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ABSTRACT Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis ( i Nmo686) and used flux balance analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and [ 13 C]bicarbonate and [ 13 C]formate tracer experiments coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings corroborate that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO 2 fixation, and we also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO 2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospira ’s in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes. IMPORTANCE Nitrospira spp. are globally abundant nitrifying bacteria in soil and aquatic ecosystems and in wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13 C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite-oxidizing bacteria. 

Abstract The symbiont “Candidatus Aquarickettsia rohweri” infects a diversity of aquatic hosts. In the threatened Caribbean coral, Acropora cervicornis, Aquarickettsia proliferates in response to increased nutrient exposure, resulting in suppressed growth and increased disease susceptibility and mortality of coral. This study evaluated the extent, as well as the ecology and evolution of Aquarickettsia infecting threatened corals, Ac. cervicornis, and Ac. palmata and their hybrid (“Ac. prolifera”). Aquarickettsia was found in all acroporids, with coral host and geographic location impacting the infection magnitude. Phylogenomic and genome-wide single-nucleotide variant analysis of Aquarickettsia found phylogenetic clustering by geographic region, not by coral taxon. Analysis of Aquarickettsia fixation indices suggests multiple sequential infections of the same coral colony are unlikely. Furthermore, relative to other Rickettsiales species, Aquarickettsia is undergoing positive selection, with Florida populations experiencing greater positive selection relative to other Caribbean locations. This may be due in part to Aquarickettsia proliferating in response to greater nutrient stress in Florida, as indicated by greater in situ replication rates in these corals. Aquarickettsia was not found to significantly codiversify with either the coral animal or the coral’s algal symbiont (Symbiodinium “fitti”). Quantitative PCR analysis showed that gametes, larvae, recruits, and juveniles from susceptible, captive-reared coral genets were not infected with Aquarickettsia. Thus, horizontal transmission of Aquarickettsia via coral mucocytes or an unidentified host is more likely. The prevalence of Aquarickettsia in Ac. cervicornis and its high abundance in the Florida coral population suggests that coral disease mitigation efforts focus on preventing early infection via horizontal transmission.