G G AGTA N G T G A .
Aeromicrobium. erythreus ...A --G G .. NN N G .AT.A G A G A .
Terrabater tumescens ...A --G GA A .G G .TA ..A A G A .
P. jensenii (Stack) TCCACGCTGT AAACGGTGGG TNCTAGGTGT CGGGTCCATT CCACGGATTC CGTGCCGTAG CTAACGCATT AAGTACCCCG CCTGGGGAGT ACGGCCGCAA [6001
P. jensenii - DSM 20535· .N .
.N. ....... G ..NNNN ... . C . .•• C ..•...P. thoenii (Stack) .•• C .•...•
P. thoenii - NCFB 568· • N .
· .N .
P. 'acidiproptonict (Stack) • N .. N .••....... N ....
P. acidipropionici - ATCC 25562· .N .
P. freudenreichii (Stack) •..••. . C •. . A .....•.. -N C GG ..
· .N .
P. freudenreichii - ATCC 6207" ••..•.. C •. • A ••..••.. -N .•••. C ••...•.. GG .. · .N .
P. cyclohexanicum •.••.• • C .. .A G ..TCT. ... . AGA.. . . .... G .....
Propioniferax. innocua .•.••. • C •. CA. ....... G GA. ... . TTC.. . C ..
• A •..••... G .•...••....•... G ..........••. ....... T ..P. acnes
"P. coccoides" - KM 252 ..N.AANAAN N A G ..TCfl TGA C AT N .
"P. coccoides" - KM375 ..... N.A.N AAAAA A N .. G ..TCA N ..NNTGA C C AT C .
Luteococcusjaponicus - ATCC(R) 51527· ....... C NAA A G ..TCA N-TNA C AT C ..C .
Luteococcusjaponicus (Tamu) · C.. .A. ....... G ..TCA. ... . TGA.. . C .. . .
Nocardioides jensenii · A. C.. . T. ... CG........ G ..ACTN ... . AG. .. . C.. ...CG. .... . .
Nocardioides /uteus .GN.ATGC.C .G.TATCA .. A--GGAACAC TGG.GAA GGCG.TTC .. T.G.AGTATC ..N TGA GGAGCGAAfI .
Nocardioides a/bus · A .C.. . T. ... CG........ G ..ACA. ... . TN. .. . C.. ...CG. .... . .
Aeromicrobium erythreus · T ..C.. . T. ... CG........ G -A. C.. . T. .. . C.. ..,CG. NNN. . .
o
\Jl
Terrabacter tumescens · T ..C.. . T. ... AA........ G ..TCTN ... ..N ..AGA.. . C.. . T ..... . .
P. jensenii (Stack) GGCTAAAACT CAAAGGAATT GACGGGGGCC CGCACAAGCG GCGGAGCATG CGGATTAATT CGATGNAACG CGAAGAACCT TACCTGGGTT TGACATGGAT (700)
P.jensenii - DSM 20535· ..... C ..
P. thoenii (Stack) .. N . ..... N ..
P. thoenii - NCFB 568· ..••• C •..•
P. acidipropionici (Stack) ..... N ..
P. acidipropionici - ATCC 25562· ..... C ..
P. freudenrichii (Stack) ..... N .. •.....••• C
..... C .. ......... CP.freudenriechii - ATCC 6207·
P. cyclohexanicum ....... C .. GC ...C . ....... TGC...... AT.C
Propionij'erax. innocua ... . N •........ CC .... ..... N ..
P.acnes ..••• C ...•
"P. coccoides" - KM 252 ... .CC ..... G ..... C .. .NNNNN- ... G C .. ...... ATGC...... ATGC
"P. coccoides" - KM 375 T C ..
Luteococcusjaponicus - ATCC(R) 51527· ....... C C .. T C . ...... ATGC...... ATGC
Luteococcus japonicus(Tamu) ....... C .. ..... C ..
Nocardioides jensenii ..... N .. ......... N N ...N.ATGC
....• N . ....G.AC.CNocardioides luteus
Nocardioides albus ..•• NN .•••
...... AC.C
....N NN . N ......... .T N .. ...... ATGCAeromicrobium erythreus
•••.. N •... ....N.AC.CTerrabacter tumescens
P. jensenii (Stack) TGGTAACG-G TNAGAGATGG CTNNCCCCCT TGATGGGCCG ATTCACAGGT CGTGCATGGC TGTCGTCAGC TCGTGTCGTG AGATGTTGGG TTAAGTCCCG (800)
P. jensenii - DSM 20535· · -. .C. ....... .CGC , ..- ....... G......... . .
P. thoenii (Stack) ·....... . .C. ....... .NNC. ..... ..- C. .. G C ..
P. thoenii - NCFB 568" · -. .CN .N ..... .CGC. ..... ..-. ...... G .
P. acidipropionici (Stack) · -. .C. ....... .NNC. ..... ..-. ...... G......... N N .. .. N .......
P. acidipropionici - ATCC 25562· · -. .C. ....... .CGC. ..... ..- ....... G......... G .
P. freudenreichii (Stack) ...A.G ..-T .C........ GCGTG ..-T .. -T -.T. G.A ....... G ..
P. freudenreichii - ATCC 6207" ·..A.G ..-T .C. ....... GCGTG ..-TN .-T - .T. G.A. ...... G N ..
P. cyclohexanicum C ..GTGGTAT .C G.GCG ..TT. --T T.-. G.A G .
Propioniferax. innocua C ..A ACC .AGAGATA .. TN-C TTA .--- ..T.-. G.NT N .
P.acnes C ..G.GT .CT CAGAGATG.. TGTG ..T .T . .TGG ..T .-. G......... G .
"P. coccoides" - KM 252 C ..A ATT CAGAGATG.A TGCC ..TTT .. --- ..T.-. G.AT G . ..... N ... C .... N .....
"P. coccoides" - KM 375 C ..A ATT CAGAGATG.A TGCC ..TTT .. --- ..T.-. G.AT G A .
Luteococcus japonicus - ATCC(R) 51527" C ..A ATT CAGAGATG.A TGCC ..TTT .. --- ..T.-. G.AT N. G . ..... N ... C .... N ... A .
Luteococcusjaponicus (Tamu) C ..A ATT CAGAGATG.A TGCC ..TTT .. --- ..T.-. G.AT G .
Nocardioidesjensenii C ..A ..GC-T CT A. A-GC -TN .-TN-.T.-. G.GT G .
Nocrdioides luteus C ..A ..GC-T GC T A-GC -TN .-TN-.T.-. G.GT G .
Nocardioides albus C ..A ..GC-C GT AC G-GC -T .. -TA-.T.-. G.GT G .
Aeromicrobium erythreus C ..A ..GC-T GC T GGCC .NN.- T- ..T. -. G.AT G.N . -o0\
Terrabacter tumescens C ..A.T.ACT CAGAGATG .. TNCGT.TT-- C.GA--CT-. G.GT G T N .
CAACGAGCCC AACCCTNGTC CACTGTTGCC AGCAATTCGN -TTCGGGACT CAGTGGAGAC CGCCGGGGTN AACTCGGAGG AAGGTGGGGA TGAGGTCAAG [9001
P. jensenii (Stack)
· G . . C. .. . G - .. G. . . . .. NNNNNNN .. C . .
P. jensenii - DSM 20535·
•...••.......... N N N - .. G T A T .
