An investigation of carbon and nitrogen metabolism through a genomic analysis of the genus Nitrobacter

The chemolithoautotrophic nitrite oxidizing bacteria (NOB) participate in the biogeochemical cycling of nitrogen by catalyzing and conserving energy from the oxidation of nitrite (NO2-) to nitrate (NO3-) via a nitrite oxidoreductase (NXR). The main objective of this work was to comparatively annotate and analyze the genome sequences of Nitrobacter winogradskyi NB255 and Nitrobacter hamburgensis X14 and use this information to extend our understanding of nitrogen and carbon metabolism in NOB. Through the analysis of the N. winogradskyi genome, genes encoding pathways for known modes of lithotrophic and heterotrophic growth were identified, including multiple enzymes involved in anapleurotic reactions centered on C2 to C4 metabolism. N. winogradskyi lacked genes encoding a complete glycolysis pathway and for the active transport of sugars. The N. hamburgensis genome harbored many genes not found in N. winogradskyi, including a complete glycolysis pathway, unique electron transport components, and putative pathways for the catabolism of aromatic, organic and one-carbon compounds. FAD-dependent oxidases were identified in the genome of N. hamburgensis which suggested that lactate could be metabolized, providing reductant and carbon to the cell. Indeed, D-lactate enhanced the growth rate and yield of N. hamburgensis in the presence of NO2- and served as a sole energy and carbon source in the absence of NO2-. Although lactate consumption occurred constitutively in lithoautotrophically grown cells, evidence was obtained for physiological adaptation to lactate. D-lactate grown cells consumed and assimilated lactate at a faster rate than NO2- grown cells, and D-lactate-dependent O2 uptake was significantly greater in cells grown heterotrophically or mixotrophically compared to cells grown lithoautotrophically. However, D-lactate could not substitute for CO 2 as the sole carbon source (lithoheterotrophy) during growth in the presence of NO2-. Through a comparative analysis of the Nitrobacter 'core' genome, many genes involved in NO2- metabolism were identified, including a dissimilatory nitrite reductase (NirK). The putative nirK in N. winogradskyi was maximally transcribed under low oxygen in the presence of NO2- and transcription was not detected under anaerobic conditions. Although production of NO under aerobic conditions was not detected, NO was consumed in a cyanide-sensitive process and reversibly inhibited NO2--dependent O2 uptake.