A metagenomic investigation of phage communities from South African deep mines

[摘要] English: Bacteriophages are viruses that infect both bacteria and archaea, and they are the mostabundant microbial communities in the ecosystem. Phages have unique applications as theyhave the ability to control the mortality rate of the hosts, and they are also useful in molecularbiology techniques as a many enzymes that are utilized in this field have a phage origin. Thoughphages 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 usingthe standard techniques and phages require these hosts for propagation and replication.Development of culture-independent techniques has managed to circumvent problemsassociated with prokaryotic diversity studies by using the 16S rDNA sequencing. Viruseshowever 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 orclades of unculturable phages. In this study tailed phages (T4-like phages and T7-likepodoviruses) were targeted because of their abundance in the environment. The major capsidprotein (g23) and DNA polymerase fragment were used to detect T4-like phages and T7-likepodoviruses, respectively. Transmission electron microscopy was also used to study anddetermine morphology of phages, though the technique does not give a true reflection of thenumber of viral like particles from the environment. The methods were first optimized with waterand sediments from Loch Logan pond where phage counts were expected to be high. Thesemethods 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 andTau Tona (level 100 and level 118) were also subjected to these two techniques. Phageparticles 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 ofphages from the mines, and the T4-like phages only in Beatrix and Tau Tona. Phylogeneticanalysis revealed that the DNA polymerase fragment from the T7-like phages is highlyconserved. The sequences were similar to the clones obtained with marine, water and soilsamples from different locations. In contrast to the T7 phages the T4-like phages were highlydiverse 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 deepmines and also to identify novel phage proteins that might have biotechnological applications.Hence shotgun libraries were also constructed to get genomic information of the viralassemblages from the mines. Sequencing revealed untapped viral communities with themajority of the clones showing no similarity to the known proteins. Very few phage proteins wereobtained and the data was not enough to identify many the novel genes from the phagecommunities. 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 largeamount of sequencing data. Biofilm from Beatrix mine was selected for pyrosequencing and 2Xquarter runs of the GS FLX were done using isolated viral DNA. The viral communities fromBeatrix mine were unique with -75% of the proteins not showing similarity to any knownproteins. Microbial analysis of Beatrix mine revealed that most the diversity from this mine isclustered within the classes Beta and Gamma Proteobacteria and the phyla Firmicutes. Hencethe portion of the known phages were all three families of dsDNA phages infectingEnterobacteria phages and few of the phage proteins were from different Bacillus sp. Proteinsfrom Acanthamoeba polyphaga mimivirus were also detected from Beatrix mine. Sevenprophages 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 and22 °C. The enzyme also ligated sticky ends at temperatures as high as 70°C. Blunt endfragments were also ligated in presence of polyethylene glycol. The expressed polynucleotidekinase had the following substitutions; D351, R38D and R126E, and R176N, no activity could bedetected, possibly a result of the substitutions. The endonuclease digested lambda DNA, andearly indications are that that the endonuclease recognizes a specific sequence and does notcut randomly. The overall results shows that uncultured phage communities, including SouthAfrican phage metagenome are the largest untapped source of genomic information in thebiosphere. In addition they are the source of novel biotechnologically important proteins.

[发布机构] University of the Free State