P. thoenii (Stack)
•..•..•• G ..••.•. C ••.•.•................ G - •. G ..••... NNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN
P. thoenii - NCFB 568·
...•....•....... N T .. -.N - .. G N N .
P. acidipropinici (Stack)
..•.•.•. G C ....•............ T .. -.G - .. G NN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN
P. acidipropionici - ATCC 25562·
• •...••.. , •..•.. C. .• •. A. . . • . .. . G N NC. G. . . . .. .. T. . . . . .. . .. N CC . N .
P. freudenreichii (Stack)
.N NG ....••• C .•.•. A GNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNCC N N .. N .
P.freudenreichii - ATCC 6207·
........ G C A G G -C.G T T AC .
P. cyclohexanicum
..•••.. . G T TA G - .. G TA T C •..•• A •.•.Propioniferax. innocua .•. C ..•..•
P. aClles ...•... • G T .. T .••........... CG.TAT GG.G C
"P. coccoides" - KM 252 · ...•••• G. • ..• N. T. .• • .A. • . . . .. . ... NNNNNN NNNG...... .. T. . . . . .. . ......•. C
••..•... N •.•. C ••.••.
.•. C •••••.
"P. coccoides" - KM 375 ..•..•• • G •...• T.C •.••. A ...•....... CG.AAT GG.G .•.••... T ..............•. C ••.•...• N ..•. C ...•..
Luteococcusjaponicus - ATCC(R) 51527· •••. • , .. G ....•.. N .••.. A CG.AAT GG.G .•...... T C . ••• C ••.•••
Luteococcusjaponicus (Tam) · ••••... G. . C... . .A. . . . . .. . ••• CG.AAT GG. G. . . . .. .• T. . . . . .. . •..•.... C .. N . C •. C •.•...
Nocardioides jensenii ....... . G CTA .. TTA CG.TAT GG.G GTAA T C ••• C .• N .••
Nocardioides /uteus ...•••• • G .•••.•. C •.•• TA ••.•••.....••..• - G .. G ..•.••.• TA .•.... T .......• N •.••••.•••••. C •• N •.•
Nocardioides a/bus ..•..•• • G .•••••. C •.•• TA ••••..••......•• - G .. G •••.•••• TA ••.•.• T •....••. N
Aeromicrobium erythreus •••.•.. • G •...••• C ••.. TA .•.....•... CG.GAT GG.G •• N ..... TA .....• T ....•.• NC
... NN ......•• C •••••.
••. C •••••.
Terrabacter tumescens •..•••. . G •...•.. C .•.. TA ..........• CG .. AT GG.G .•..... NTN T ...••... C
TCATNATGCC CCTTATGTCC AGGGCTTCAC GCATGCTACA ATGGCTGGTA CAAAGAGTTG CGACACT-GT GAGGTGGAGC GAATCTCAAA AAGCCAGTCT [10001
P.jensenii (Stack) •••• C ••••••••••••••••••••.••.••.•••.••• N ••••.•••.•••••••••..•.••.•. - ••.•••••...••••.•••.•••••.•••••.
P. jensenii - DSM 20535·
P. thoenii (Stack) .. , .T ...•. .............................. •••• • C ••••
..... G ..
NNNNNNNN •. .............................. •.•• • C ••.. .. ... G ..P. thoenii - NCFB 568· .... . C G TC- .. . ....... G ...... G.N ..
P. acidipropionici (Stack) ..• • N ...•. · N C. . .. . G . . TC - .. . T . . .. TTG .
P. acidipropionici - ATCC 25562· NNNNNNNN .. ............... C G- .CT GT G G ..
Pi freudenreichii (Slack) ••• • C .•••. · . . . . . . . .. . C. . .. . .. G- . CT.. . GT. . .. . G. . G .
P. freudenreichii - ATCC 6207· ••• • C ••••• · . . . . . . . .. . C.. .,. G- . CT.. . GT. . .. . G. . .
P. cyclohexanicum .... C C . · TC T.G.C A-.CT.C .. A.GTA C .. CN NNNNNNNNNN
Propioniferax. innocua .... C .. · . . . . . . . .. .. G -.. GCGAG . CT.. . GT. . .. . GG . . G .
P. acnes .... C .. · .... C. . •. . G. C.. . .. A-. GT. CA GT. . .. . C. . .. . G .
"P. coccoides" - KM 252 . " .C . ..... C G.C ...•. A-.CT.C A CT C G .
"P. coccoides" - KM 375 . " .C . ..... C G.C A-.GT.C A GT C G ..
Luteococcus japonicus - ATCC(R) 51527· .••. C •..•. . C G.C A-.CT.C A GT C .•....... G .
Luteococcus ioponicus (Tamil) .... C .. A CA G .. G.C T .. C-.C A C G .. A .
Nocardioides jensenii .... C .
.... C . · . . . . . . . .. . C. . .. . N ..• G . C.. . .. TC . C- .. . GT. . .. . C. . .. . G ....Nocardioides luteus ..... C N G.C TC.C- .. N GT C G ....
Nocardioides a/bus .... C . ........... C G.C A .. C- .. A T.T C G.N .. o
Aeromicrobium. erythreus .. T.C N A -..J
..•.. C G.C A •. C-.C ..... C ...... A .. G ....
Terrabacter tumescens .... C C ..
P. jensenii (Stack) CAGTTCGGAT TGGGGTCTGC AACTCGACCC CATGAAGTCG GAGTCGCTAG TAATCGCAGA TCAGCAACGC TGCGGTGAAT ACGTTCCCGG GNCTNGTACA [l100J
P.jensenii - DSM 20535· NN. ....... .N .NNN. ... .NN. ...... . NN . . NN .N .G ..T .
P. thoenii (Stack) ..............•......... G ..................•............................................... C .. T .
P. thoenii - NCFB 568· ............................................................ .C .. T" .
P. acidipropionici (Stack) ............................................................ .C .. T .
.NNN NNNNNN N NNN NNNNG N . ...NNNNNNN NNNNNNNNNNP. acidipropionici - ATCC 25562·
P. freudenreichii (Stack) ........................................
.N .. T .
........................................ •.•. •. . N •• .G .. T .P. freudenreichii - ATCC 6207·
P. cyclohexanicum .......... . .G .. T .
Propioniferax. innocua NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN.N .. T .....
P. acnes ......•. . T
....•.. . N . •• N ••.•••.• G .• T •.•••
"P. coccoides" - KM 252 ...... .NN. •• N •••••..• G •• T .••••
"P. coccoides" - KM 375
Luteococcus japonicus - ATCC(R) 51527· •..••• •NN. •...... . N .
.G .. TC .
•••. . TT ... .G .. T .
Luteococcusjaponicus (Tamu) .... .CG . .C .. T .
Nocardioides jensenii .... .CG . .C .. T ...•.
Nocardioides luteus .... .CG . .c .. T •....
Nocardioides albus .C .. T •..•.
Aeromicrobium erythreus •. . N •...•. •..... . N ..
.NN.N .....
.... .CG ... .C .. T .....
