An investigation into the bacterial phylum, candidate division TM7

• Main Author: Winsley, Tristrom
• Format: Doctoral or Postdoctoral Thesis
• Language: unknown
• Published: UNSW Sydney 2014
• Subjects: Uncultured bacteria; Candidate division TM7; Bacterial cultivation; Bacterial phylogeny; Soil bacteria; Antarctic; The Antarctic; Antarc*
• Online Access: https://dx.doi.org/10.26190/unsworks/16668; http://hdl.handle.net/1959.4/53315

Candidate division TM7 is a highly ubiquitous phylum and 16S rRNA gene sequences have been detected in soils, sediments, seawater, wastewater treatment plants, activated sludges, as well as a host of clinical environments. Several studies have investigated the TM7 phylum resulting in insight into the members of this enigmatic division, yet they lack a pure culture representative. In this thesis both culture-independent and culture-dependent approaches were used to investigate the TM7 phylum. Reported is the design and validation of a ‘universal’ bacterial PCR primer set, specifically designed to capture greater diversity from the candidate phyla. Two primers, 356F and 1064R were designed and in silico performance revealed an extrapolated coverage of 98 % and 95 % of the Bacteria. The primers were then applied in a pyrosequencing assay to survey soils from the Antarctic, sub-Antarctic and Australian Desert and a direct comparison to the commonly used ‘universal’ PCR primers 27F and 519R was made. Assessment of the diversity captured revealed that 356F/1064R recovered 3-fold more candidate phyla and 2-3 times the abundance of TM7 compared to 27F/519R. Additionally, the evenness of species recovered with 356F/1064R was greater than that of 27F/519R, reducing the skewness of the data towards so-called ‘dominant’ taxa. The second chapter describes the design of PCR primers to target the TM7 phylum only. Two primers, TM7-590F and TM7-965R were designed, which when analysed in silico showed they exhibited 95 % and 93 % coverage of the TM7 phylum and combined resulted in 100 % specificity, with no homology to any other phyla observed. These primers were then used to recover 2805 TM7 OTUs from 6 diverse environments via barcode error tag pyrosequencing, double that which was currently available in databases. These and existing TM7 sequences were used to construct a phylogeny for the TM7 candidate division, resulting in the identification of 4 major paraphyletic subdivision groups and confirmation that clinically sourced TM7 OTUs are all only affiliated with subdivision 3. Finally, a single-cell based cultivation approach combined with the soil substrate membrane system (SSMS) was used to target presumptive filamentous bacteria from the candidate division TM7. Soils and SSMS enrichments were screened using the newly validated TM7-specific primers to identify those highly abundant with TM7 bacteria. Subsequently, target soils and growth membranes were then used for pure cultivation attempts using two strategies, cell sorting or micromanipulation. Micromanipulation of presumptive TM7 microcolonies resulted in the recovery of 9 isolates of TM7 bacteria spanning both subdivisions 1 and 3. Whilst co-cultures on 0.1 X R2A media were obtained, further work is required to isolate these into pure culture to characterise the phylum in more depth.