|dc.description.abstract||Bacteria are one of the three taxonomic domains and play a major role in the biological processes on Earth, yet their taxonomy and constituent species are poorly understood. OP10 is a candidate division within the domain Bacteria that up until recently has no cultivated representative. Until the recent isolation of the cultivated OP10 strains T49 and P488 from the Taupo Volcanic Zone in New Zealand (Stott et al. 2008), little was known about this putative phylum despite the fact that its presence was detected in various environmental surveys.
This study aims to (i) characterise the physiology of one of the first isolated OP10 species, strain T49; and (ii) investigate and expand upon known OP10 diversity by using targeted oligonucleotide primers.
Optimal growth temperature and pH of T49 was determined by cultivation in liquid medium. T49 substrate utilisation was investigated by culturing T49 with various substrates, with a focus on its sugar utilisation profiles. T49 was also characterised for its cell morphology through transmission and scanning electron microscopy as well as phase contrast microscopy. The total fatty acid profile of T49 was characterised through gas chromatography and mass spectroscopy.
OP10 diversity was investigated at several geothermal sites using OP10-specific primers designed in the course of this study. The primers target conserved regions of the 16S rRNA gene sequence specific to clades within OP10. The primers were designed in silico by obtaining publically available OP10 16S rRNA gene sequences (through the Greengenes and SILVA databases) as references and dividing the candidate division into distinct clades through phylogenetic analysis with ARB. Primers were selected for specificity and coverage of the reference sequences within each of the clades. The performance of the primers were investigated by applying the primers against environmental DNA samples where OP10 sequences were previously identified, as well as various negative controls. The results were validated by sequencing PCR products generated to identify positive OP10 hits and false positive non-OP10 hits.
This study found that T49 had a rod-shape morphology with the dimensions of 0.5-0.7 m by 2.5-3.0 m, more than half (67.4%) of the total fatty acids of the bacteria consisted of stearic acid (18:0 ) and palmitic acid (16:0), and GC content of 54.6%. Furthermore, T49 was found to have a growth temperature range of less than 50 degrees C to 73 degrees C and optimum at 68 degrees C, growth pH range of 4.9 to 5.8 and optimum at pH 5.3. Substrate utilisation experiments showed that strain T49 grew optimally using mono- and di- saccharides such as arabinose, mannose, ribose, galactose, and maltose, as well as amorphous polysaccharides including starch, glycogen, and dextrin.
The OP10-specific primers successfully identified the presence of OP10 in environmental samples from Waikite, Waipahihi, and Mount Ngauruhoe , confirming previous unpublished 16S rRNA gene sequence surveys. Furthermore, the OP10-specific primers also identified previously undetected genetic diversity in Tikitere and Mount Ngauruhoe, demonstrating the advantage of the primers over traditional universal primers with their high specificity for OP10 DNA sequences which enabled more sensitive surveys of OP10 in the environment.
The characterisation of the first OP10 species and the development of OP10-specific primers enable further investigation into this major taxonomic group within the Bacteria domain. T49 and its related strains may play important role within their environments, which can now be investigated based on these known physiologies. The effectiveness of the OP10-specific primers experimented may be use to detect presence of OP10 species in the environment at a higher sensitivity and selectiveness than traditional primers. The results from this research have expanded our knowledge on this previously unknown phylum. By increasing our knowledge of the OP10 candidate division, we also increase our understanding of the global bacterial diversity and this may help bring about insights into biology and global climate processes as well generating practical solutions in these fields.
Many of these initial observations of T49 merit further investigation. These include: The production of pigments and lipids, two distinct cytosolic structures, and interesting growth behaviours such as quorum sensing and biofilm formation. OP10-specific primers developed in this study can be improved upon through further validations with environmental DNA representing clades that were not tested during this study due to unavailability. The improved primers can be developed as a rapid diagnostic tool to detect OP10 in the environment for isolation efforts. The primers may also act as fluorescent probes to identify OP10 in microbial consortia such as biofilms while preserving the structure of the microenvironments.||en_NZ