Terrabacter tumescens
P. jensenii (Stack) CACCGCCCGT CAAGTCATNA AAGTCGGTAA CACCCGA [l137J
P. jensenii - DSM 20535· .NNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNN
P. thoenii (Stack) •••.••..•• T .•. N ••. G •..•.••••..
P. thoenii - NCFB 568· ...... NNNN NNNNNNNNNN NNNNNNNNNN NNNNNNN
P. acidipropionici (Stack) ., ......••..•..... N ....•...•..
P. acidipropionici - ATCC 25562' NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNN
P. freudenreichii (Stack) ••.••...•••••.•••. G •..••.•••..
Pi freudenretchii - ATCe 6207· .NNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNN
P. cyclohexanicum •••.••••••..••.••• G ....••..•.•
Propioniferax. innocua NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNN
P. acnes •..••..••.••.•••.• G •.••• T ••...
"P. coccoides" - KM 252 •••••••. G •.. N •.•••..•..•• A.
"P. coccoides" - KM 375 .N NN N A.
Luteococcus japonicus - ATCe(R) 51527· ••••••.• G •.•••.• N ••••..•. A.
Luteococcusjaponicus (Tamu) •..••.•• G .•• C .••.••.
Nocardioidesjensenii ............ N CG C ..
Nocardioides luteus ..... NN. .. ..C CG . . C ..
Nocardioides albus ..... GN. .. ..C CG . ..C C ..
..C.N ... c .. o-Aeromicrobium erythreus G .•....•. 00
Terrabacter tumescens ....... CG.
109
Fig. 7. Rooted phylogenetic tree showing the intrageneric relationships of the classical
Propionibacterium species relative to that of the two "P. coccoides" strains, the
1.japonicus type strain, as well as phylogenetically closely related genera. The
percentage values indicated on the tree are the frequencies with which branching occurred
in 1000 bootstrap replications.
110
1'. jensemi - (Charfreitag and Stackebrandt, 1989)
P.jellsenii DSM 20535'
99%
P. thoenn - iCharfreitag and Stackebrandt, 1989)
99% P. thoenii NCFB 568'
P. acidipropionici - tCharfreitag and Stackebrandt, 1989)
P. acidipropionici AT CC 25562'
100%
P. freudenreichii - (Charfreitag and Stackebrandt, 1989)
98% Ps freudenreichii ATCC 6207'
P. cyclohexanicum
72%
Propioniferax innocua
89%
"P. coccoides" KM 252
Luteococcus japonicus A TCC(!t) 51527'
97% ·P. coccoides" KM 375
Luteococcus japonicus (Tomura et al., 1994)
Terrabacter tumescens
Aeromicrobium erythreus
Nocardioides jensenii
Noc:ardioides albus
Nocardioides luteus
111
obtained during this study was identical to that as reported by Riedel (1997) and
confirmed the disparities reported by Riedel (1997) concerning the published sequence
data of the classical Propionibacterium species. Despite numerous repetitions, the
sequence data obtained during this study for the Luteococcus japonicus type strain
was also observed to differ in 24 bp from the published sequence data of Tamura et al.
(1994).
The two "P. coccoides" strains and the L. japonicus type strain evaluated
during this study were observed to group together forming a phylogenetic tight cluster
which could clearly be differentiated from the members of the genus
Propionibacterium and the various outgroups included in this study. A tighter
phylogenetic relationship was, however, observed between the "P. coccoides" (KM
252) strain and the L. japonicus type strain than between the latter and "P. coccoides"
(KM 375), confirming the overall phenotypic similarity observed previously (Chapter
3).
DNA:DNA Probe Hybridisation. Reciprocal hybridisation percentages were
averaged to obtain a final percentage of DNA reassociation. These values are
presented in Table 3. Despite numerous problems encountered with the application of
this technique and the high standard deviation values obtained when two strains were
closely related, five major groups could be differentiated on the basis of their high
DNA:DNA homology values. Four of the major groups represented the various
classical Propionibacterium species as described by Moore and Holdeman (1974) and
Cummins and Johnson (1986). Whilst the fifth group contained the two
"P. coccoides" strains and the Luteococcus japonicus type strain.
The first major group consisted of the two P. acidipropionici strains. A 91 ±
21% DNA:DNA homology was observed between these strains. The second major
group consisted of the two P. freudenreichii strains with a 95 ± 15% DNA:DNA
homology. All the strains which were phenotypically and according to their
PCRfRFLP as well as ribotyping profiles identified as P. jensenii grouped together.
The DNA:DNA homology values of this group varied from 60 ± 14 to 88 ± 21%. The
three P. thoenii strains displayed DNA:DNA homology values ranging from 59 ± 15
to 68 ± 1%. Very high homology values were observed between the two
"P. coccoides" strains and the L. japonicus type strain. Homology calculated between
Table 3. The average normalised percentage hybridisation observed between the different bacterial strains.
1 100%
2 91±21 100%
3 6±2 7±2 100%
4 7±2 6±4 ·95±15 100%
5 13±5 19±5 7±3 6±2 100%
6 22±1 18±6 12±7 9±2 86±12 100%
Bacterial 7 19±2 19±5 9±0.5 11±7 60±14 88±18 100%
strains 8 14±6 12±5 10±2 4±1 82±17 88±21 75±3 100%
(No.) 9 19±2 23±5 11±6 9±3 37±0.5 34±1 36±3 39±13 100%
10 19±7 19±12 7±5 5±0.5 20±7 27±0.25 26±15 29±11 66±10 100%
11 19±8 20±13 14±5 20±15 25±9 35±6 30±18 39±21 59±15 68±1 100%
12 8±3 8±1 5±2 5±3 4±0.4 6±1 5±1 4±2 6±2 5±2 7±3 100%
13 6+3 5+1 4±2 8±6 6±3 8±4 6±2 7±3 9±6 9±7 12±10 95±15 100%
14 10±7 6±1 6±3 10±3 9±7 11±7 6±3 7±2 9±6 8±6 12±10 93±21 91±11 100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Bacterial strains (No.)
No. Lab. No. Name Source Strain Isolated from Identification key# Abbreviations
1 424 P. acidipropionici 1 ATCC 25562* - P. acidipropionici *: Type strain
2 421 "P. pentosaceum" 4 NCFB 570 Emmentaler P. acidipropionici #: Identification key of Johnson and Cummins (1986)
3 423 P. freudenreichii 1 ATCC 6207* Swiss cheese P. freud. ss .. freudenreichii
4 453 "P. shermanii" 5 P 67 - P. freud. ss .. freudenreichii 1: ATCC, American Type Culture Collection, Rockville
5 80 P.jensenii 2 DSM 20535* Buttermilk P. jensenii Maryland, USA.
6 130 "P. intermedium" 5 NCIB 8728 - P.jensenii 2: DSM, Deutsche Sammlung Von Mikroorganismen und
7 427 "P. pituitosum" 4 NCFB 1077 - P. jensenii Zelkulturen, Braunschweig, Germany.
8 75 "P. technicum" 6 P 74 Emmentaler P. jensenii 3: L.I. Vorobjeva, Moscow State University, Russia.
9 419 P. thoenii 4 NCFB 568* Emmentaler P. thoenii 4: NCFB, National Collection of Food Bacteria, Shinfield,
10 294 P. thoenii 7 - Anaerobic digester P. thoenii Reading, UK.
Il 297 P. thoenii 7 - Anaerobic digester P. thoenii 5: NCIB, National Collection of Industrial Bacteria, UK.
12 362 "P. coccoides" 3 - Soviet cheese - 6: G.W. Reinbold, Iowa State University, Iowa, USA.
13 363 "P. coccoides" 3 VKMAC Soviet cheese - 7: Environmental Microbiology Culture Collection, .-...
