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

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 CO₂ 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 CO₂ assimilation during cultivation with both CH₄ and CO₂ as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO₂-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. ¹³CO₂ tracer analysis showed that CH₄ and CO₂ enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO₂ assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate ¹³C 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 CO₂ play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO₂ 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 CO₂-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH₄ and CO₂. IMPORTANCE The importance of RubisCO and CO₂ in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO₂ and RubisCO are essential for M. capsulatus Bath growth. ¹³CO₂ tracing experiments supported that RubisCO mediates CO₂ 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 CH₄/CO₂-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO₂ assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH₄ and CO₂.