Charactt[sic]erization of a plasmid conferring NAD independence in Haemophilus paragallinarum - Van Zyl, Anna Elizabeth (University of the Free State, 2003)
Members of the family Pasteurellaceae are classified in part by whether or not they require NAD+ supplement for growth on laboratory media. It is known that this phenotype is determined by a plasmid whose presence allows NAD+-independent growth of Haemophilus paragallinarum. In this study, this 6-kb plasmid, which was previously shown to be responsible for NAD+ independent growth of H. paragallinarum on defined media, was isolated. Isolated plasmid DNA was shredded by sonification and subcloned into vector PGEM-T easy. The recombinant plasmid was transformed into E.coli the transformants isolated were sequenced. Sequence analysis revealed one open reading frame of 1119bp that is predicted to encode a protein with a molecular mass of 43kD. Compared with the sequence databases, this protein was found to have significant sequence homology to Quinolinate phosphoribosyltransferase of Bacillus anthracis this enzyme is responsible for the production of nicotinic acid mononucleotide (NAMN) from nicotinate and quinolinate. A 3284bp nucleotide fragment of the plasmid revealed four additional open reading frames. Proteins encoded on this fragment of the plasmid all have significant homology to proteins from H. influenzae of which all have functions related to production and immunity of the bacteriocin haemocim. This bacteriocin produced by most type b-encapsulated strains of H. influenzae, is toxic to virtually all non-type b strains of H. influenzae idependent of encapsulation. This bacteriocin is thought to inhibit DNA synthesis, of susceptible strains. Purification of this bacteriocin and testing its toxicity to other pathogens as a possible antimicrobial drug might form the bases of a future study. Previous work has indicated that plasmid bearing strains of H. paragallinarum are less virulent, thus creating the possibility that more virulent wild type strains can be transformed and used as live vaccines. The influence of transformation with this plasmid on other members of the family Pasteurellaceae and the possibility of creating live vaccines should be further investigated Since species of the genus Haemophilus cannot easily be transformed with plasmid, this naturally occurring plasmid could be modified to create a vector, which has specific application in the transformation of Haemophilus species.

Die klassifikasie van bakteriese spesies van die familie Pasteurallaceae word gedeeltelik gebaseer op die afhanklikheid van NAD+ vir groei op laboratorium media. Dit is bekend dat die NAD+ onafhanklike fenotipe van Haemophilus paragallinarum onderskei kan word deur die teenwoordigheid van ’n plasmied. In hierdie studie is die plasmied van ongeveer 6kbp wat NAD+ onafhanklike groei op bepaalde media bevorder, geïsoleer. Plasmied DNA is deur middel van sonifikasie gefragmenteer en in vektor pGEM-T Easy gekloneer. Rekombinante plasmiede in E. coli getransformeer, geïsoleer en aan basisbaarvolgordebepaling onderwerp. Ontleding van die basispaarvolgorde toon dat die plasmied ‘n oopleesraam van 1119bp bevat, wat kodeer vir ’n proteïen met ‘n geraamde molekulêre massa van 43kD. Na vergelyking met basispaarvolgorde databasisse is gevind dat hierdie proteïen betekenisvolle homologie met quinoliensuur fosforibosieltransferase, geïsoleer uit Bacillus anthracis, toon. kwinolienaat fosforibosieltransferase is verantwoordelik vir die katalitiese produksie van nikotiensuur mononukleotied vanaf nikotienaat en kwinolienaat. Daar is verder bevind dat die plasmied ’n 3284bp fragment bevat waarop vier oopleesrame aangetref word. Die proteïne waarvoor die leesrame op hierdie fragment kodeer toon betekenisvolle homologie met proteïne geïsoleer vanuit Haemophilus influenzae, wat betrokke is by die produksie van en immuniteit teen die bakteriosien haemosien. Die meerderheid tipe b-gekapsuleerde stamme van H. influenzae produseer haemosien, terwyl die bakteriosien die groei van bykans alle nie-tipe b, beide gekapsuleerde en nie-gekapsuleerde, stamme belemmer. Daar word vermoed dat die inhibisie effek deur haemosien bewerkstellig word deurdat die bakteriosien die DNS sintese van vatbare stamme onderdruk. In ‘n verdere studie kan gefokus word op die isolasie en suiwering van haemosien en die bepaling van die mate waarin die bakteriosien die groei van ander patogeniese bakterieë belemmer. Verdere ondersoek kan ook ingestel word na die moontlike aanwending van haemosien as ‘n anti-mikrobiese middel. Daar is vantevore bevind dat stamme van H. paragallinarum waarin plasmiede voorkom minder virulent is na aanleiding van die waarneming dat hoenders wat met hierdie stamme besmet is, minder intense simptome vertoon. Hierdie waarneming kan moontlik verklaar word in terme van die onderdrukking van sekondêre invektiewe mikro-organismes deur haemosien. Verdere ondersoeke kan dus gefokus word op die relatiewe dominansie van plasmied bevattende stamme van H. paragallinarum, om bogenoemde hipotese te toets. Dit is ook moontlik dat plasmied bevattende stamme inherent minder virulent is, wat dit moontlik maak om meer virulente wilde stamme te transformeer en sodoende lewende entstowwe te skep. Die uitwerking van transformasie met die geïsoleerde plasmied op ander spesies in die familie Pasteurellaceae en die moontlike produksie van lewende entstowwe behoort verder ondersoek te word. Spesies in die genus Haemophilus kan slegs met moeite met plasmiede getransformeer word. Die plasmied geïsoleer vanuit H. paragallinarum kan gemodifiseer word en sodoende dien as ‘n vektor vir die transformasie van Haemophilus spesies.
Dissertation (M.Sc.Agric. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2003
Keywords: Haemophilus infections; Veterinary microbiology