1910 University of the Orange Free State, Souuth Africa. N
14 364 L. japonicus I ATCC(R) Ground and water
51527*
113
the two "P. coccoides" strains was 95 ± 15%, between "P. coccoides" (KM 252) and
L. japonicus was 93 ± 21%, and between "P. coccoides" (KM 375) and L. japonicus
was 91 ± 11%. Very low DNA:DNA homology values (ranging from 4 ± 0.4% to 39
± 21%) were observed between the different classical Propionibacterium species and
the "P. coccoides" / L. japonicus strains.
Discussion
Due to the higher degree of overall phenotypic similarity observed between the
"P. coccoides" strains and the L. japonicus type strain, than between the latter and
members of the genus Propionibacterium, a molecular and phylogenetic analysis of
the various strains was undertaken. Using the polymerase chain reaction as described
by Riedel et al. (1994), the predicted 1110bp fragment of the 16S rRNA gene was
successfully amplified. All four the classical Propionibacterium species could be
differentiated from each other on the basis of their restriction fragment length
polymorphism data using the restriction endonucleases HaelI and Smal, confirming
the results as reported by Riedel et al. (1994: 1998). Although the "P. coccoides" and
Luteococcus japonicus strains could be differentiated from the various classical
Propionibacterium species on the basis of their restriction endonuclease profiles, it
was not possible to differentiate them from each other, despite the evaluation of
numerous restriction endonucleases. These results subsequently confirm the high
degree of overall phenotypic similarity observed between the "P. coccoides" strains
and the Luteococcus japonicus type strain (Chapter 3).
It was only possible to differentiate the "P. coccoides" and L. japonicus strains
via ribotyping on the basis of their BstEII restriction endonuclease profiles. The
hybridisation pattern obtained for the two "P. coccoides" and L. japonicus strains
using the 16S rDNA probe of P. freudenreichii subsp. freudenreichii (ATCC 6207)
and the BstEII digested DNA, revealed 3 distinct ribotypes. This data thus confirmed
the phenotypic differentiation observed between these three species in chapter 3. An
82% similarity was, however, observed between the P. jensenii species and the two
"P. coccoides" strains as well as the L. japonicus type strain when a numerical
analysis was performed on the ribotyping data as obtained during this study and that
reported by Riedel and Britz (1996). These results subsequently confirm the high
114
degree of similarity observed by Vorobjeva et al. (1983) between "P. coccoides" and
P. jensenii based on DNA:DNA homology.
Based on the 16S rDNA sequence data and the branching pattern observed in
the phylogram, the classical Propionibacterium species could be divided into two
major phylogenetic groups. The larger major group containing the P. acidipropionici,
P. jensenii, and P. thoenii species could be divided into two smaller subdivisions.
The P. jensenii and P. thoenii subdivision, which clustered together with a 99%
confidence interval was observed to be the most closely related phylogenetically.
Together with the P. acidipropionici subdivision, the P. jensenii and P. thoenii
subcluster formed a phylogenetic cluster (99% confidence interval) which embraced
the classical propionibacteria containing the LL-isomer of diaminopimelic acid as the
diamino acid of their cell wall peptidoglycan. Based on the 16S rDNA relatedness,
this major group is phylogenetically well separated from the P. freudenreichii and P.
cyclohexanicum major group with a 93% confidence interval value.
Propionibacterium acnes, the only member of the cutaneous group evaluated during
this study, as well as Propioniferax innocua were observed to be phylogenetically
closely related to the classical Propionibacterium species confirming the report of
Johnson and Cummins (1972). Of the various phylogenetically related genera
evaluated during this study, L. japonicus was observed to be the most closely related
to the classical Propionibacterium species, with a 89% confidence interval confirming
the report of Tamura et al. (1994). Both "P. coccoides" strains clustered with the L.
japonicus type strain, confirming the high degree of phenotypic similarity observed
between these strains. The "P. coccoides" / L. japonicus clade was, however, clearly
separated from the cluster representing the various Propionibacterium species. With
only a 16 bp difference between the sequence data of "P. coccoides" (KM 252) and
the L. japonicus type strain, a tighter phylogenetic relationship with a confidence
interval of 93% was observed than between "P. coccoides" (KM 375) and L.
japonicus. The confidence interval value of 98% between these two strains was based
on a 23 bp difference in their 16S rRNA sequence data. This divergence 16S rDNA
sequence in data subsequently also confirms the variation observed in the phenotypic
characteristics of these strains.
Despite numerous precautionary measures, including adaptations during the
quantification of the DNA, especially since the lambda phage DNA, which was used
115
as the concentration standard, had a lower mol% G + C content than the
Propionibacterium and Luteococcus genera and the optimisation of the DNA
hybridisation temperatures, numerous difficulties were experience with the application
of the DNA:DNA probe hybridisation technique as described by Viljoen (1996).
Excessive problems were experience with the quantification of the DNA, which could
possibly have contributed to the large standard deviation observed. It is subsequently
imperative that this technique should be optimised before any conclusive results can
be made. Five DNA:DNA homology groups, four resembling the various classical
propionibacteria species and one containing the "P. coccoides" and L. japonicus
strains could, however, be differentiated. These results subsequently confirm the
phylogenetic analysis of the 16S rDNA sequence data. The P. jensenii and P. thoenii
homology groups showed about 20 to 40% DNA:DNA homology with each other,
whilst the P. acidipropionici homology group showed from 12 to 23% homology to
the P. jensenii and P. thoenii reference strains, respectively. The Propionibacterium
freudenreichii homology group showed from 6 - 7, 4 - 12, and 5 - 20% DNA:DNA
homology with the P. acidipropionici, P. jensenii and P. thoenii homology groups,
respectively. The homology group consisting of the "P. coccoides" strains and the
L. japonicus type strain displayed between 4 - 12% DNA:DNA homology to the four
classical Propionibacterium homology groups.
Although the four DNA:DNA homology groups, representing the four
classical Propionibacterium groups as determined by Johnson and Cummins (1972)
could be confirmed during this study, the homology values obtained during this study
were approximately 15% lower than those as reported by Johnson and Cummins
(1972). According to Johnson and Cummins (1972) the P. jensenii group showed
about 50% homology to the P. thoenii group and about 30 to 35% to the P.
acidipropionici group. During this study, the average DNA:DNA homology between
the P. jensenii and P. thoenii groups was 31%, while the average DNA:DNA
homology between the P. jensenii and P. acidipropionici groups was observed to be
17%. Johnson and Cummins (1972) also reported that the P. .freudenreichii strains
showed a rather lower level of similarity (8 to 25%) to the other homology groups.
Similar results were observed during this study. The statement of Vorobjeva et al.
(1983) that a high degree of DNA:DNA homology existed between P. jensenii and "P.
coccoides" could, however, not be validated during this study.
