Genetics and physiology of ammonia catabolism by the methane-oxidizing bacterium, Methylococcus capsulatus Bath

Methanotrophic bacteria and ammonia-oxidizing bacteria (AOB) share phylogenetic history, physiological capabilities, and perform ecologically analogous roles in the global carbon and nitrogen cycles. Many of the characteristics shared by methanotrophs and AOB arise from their possession of the homologous enzymes, particulate methane monooxygenase and ammonia monooxygenase, respectively, which they use to oxidize both methane (CH4) and ammonia (NH 3). Studies described here address several questions related to NH 3 oxidation by methanotrophs and CH4 metabolism by AOB. These include analyses of the transcription of genes encoding a putative hydroxylamine oxidoreductase (HAO) and other proteins with putative functions in NH 3 catabolism by the methanotrophic bacterium, Methylococcus capsulatus Bath. AOB use HAO to oxidize hydroxylamine to nitrite and transcribe hao in response to NH3. M. capsulatus Bath significantly increased steady state mRNA levels of hao and cytS (encodes cytochrome c'-beta) in the presence of NH3, while transcription of genes for cytochrome P460 (cytL; previously identified hydroxylamine-oxidizing enzyme) and a cytochrome c nitric oxide reductase (cNOR; norB) remained unchanged. As hao transcription increased in response to NH3, the physiology of M. capsulatus Bath cultures pre-exposed to CH4-only and pre-exposed to CH4 and NH3 was compared in Chapter III. Nitrite production from 5 mM (NH4)2SO4 by M. capsulatus Bath cultures pre-exposed to CH4-only was consistently greater than nitrification by cultures pre-exposed to CH 4 and NH3. This difference in nitrification ability was likely due to decreased NH3 oxidation by M. capsulatus Bath cultures pre-exposed to NH3, as these cultures were less sensitive to NH3-mediated inhibition of CH4 oxidation. The results presented in Chapters II and III suggest that methanotrophs exhibit a complex physiological response to NH3. In Chapter IV, the expression of genes with potential functions in nitric oxide metabolism by M. capsulatus Bath was assessed. M. capsulatus Bath increased transcription of norB by 3.7- to 5.5-fold in response to sodium nitroprusside (SNP; releases nitric oxide) and 22-fold in response to NaNO2, which when combined with previous physiological studies suggests that M. capsulatus Bath has a nitric oxide-responsive functional cNOR. Finally, Chapter V describes possible CH4 metabolism pathways in an NH3-oxidizing bacterium, Nitrosococcus oceani, identified through comparison of its genome with that of M. capsulatus Bath, and proposes that ammonia monooxygenase, glutathione-dependent formaldehyde dehydrogenase, and tetrahydrofolate have roles in CH4 oxidation and CH4-carbon assimilation. Studies presented in this dissertation contribute to understanding the genetic basis for non-canonical roles of microorganisms in biogeochemical cycles, particularly the role of methanotrophs in nitrogen cycle transformations.