Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath

The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO(2) was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO(2) assimilation during cultivation with both CH(4) and CO(2) as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO(2)-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. (13)CO(2) tracer analysis showed that CH(4) and CO(2) enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO(2) assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate (13)C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO(2) play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO(2) via RubisCO, which may play a more pivotal role in the Earth’s biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO(2)-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH(4) and CO(2). IMPORTANCE The importance of RubisCO and CO(2) in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO(2) and RubisCO are essential for M. capsulatus Bath growth. (13)CO(2) tracing experiments supported that RubisCO mediates CO(2) fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH(4)/CO(2)-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO(2) assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH(4) and CO(2).