116
It is evident from the results of this study, that a higher degree of molecular
and phylogenetic similarity exists between the genus Luteococcus and the "P.
coccoides" strains than between the latter and the genus Propionibacterium. The
results of this study subsequently also confirm the high degree of phenotypic
similarity observed between these strains. During this study the conclusions made
during the phenotypic characterisation of "P. coccoides" (Chapter 3) could be
confirmed. Since no genera of cocci with LL-DAP in their cell wall and with a high
mol% G + C content have ever been described, Tamura et al. (1994) only compared
their data to the genera Propionibacterium, Nocardioides, Aeromicrobium and
Terrabacter. The genus Sarcina, which is the only bacterial coccus containing LL-
DAP in their cell wall has a low mol% G + C content in its DNA and due to the fact
that it is a strict anaerobe, was not included (Tamura et al., 1994). With all the above
mentioned high mol% G + C content genera included in this study, it is thus
indisputable that the proposal of Vorobjeva et al. (1983) to include "P. coccoides" as a
new species within the genus Propionibacterium is incorrect. The "P. coccoides"
strains should subsequently be transferred to the genus Luteococcus as putative
subspecies. The genus description of Luteococcus japonicus should subsequently also
be revised to include the phenetic and molecular description of these strains.
117
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and their systematic position. Microbiol. 52,368-373 (1983).
Wilson, K., Goulding, K.H.: A biologists guide to principles and techniques of
practical biochemistry, pp. 333-334. Edward Arnold, UK 1986.
120
Chapter 5
General discussion and conclusions
Ever since the description of the first propionibacteria by Von Freudenreich
and Orla-Jensen in 1906, this group of organisms has been the centre of many
controversies in terms of their identification and taxonomic status. This is not only
evident for species already included within the genus Propionibacterium but also
when newly proposed species are considered. Even after the compilation of
classification keys by Moore and Holdeman (1974) as well as Cummins and Johnson
(1986), several reports can still be found in the literature where difficulties with the
identification of isolates were experienced. With the advance of genetically based
identification systems, only more questions started arising. In addition to the
problems experienced in some publications concerning the identification and the
differentiation of the various subspecies within Propionibacterium freudenreichii
(Riedel and Britz, 1992; Bear and Ryba, 1988; Johnson and Cummins 1972), 16
distinct ribotype profiles were reported by Riedel and Britz (1996) within the four
classical Propionibacterium species. Another typical example concerns the bacterium
P. propionicum which was proposed as a new species within the genus
Propionibacterium by Charfreitag et al. (1988). Despite numerous publications
(O'Donnell et al., 1985; Cummins and Johnson, 1992; Cummins and Moss, 1990)
confirming this proposal, P. propionicum was retained as Arachnia propionica in
Bergey's Manual of Determinative Bacteriology (Halt et al., 1994). Reinbold's (1978)
perspective that the consolidation of various "old" classical species to form the P.
acidipropionici, P. freudenreichii, P. jensenii and P. thoenii species by Moore and
Holdeman (1974), only worsened the taxonomy of the classical propionibacteria, is
thus still very appropriate.
In 1983, Vorobjeva et al. described a new species within the genus
Propionibacterium. Based on the high degree of similarity between this bacterium
and members of the classical Propionibacterium group, both on a phenotypic as well
as on a genetic level, Vorobjeva et al. (1983) designated the name "Propionibacterium
121
coccoides" to this newly described Gram-positive coccus. This bacterium not only
displayed the same phenotypic similarities, including the formation of propionic acid
as the major metabolite of lactate fermentation and the same electrophoretic mobility
of the various enzymes (catalase, superoxide dismutase and peroxidase), to those
already described for the genus Propionibacterium, but it was also found to be
genetically related to the genus Propionibacterium. The mol% G + C content of the
DNA of"P. coccoides" was determined to be 63.4 mol%, which was within the range
of that published for the genus Propionibacterium (Vorobjeva et al., 1983). Based on
their DAN:DNA homology values, the highest similarity (49%) was observed between
"P. coccoides" and that of the propionic acid bacterium P. jensenii.
According to the definition of the genus Propionibacterium as given by Moore
and Holdeman (1974), this propionic acid producing coccus should subsequently be
classified within this genus. Although numerous taxonomic studies have been
undertaken (Britz and Riedel, 1991: 1994; Riedel and Britz, 1993; Riedel et al.,
1992: 1994) in an attempt to solve the taxonomic dilemma within the genus
Propionibacterium, none have ever included "P. coccoides". Subsequently the correct
systematic position of this organism remained unresolved. Although this proposal of
Vorobjeva et al. (1983) was mentioned as well as fertified by Cummins and Johnson
(1992), it was never even mentioned in either the Bergey's Manual of Systematic
Bacteriology (Cummins and Johnson, 1986) or Bergey's Manual of Determinative
Bacteriology (Holt et al., 1994). Based on these reasons, it was thus evident that the
taxonomic status of "P. coccoides" relative to that of the classical Propionibacterium
species would have to be re-examined.
The first objective of this study was, therefore, to further characterise
"P. coccoides" phenotypically and to determine it's systematic position relative to that
of the already described classical Propionibacterium species. A Gram-positive, high
mol% G + C content coccus, namely Luteococcus japonicus, which was previously
determined to be the closest phylogenetic neighbour of the genus Propionibacterium
(Tamura et al., 1994), was also included during this study as an outgroup. On the
basis of the results obtained in chapter 3, it was evident that the "P. coccoides" strains
as described by Vorobjeva et al. (1983) could not be identified as any of the classical
122
Propionibacterium species as described by Cummins and Johnson (1986).
Furthermore, the "P. coccoides" strains clustered together with the L. japonicus type
strain in a separate cluster, which was clearly delineated from the various clusters
representing the classical Propionibacterium species. Based on the results obtained
during this study (Chapter 3), it was possible to differentiate the two "P. coccoides" as
well as the L. japonicus strains from P. acidipropionici, P. freudenreichii, P. jensenii
and P. thoenii on their ability to ferment D-mannose and sorbitol, as well as their
characteristic bright yellow pigmentation. Although the "P. coccoides" / L. japonicus
cluster coalesced at a 80% similarity level, it was still possible to phenotypically
differentiate between L. japonicus and the two "P. coccoides" strains themselves.
Based on the results of the numerical analysis of the phenotypic data, it was apparent
that a higher degree of phenotypic similarity existed between L. japonicus and
"P. coccoides" than between the latter and the members of the genus
Propionibacterium. Itwas subsequently apparent that the proposal of Vorobjeva et al.
(1983) to include "P. coccoides" as a new species within the genus
I Propionibacterium, could possibly be incorrect.
In order to validate the results observed during the numerical analysis of the
phenotypic data, a molecular and phylogenetic analysis of the classical
propionibacteria, "P. coccoides" and L. japonicus was undertaken. The fist objective
of this study was to determine the relationship between the "P. coccoides" strains and
known marker strains using molecular techniques based on the 16S ribosomal RNA
genes as well as the flanking regions. Using the rapid yet sensitive restriction
fragment length polymorphism technique as described and optimised by Riedel et al.
(1994: 1998), an identical RFLP profile was obtained for both the "P. coccoides"
strains and the L. japonicus type strain. These RFLP patterns were obtained after
restriction endonuclease digestion of the PCR products, obtained using primers
16sP 1-16sP4, with the restriction endonucleases HaeIII and AluI. Although it was
possible to differentiate between the four classical Propionibacterium species,
confirming the data obtained by Riedel et al. (1994: 1998), and the RFLP profile
obtained for the two "P. coccoides" and the L. japonicus strains, differentiation
between the latter two species, using various other restriction endonucleases, was,
however, not possible.
