Publication Date

Fall 2010

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Advisor

Cleber Ouverney

Keywords

16S, phylogenetics, qPCR, TM7, uncultivated

Subject Areas

Molecular Biology; Microbiology

Abstract

The TM7 bacterial phylum has no cultivated species and includes members that span a broad range of environmental and human habitats, some of which are associated with human periodontitis. In this project, activated wastewater TM7 bacteria were analyzed and their relatedness compared to human-associated TM7 bacteria for the long-term goal of using an environmental TM7 to better understand TM7 pathogenesis in humans. DNA was extracted from activated wastewater and PCR amplified using TM7 16S rRNA gene- specific primers. The ~1,170 base pair PCR products were then cloned and sequenced. DNA sequencing and phylogenetic analysis identified environmental TM7 clones with high 16S rRNA gene similarity (99%) and mask coverage (98%) with the human oral TM7, sub-gingival clone 3 (SBG3). Total bacterial, total TM7, and environmental SBG3 populations were quantified over a two-year period using real-time quantitative PCR. Results indicated TM7 bacteria were present year-round in the six sample sites studied, while environmental SBG3 levels varied between sample sites and sample dates. The relative abundance of TM7 ranged from 2.72% to 3.75% of the total sludge prokaryotic community. Based on qPCR, environmental SBG3 ranged from 0.001% to 0.100% of the total sludge prokaryotic community. TM7 cell morphology determined via fluorescent in situ hybridization was consistent with previously described morphologies. Given these results, this wastewater can serve as a reservoir to study TM7 and to better understand TM7 pathogenesis in human diseases, including periodontitis.

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