A metagenomic investigation of phage communities from South African deep mines

English: Bacteriophages are viruses that infect both bacteria and archaea, and they are the most abundant microbial communities in the ecosystem. Phages have unique applications as they have the ability to control the mortality rate of the hosts, and they are also useful in molecular biology techniques as a many enzymes that are utilized in this field have a phage origin. Though phages are the most abundant they still the most unexplored, especially from the environment. This is due to the fact that approximately 99% of their bacterial hosts cannot be cultured using the standard techniques and phages require these hosts for propagation and replication. Development of culture-independent techniques has managed to circumvent problems associated with prokaryotic diversity studies by using the 16S rDNA sequencing. Viruses however do not have a common gene or sequence fragment that can be used for phylogeny. Development of the phage proteomic tree has facilitated PCR detection of different families or clades of unculturable phages. In this study tailed phages (T4-like phages and T7-like podoviruses) were targeted because of their abundance in the environment. The major capsid protein (g23) and DNA polymerase fragment were used to detect T4-like phages and T7-like podoviruses, respectively. Transmission electron microscopy was also used to study and determine morphology of phages, though the technique does not give a true reflection of the number of viral like particles from the environment. The methods were first optimized with water and sediments from Loch Logan pond where phage counts were expected to be high. These methods were used preliminarily as a way to check the presence of phages in the samples. Water samples collected from four South African mines, Masimong, Beatrix, Star Diamonds and Tau Tona (level 100 and level 118) were also subjected to these two techniques. Phage particles were only observed with Beatrix mine when using transmission electron microscopy. The samples were further characterized as the TEM requires high viral counts. Uncultured T7- like podoviruses were detected with all mine samples indicating the presence of these groups of phages from the mines, and the T4-like phages only in Beatrix and Tau Tona. Phylogenetic analysis revealed that the DNA polymerase fragment from the T7-like phages is highly conserved. The sequences were similar to the clones obtained with marine, water and soil samples from different locations. In contrast to the T7 phages the T4-like phages were highly diverse with very few clones showing similarity to the known capsid protein. The main focus of the project however was the genomic analysis of viral communities from deep mines and also to identify novel phage proteins that might have biotechnological applications. Hence shotgun libraries were also constructed to get genomic information of the viral assemblages from the mines. Sequencing revealed untapped viral communities with the majority of the clones showing no similarity to the known proteins. Very few phage proteins were obtained and the data was not enough to identify many the novel genes from the phage communities. This is due to the fact that only 20 clones from each mine were sequenced. Therefore the use of high throughput sequencing technique was necessary to obtain large amount of sequencing data. Biofilm from Beatrix mine was selected for pyrosequencing and 2X quarter runs of the GS FLX were done using isolated viral DNA. The viral communities from Beatrix mine were unique with -75% of the proteins not showing similarity to any known proteins. Microbial analysis of Beatrix mine revealed that most the diversity from this mine is clustered within the classes Beta and Gamma Proteobacteria and the phyla Firmicutes. Hence the portion of the known phages were all three families of dsDNA phages infecting Enterobacteria phages and few of the phage proteins were from different Bacillus sp. Proteins from Acanthamoeba polyphaga mimivirus were also detected from Beatrix mine. Seven prophages were detected with possible genome sizes ranging from 5 kb to more than 12 kb. These prophages contained high number of hypothetical proteins. Novel proteins were identified from the Beatrix mine sequencing and three proteins; DNA ligase, SegB homing endonuclease and polynucleotide kinase were selected for expression studies. The ATP-dependent DNA ligase was able to ligate sticky ends at temperatures 4°C, 16°C and 22 °C. The enzyme also ligated sticky ends at temperatures as high as 70°C. Blunt end fragments were also ligated in presence of polyethylene glycol. The expressed polynucleotide kinase had the following substitutions; D351, R38D and R126E, and R176N, no activity could be detected, possibly a result of the substitutions. The endonuclease digested lambda DNA, and early indications are that that the endonuclease recognizes a specific sequence and does not cut randomly. The overall results shows that uncultured phage communities, including South African phage metagenome are the largest untapped source of genomic information in the biosphere. In addition they are the source of novel biotechnologically important proteins.