123
The next objective of this study was to investigate the relative homogeneity
observed within each of the two "P. coccoides" strains and the L. japonicus type strain
based on the RFLP data, which was in total contrast to the phenotypic heterogeneity
observed, using ribosomal RNA gene restriction patterns (ribotyping). The
combination of the HaeII and Smal restriction endonuclease patterns resulted in the
generation of a unique ribotype profile (profile Q) which was observed for both the
"P. coccoides" strains as well as the L. japonicus type strain. This ribotyping profile
could clearly be differentiated from the 16 distinct ribotype profiles as determined for
the classical Propionibacterium strains by Riedel and Britz (1996). Despite the
evaluation of 12 other restriction endonucleases using the ribotyping technique,
differentiation between the "P. coccoides" strains and the L. japonicus type strain was
only possible using the 16S rDNA probe of the P. freudenreichii subsp.freudenreichii
(ATCC 6207) and the restriction endonuclease BstEII. Although the three distinct
ribotypes obtained for the two "P. coccoides" strains and the L. japonicus type strain
confirmed the phenotypic heterogeneity observe between these two species in chapter
3, it is evident that the organisms were very closely related on a molecular level.
Due to the large degree of phenotypic diversity observed between the two
"P. coccoides" strains and the L. japonicus type strain (Chapter 3) and since
confirmation of this phenomenon using the RFLP and ribotyping techniques were
lacking, the next objective of this study was to determine the intrageneric relationship
between these three strains, the type strains of the various classical Propionibacterium
species and phylogenetically related genera. This phylogenetic relationship was
determined by comparative analysis of their 16S ribosomal DNA sequence data. The
various Propionibacterium species were observed to coalesce in a phylogenetic clade
which was clearly delineated from the clade containing the "P. coccoides" and
L.japonicus strains. A 16 and 23 base pair difference was observed to exist in the
16S rDNA sequence data of the L. japonicus, "P. coccoides" (KM 252) and the
"P. coccoides" (KM 375) strains, respectively. Four clusters, each representing one of
the current classical species (Cummins and Johnson, 1986) could be delineated in the
major Propionibacterium clade after PAUP analysis of the sequence data (Chapter 4).
Two major groups within the genus Propionibacterium were, however, apparent
124
confirming the results obtained by Riedel (1997). The P. acidipropionici, P. jensenii
and P. thoenii strains formed a phylogenetic cluster which was clearly separated from
the P. freudenreichii species.
The last objective of this study was to investigate the heterogeneity observed
between various classical Propionibacterium species and the two "P. coccoides" and
L. japonicus type strain, using the entire genome structure. DNA:DNA probe
hybridisation (Viljoen, 1996) was subsequently evaluated as a possible reliable species
differentiation technique. Five major DNA:DNA homology groups, four resembling
the various classical propionibacteria species and one containing the two
"P. coccoides" strains and the L. japonicus type strain could be differentiated. A very
high degree of DNA:DNA homology (>90%) was observed between the L. japonicus
type and the two "P. coccoides" strains. These results subsequently confirm the
phylogenetic analysis of the 16S rDNA sequence data, where the L. japonicus /
"P. coccoides"strains clustered together in a clade which was phylogenetically clearly
distinct from the clade containing the various Propionibacterium species. A low
degree of DNA:DNA homology was observed between the various Propionibacterium
species and the group containing L. japonicus and the "P. coccoides" strains,
confirming the phenotypic, molecular and phylogenetic analyses.
In conclusion, the results of this study confirm the varIOUSdifferentiation
techniques as described for the classical Propionibacterium species (Riedel, 1997).
This not only included phenotypic characterisation but also molecular
characterisation, which included analysis of the polymerase chain reaction, restriction
fragment length polymorphism, ribotyping as well as the 16S rRNA sequencing
techniques. The data obtained using these optimised techniques, clearly indicated that
the proposal of Vorobjeva et al. (1983) to include "P. coccoides" as a new species
within the genus Propionibacterium was incorrect and that this proposition requires
some revision. Based on the higher degree of overall phenotypic and molecular
similarity between L. japonicus and "P. coccoides" than between the latter and the
various species of the genus Propionibacterium, as observed during this study, it is
subsequently proposed that the "P. coccoides" strains be transferred to the genus
Luteococcus as putative subspecies. Furthermore it is suggested that the genus
125
description (Tamura et aI., 1994), as stated below, should be revised to include the
phenetic and molecular description of these strains.
The genus Luteococcus was first described in 1994 by Tamura et al. This
Gram-positive cocci was first known as "Micrococcus aurantiacus" IFO 12422 and
Micrococcus sp. IFO 15385. Originally isolated from soil on Tokara island and from
water for brewing "rniyamizu", respectively (Oda, 1935), they have been maintained
in the culture collection of the Institute for Fermentation, Osaka. The taxonomic
position of these strains has, however, remained uncertain due to the fact that they
contain LL-diaminopimelic acid (LL-OAP) in their cell wall, instead of lysine which
is characteristic of other Micrococcus species. Since, no Gram-positive cocci with a
high mol% G + C content and containing LL-DAP had been described previously,
these organisms were compared to the genera Propionibacterium, Aeromicrobium,
Terrabacter and Nocardioides (Tamura et aI., 1994). Analysis of the partial 16S
rRNA sequence indicated that the genus Luteococcus represented a distinct line of
descent among the Gram-positive bacteria with a high G + C content. Luteococcus
japonicus was further revealed to be phylogenetically more closely related to rod-
shaped P. innocuum than to the other genera of Gram-positive cocci, rods and
nocardioforms with high mol% G + C contents.
The description of Luteococcus japonicus as described by Tamura et al. (1994)
is as follows: cells are spherical and 0.7 to 1.0 urn in diameter and occur singly, in
pairs, or in tetrads. Endospores are not formed. Gram-positive. Colonies are circular
and smooth and may be cream coloured to yellow. Facultative anaerobic. Catalase
and oxidase positive. Urease negative. The cells do not reduce nitrate to nitrite.
Oxidation-fermentation is fermentative. Acid is produced from D-glucose, O-ribose,
O-galactose, D-mannose, D-fructose, sucrose, maltose, trehalose, raffinose, glycerol,
mannitol, inositol and L-arabinose but not from D-xylose, O-arabinose or L-rharnnose.
Starch is hydrolysed. Tween 20, 40, 60 and 80 are not hydrolysed. Propionic acid is
formed from glucose as a major product. Optimum growth temperature is 26 to 28 C.
Cell wall peptidoglycan contains LL-diaminopimelic acid, alanine, glycine and
glutamic acid (ea 1:2: 1:1). The major menaquinone is MK -9(~). Mycolic acid is not
126
present. The major cellular fatty acid is 16:1 and among the minor components a
small amount of 2-0H iso-18:0 is also present. Arabinose is present as a diagnostic
sugar in the cell wall. As polar lipids, phosphatidylinositol, diphosphatidylglycerol
and phosphatidylglycerol are present. The DNA base composition of the type strain is
67% as determined by HPLC. Distribution: soil and water. The type species is
Luteococcus japonicus (IFO 12422).
A comparison of the phenotypic characteristics as described by Tarnura et al.
(1994) to those as obtained for "P. coccoides" during this study, as well as those
published by Vorobjeva et al. (1983), revealed that the only difference between
L. japonicus and the two "P. coccoides" strains examined during this study, was the
inability of the two "P. coccoides" strains to ferment inositol. Nitrate reduction was
also determined to be positive for "P. coccoides" (KM 375) and yet negative for both
"P. coccoides" (KM 252) and L. japonicus (ATCC(R) 51527).
