M.S.--University of Oklahoma,2014Includes bibliographical references (leaves 45-50).Previously, metagenomic studies of Zodletone Spring (south-western Oklahoma) revealed uncultured and poorly characterized bacterial phyla. Among them, a bacterial phylum "Candidate Division TM6" is present. It is widespread in a variety of natural environments and may contribute to geochemical cycling processes. We used a single-cell approach for targeting and separation of cells belonging to candidate division TM6. This approach includes design of specific primers, fluorescence in situ
hybridization and fluorescence activated cell sorting, to obtain bacteria within this novel phylum. Extracted DNA from sorted cells was amplified using multiple displacement amplification and phylogenic analysis of sorted cells demonstrated the presence of candidate division TM6 DNA, based on 16SrRNA analysis. Amplified DNA was used for whole-genome sequencing on a MiSeq Illumina platform and generated 250 bp
paired end reads. Genome de nova assembly was done using SPAdes 2.5.0 and Ray 2.2.0 algorithms, with subsequent phylogenetic binning of scaffolds into the TM6 assembly of 64,034 bp. Assembled genes were annotated on the RAST server and manually with the NCBI database. There were nine proteins encoded by assembled
genes: ATP-dependent DNA helicase PcrA (EC 3.6.4.12), DNA polymerase III subunit gamma/tau (EC 2.7.7.7), transcriptional regulator OmpR, histidine kinase (EC 2.7.13.3), lysophospholipase (EC 3.1.1.5), cyanophycin synthase (EC 6.3.2.29), acetyltransferase 3 (EC 2.3.-.-), allophanate hydrolase (EC 3.5.1.54), RND multidrug efflux transporter and beta-lactamase (EC 3.5.2.6). All the proteins clustered distinctly from those in other different phyla. Given these results, we suggest that CD TM6 is able to use nitrogen, stored in a form of a cyanophycin polymer, by utilizing it in urea cycle
