Physiological and transcriptomic response to methyl-coenzyme M reductase limitation inMethanosarcina acetivorans

Abstract

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis, the microbial metabolism responsible for nearly all biological methane emissions to the atmosphere. Decades of biochemical and structural studies have generated detailed insights into MCR functionin vitro, yet very little is known about the interplay between MCR and methanogen physiology. For instance, while it is routinely stated that MCR catalyzes the rate-limiting step of methanogenesis, this statement has not been categorically tested. Here, to gain a more direct understanding of MCR’s control on the growth ofMethanosarcina acetivorans,we generate a strain with an induciblemcroperon on the chromosome, allowing for careful control of MCR expression. We show that MCR is not growth rate limiting in substrate-replete batch cultures. However, through careful titration of MCR expression, growth-limiting state(s) can be obtained. Transcriptomic analysis ofM. acetivoransexperiencing MCR-limitation reveals a global response with hundreds of differentially expressed genes across diverse functional categories. Notably, MCR limitation leads to a strong induction of methylsulfide methyltransferases, likely due to insufficient recycling of metabolic intermediates. In addition, themcroperon does not seem to be transcriptionally regulated, i.e., it is constitutively expressed, suggesting that the overabundance of MCR might be beneficial when cells experience nutrient limitation or stressful conditions. Altogether, we show that there is wide range of cellular MCR concentrations that can sustain optimal growth, suggesting that other factors like anabolic reactions might be rate-limiting for methanogenic growth.ImportanceMethane is a potent greenhouse gas that has contributed toca.25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. In methanogens, methyl-coenzyme M reductase (MCR) catalyzes methane formation. Even though MCR comprisesca.10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. Here, we show thatMethanosarcina acetivoransgrown under standard laboratory conditions produces more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher throughput manner than isolation and biochemical characterization of pure protein.