With the results obtained and presented in this study, it is evident that the
"P. coccoides" species closely resembles the species description of Luteococcus
japonicus. Controversy, however, still exists concerning the spelling of the genus
Luteococcus and according to rule 61 of International Code of Nomenclature of
Bacteria the spelling should be changed to Luteicoccus (L. adj. Luteus a urn, yellow;
Gr. N. coccus, grain, berry; M.L. mase. N. Luteicoccus, yellow coccus). Until this
controversy is resolved it is, however, proposed that the two "P. coccoides" strains
examined during this study, namely "P. coccoides" (KM 252) and "P. cocoides" (KM
375) be reclassified as "Luteococcus japonicus subspecies casei" and "Luteococcus
japonicus subspecies tyrophilus", respectively. A thorough description of these two
subspecies as determined during this study and as described by Vorobjeva et al.
(1983), is as follows:
Description of "Luteococcus japonicus subspecies casei" subsp. nov.
Cells are Gram-positive cocci, facultative anaerobic, non-motile, non-spore-
forming and are spherical at all stages of growth. Colonies on YEL plates are usually
opaque, circular and slightly raised yellow coloured cells. The organism is a
facultative anaerobe which ferments lactate with the production of propionate, acetate
127
and C02 as principal end products, apparently by the succinate-methylmalonyl CoA
pathway. It exhibits catalase, superoxide disrnutase, peroxide, arginine dihydrolase,
lysine dihydrolase, ornithine dihydrolase and urease activity. Citrate is not utilised
and no ortho-nitro-phenyl-galactoside, tryrophane desaminase, indole, acetoin
production, gelatin liquefaction, L-diaminopimelic acid and meso-diaminopimelic
acid activity is detectable. Acid is produced from glycerol, erythritol, D-arabinose,
galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, maltose, melibiose,
trehalose, inulin, melezitose, D-raffinose, amidon, glycogene, D-turanose, D-arabitol
and L-arabitol. Acid is not produced from L-arabinose, ribose, D-xylose, L-xylose,
adonitol, p-methyl xyloside, L-sorbose, rhamnose, dulcitol, inositol, a-methyl-D-
mannose, a-methyl-D-glucoside, N-acetyl-glucosamine, amygdalin, arbutin, esculin,
salicin, cellobiose, lactose, xylitol, p-gentiobiose, D-Iyxose, D-tagatose, gluconate, 2
keto-gluconate and 5 keto-gluconate. Growth most rapid at 22-30cC, but also down to
8-10cC. This bacterium is also halo-tolerant up to 6.5% NaCI. The designated type
species is "Luteococcus japonicus subspecies casei" (KM 252). The mol% G+C
content of the DNA is 63.4 mol% (Tm).
Description of "Luteococcus japonicus subspecies tyrophilus" subsp. nov.
Cells are Gram-positive cocci, facultative anaerobic, non-motile, non-spore-
forming and are spherical at all stages of growth. Colonies on YEL plates are usually
opaque, circular and slightly raised yellow coloured cells. The organism was
facultative anaerobe which ferments lactate with the production of propionate, acetate
and C02 as principal end products, apparently by the succinate-methylmalonyl CoA
pathway. It exhibits catalase, superoxide disrnutase and peroxide activity. Citrate is
not utilised and no arginine dihydrolase, lysine dihydrolase, ornithine dihydrolase,
urease, ortho-nitro-phenyl-galactoside, tryrophane desaminase, indole, acetoin
production, gelatin liquefaction, L-diaminopimelic acid and meso-diaminopimelic
acid activity is detectable. Acid is produced from glycerol, erythritol, D-arabinose,
galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, maltose, trehalose,
melezitose, D-raffinose, D-turanose and D-arabitol. Acid is not produced from L-
arabinose, ribose, D-xylose, L-xylose, adonitol, p-methyl xyloside, L-sorbose,
rhamnose, dulcitol, inositol, a-methyl-D-mannose, a-methyl-D-glucoside, N-acetyl-
128
glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, lactose, melibiose,
inulin, amidon, glycogene, xylitol, p-gentiobiose, D-Iyxose, D-tagatose, L-arabitol,
gluconate, 2 keto-gluconate and 5 keto-gluconate. Growth is most rapid at 22-30DC,
but growth also occurs at 8-10DC. This bacterium is halo-tolerant up to 6.5% NaCI.
The designated type species is "Luteococcus japonicus subspecies tyrophilus" The
mol% G+C content of the DNA is 63.4 mol% (Tm).
Based on the results as presented during this study it is recommended that
future research be conducted in the following directions:
a) More diverse ecological niches be examined for the presence of Luteococcus
japonicus strains. This will increase the diversity of the strains available for
further phenotypic and molecular studies;
b) The phenotypic relatedness between the various L. japonicus strains should be
re-examined using more phenotypic properties;
c) A more extensive DNA:DNA hybridisation study, encompassing more strains
of Luteococcus japonicus and the classical Propionibacterium species should
be undertaken;
d) The significance and distribution of Luteococcus japonicus strains in the dairy
industry should be investigated; and that
e) Species specific probes, based on the 16S rRNA gene sequence data should be
developed. This would enable the rapid identification and enumeration of the
genus Luteococcus.
129
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samples. Int. Dairy J. 2, 299-310 (1992).
Britz, T.l, Riedel, K-H.l: A numerical taxonomic study of Propionibacterium
strains from dairy sources. J. Appl. Bacteriol. 71,407-416 (1991).
Britz, T.J., Riedel, K-I--I.J.: Propionibacterium species diversity in Leerdammer
cheese. Int. J. Food. Microbiol. 22, 257-267 (1994).
Charfreitag, 0., Collins, M.D., Stackebrandt, E.: Reclassification of Arachnia
propionica as Propionibacterium propionicus comb. nov. Int. J. Syst.
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Johnson, J.L., Cummins, C.S.: Cell wall composition and deoxyribonucleic acid
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132
Chapter 6
Summary
A study was undertaken to re-evaluate the systematic position of
"Propionibacterium coccoides" relative to that of other classical Propionibacterium
species. Two "P. coccoides" strains were evaluated by numerically relating them to
the type and reference strains of the genus Propionibacterium as well as the type strain
of the genus Luteococcus (as outgroup) by comparison of a wide range of phenotypic
characters. Numerical clustering revealed five major groups, four of which
corresponded to the existing classical species, while the fifth cluster grouped the two
"P. coccoides" strains with the type strain of the species Luteococcus japonicus.
These results clearly indicated that a higher degree of overall phenotypic similarity
existed between L. japonicus and the "P. coccoides" strains than between the latter
and the genus Propionibacterium.
Using the polymerase chain reaction, the various 16S ribosomal RNA genes of
the four Propionibacterium type strains, the two "P. coccoides" strains and the type
strain of L. japonicus were successfully amplified. Visual differentiation between the
four classical Propionibacterium type strains was possible after restriction
endonuclease digestion of the peR products obtained using primers 16sP 1-16sP4 with
the restriction endonucleases HaeIII and A/uI. Although a unique pattern was
obtained for the two "P. coccoides" and the L. japonicus strains when compared to
those as obtained for the four classical Propionibacterium type strains, differentiation
between the two "P. coccoides" and the L. japonicus strains, even after the evaluation
of numerous restriction endonucleases, was essentially still problematic.
