Date of Award
Doctor of Philosophy
Alison Buchan, Robert Hettich, Chris Schadt, John Biggerstaff
Microbial communities associated with the human oral cavity are complex, and many oral microbes have yet to be cultured. These uncultured community members are of interest ecologically and phylogenetically, and a number of uncultured species have been positively correlated with oral diseases such as periodontitis. Thus, an approach was adapted to selectively separate single cells from mixed populations of oral bacteria and obtain genomic information for uncultured community members. A combination of fluorescent labeling, cell sorting with flow cytometry and multiple displacement amplification was used to obtain sufficient genomic material for whole-genome pyrosequencing.
The first targets were from uncultured oral lineages within Deltaproteobacteria groups Desulfobulbus and Desulfovibrio, and cells were selectively isolated with group-specific, fluorescent in situ hybridization probes. Deltaproteobacteria were targeted to better understand their increased abundance in periodontitis patients. The resulting oral Deltaproteobacteria genomes encoded several unique proteins that appear to function in survival in a host environment, including proteins similar to virulence factors of known human pathogens. Thus, it is likely that the oral Deltaproteobacteria are not just opportunistic community members of periodontitis sites but also have the potential to play a role in disease etiology.
A second target was a low abundance community member belonging to the class Anaerolineae, within Chloroflexi subphylum I. This diverse group of organisms has only a few cultured isolates, and limited genomic information is available, making it difficult to discern their ecological significance. Genomic data suggest that oral Anaerolineae are anaerobic heterotrophs with an abundance of carbohydrate transport and metabolism genes. The presence of a unique phosphotransferase system and other genes necessary for N-acetylglucosamine metabolism suggests this organism grows by scavenging material from surrounding lysed bacterial cells.
These single-cell genomic studies have provided insight into the ecological niches of two uncultured groups within the oral environment, revealed differences between environmental and host-associated organisms within this groups and provided possible gene targets for future studies. Ultimately, methods employed in these studies could be readily applied to gain information on other uncultured microbes from any environment.
Campbell, Alisha Gail, "Characterization of uncultured, human oral microbiota using a targeted, single-cell genomics approach. " PhD diss., University of Tennessee, 2013.