RubisCO-encoding methanotrophs elicit a coordinated transcriptional response to carbon dioxide limitation.

The methanotroph Methylococcus capsulatus encodes the carbon dioxide (CO2)-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which, along with CO2 and methane (CH4), are required for growth of this bacterium. This unique, dual one-carbon metabolism is present in phylogenetically diverse methanotrophs and is likely widespread in nature, but the connection between organic and inorganic carbon metabolism in these methanotrophs is unclear. Here we used a systems approach to investigate the requisite role of CO2 in M. capsulatus metabolism and physiology. We show that atmospheric CO2 levels are sufficient for M. capsulatus cultivation, and that CO2 assimilation is linked to cellular redox balance. In the absence of CO2, M. capsulatus elicits a global transcriptional response that includes induction of RubisCO- and other inorganic carbon assimilation-encoding genes with a concomitant decrease in ribosome biogenesis and nitrogen assimilation genes typical of a bacterial stringent response. This CO2 starvation response balances biosynthesis with carbon availability. Genetic and biochemical evidence show that a LysR-type transcriptional regulator (CbbR) activates RubisCO and other CO2 assimilation gene transcription via direct promoter binding in response to elevated intracellular RubisCO substrate, ribulose-1,5-bisphosphate, in CO2-starved cells. These results offer insights into the essentiality of CO2 for M. capsulatus metabolism and establish a function for CbbR-family regulators in methanotrophs beyond their canonical association with CO2 fixation in autotrophic bacteria.