Due to the success obtained during the application of ribotyping as a technique
to enable species differentiation within the genus Propionibacterium, this technique
was also utilised during this study. The combination of the HaeII and Smal restriction
endonuclease patterns resulted in the elucidation of the four classical
Propionibacterium type strains as well as the two "P. coccoides" and the L. japonicus
strains. A unique yet identical ribotyping profile was obtained for the two
"P. coccoides" strains and the L. japonicus type strain. In an attempt to justify the
133
phenotypic heterogeneity observed between the two "P. coccoides" strains and the
L. japonicus type strain, numerous other restriction endonucleases were also evaluated
using ribotyping. The two "P. coccoides" strains and the L. japonicus type strain
could only be differentiated after digestion of the genomic DNA with the restriction
endonuclease BstEII.
The intrageneric phylogenetic relationship of the four classical
Propionibacterium type strains, the two "P. coccoides" strains as well as the
L. japonicus type strain and various other phylogenetic related genera was determined
by comparative analysis of their 16S ribosomal RNA gene sequence data. The
"P. coccoides" strains were observed to cluster together with the type strain of the
genus Luteococcus in a clade which was phylogenetically clearly delineated from the
cluster containing the various Propionibacterium species. This data subsequently
confirmed the results obtained during the numerical analysis of the phenotypic
characteristics.
Finally DNA:DNA probe hybridisation was evaluated as a possible reliable
species differentiation technique. Five major DNA:DNA homology groups could be
distinguished. Four of these homology groups resembled the various classical
Propionibacterium species, while the fifth DNA:DNA homology group consisted out
of the "P. coccoides" strains and the L. japonicus type strain. The high degree of
DNA:DNA homology between the "P. coccoides" strains and the L. japonicus type
strain subsequently confirmed the phylogenetic analysis of the 16S rDNA sequence
data.
The results of this study subsequently indicate that the proposal by Vorobjeva
et al. (1983) to include "P. coccoides" as a new species within the genus
Propionibacterium is incorrect due to the higher degree of phenetic, molecular and
phylogenetic similarity between Luteococcus japonicus and "P. coccoides" than
between the latter and the genus Propionibacterium. It is subsequently proposed that
the "P. coccoides" strains be putatively reclassified as Luteococcus japonicus
subspecies casei and Luteococcus japonicus subspecies tyrophilus and that the genus
description of Luteococcus be revised to include the phenetic and molecular
description of these strains.
134
Hoofstuk 6
Samevatting
'n Studie was onderneem om die sistematiese posisie van "Propionibacterium
coccoides" te herevalueer relatief tot dié van ander klassieke Propionibacterium
spesies, Twee "P. coccoides" stamme was geevalueer deur hulle numeriese
verwantskappe aan die tipe- en verwysingsstamme van die genus Propionibacterium
sowel as the tipe stam van die genus Luteococcus (as buite-groep) te bepaal, deur 'n
verskeidenheid fenotipiese karakters te vergelyk. Numeriese groepering het op vyf
hoofgroepe gedui, waarvan vier ooreenstem met die bestaande klassieke spesies en
die vyfde groepering wat die twee "P. coccoides" stamme saamgroepeer met die tipe
stam van die spesie Luteococcus japonicus. Hierdie resultate dui duidelik aan dat 'n
hoër graad van algehele ooreenkoms tussen L. japonicus en die "P. coccoides"
stamme bestaan, as tussen laasgenoemde en die genus Propionibacterium.
Deur gebruik te maak van die polimerase ketting reaksie (PKR), was die verskeie 16S
ribosomale RNA gene van die vier Propionibacterium tipe stamme, die twee
"P. coccoides" stamme en die tipe stam van L. japonicus suksesvol vermeerder.
Visuele differensiasie tussen die vier klassieke Propionbacterium tipe stamme was
moontlik na vertering van die PKR produkte verkry, deur gebruik te maak van die
voorvoerders 16sPl-16sP4, met die endonukleases HaelII en Alul. Alhoewel 'n
unieke profiel verkry is vir die twee "P. coccoides" en die L. japonicus stamme, as dit
vergelyk word met dié verkry vir die vier klassieke Propionibacterium tipe stamme,
was die differensiasie tussen die twee "P. coccoides" en die L. japonicus stamme,
selfs na evaluasie van verskeie restruksie endonukleases, essensieel steeds
problematies.
As gevolg van die sukses behaal gedurende die aanwending van ribotipering as
tegniek om spesies differensiasie in staat te stel binne die genus Propionibacterium,
was hierdie tegniek ook aangewend gedurende hierdie studie. Die kombinasie van die
Haell en Smal restruksie endonuklease pofiele het die verduideliking van die vier
klassieke Propionibacterium tipe stamme asook die twee "P. coccoides" en die
L. japonicus stamme tot gevolg gehad. 'n Unieke tog identiese ribotiperings profiel
was verkry vir die twee "P. coccoides" stamme en die L. japonicus tipe stam. In 'n
135
poging om die fenotipiese heterogenitiet waargeneem tussen die twee "P. coccoides"
stamme en die L. japonicus tipe stam te verdedig, was talryke ander restruksie
endonukleases ook geevalueer deur die gebruik van ribotipering. Die twee
"P. coccoides" stamme en die L. japonicus tipe stam was slegs differensieerbaar na
snyding van die genomiese DNA met die restruksie endonuklease BstEII.
Die intrageneriese filogenetiese verwantskap van die VIer klassieke
Propionibacterium tipe stamme, die twee "P. coccoides" stamme asook die
L. japonicus tipe stam en verskeie ander filogeneties verwante genera is bepaal deur
vergelykende analise van hulle 16S ribosomale RNA geenopeenvolging. Die
"P. coccoides" stamme is bepaalom saam te groepeer met die tipe stam van die genus
Luteococcus in 'n groep wat filogeneties duidelik afbeeldbaar was van die groepering
wat die verskeie Propionibacterium spesies bevat. Hierdie data bevestig vervolgens
die resultate verkry gedurende die numeriese analise van die fenotipiese
karaktertrekke.
Ten slotte is DNA:DNA kruising ondersoek as 'n moontlike betroubare spesies
differensieerbare tegniek. VyfhoofDNA:DNA homologie groepe was onderskeibaar.
Vier van hierdie groepe kom met die verskeie klassieke Propionibacterium spesies
ooreen, terwyl die vyfde DNA:DNA homologie groep uit die "P. coccoides" stamme
en die L. japonicus tipe stam bestaan. Die hoë graad van DNA:DNA homologie
tussen die "P. coccoides" stamme en die L. japonicus tipe stam bevestig vervolgens
die filogenetiese analise van die 16S rDNA geenopeenvolging data.
Die resultate van hierdie studie dui eerstens aan dat die voorstel van Vorobjeva et al.
(1983) om "P. coccoides" in te sluit as 'n nuwe spesie binne die genus
Propionibacterium verkeerd is as gevolg van die hoë graad van fenetiese, molekulêre
en filogenetiese ooreenkomste tussen Luteococcus japonicus en "P. coccoides" as
tussen laasgenoemde en die genus Propionibacterium. Gevolglik word dus voorgestel
dat die "P. coccoides" stamme putatief heringedeel word as Luteococcus japonicus
subspecies casei en Luteococcus japonicus subspecies tyrophilus en dat die genus
beskrywing van Luteococcus hersien moet word om die fenetiese en molekulêre
beskrywing van hierdie stamme in te sluit.
uo