OXYGEN-LIMITED METABOLISM IN THE METHANOTROPH METHYLOMICROBIUM BURYATENSE 5GB1C

The bacteria that grow on methane aerobically (methanotrophs) support populations of non-methanotrophs in the natural environment by excreting methane-derived carbon. One group of excreted compounds are short-chain organic acids, generated in highest abundance when cultures are grown under O2-starvation. We examined this O2-starvation condition in the methanotroph Methylomicrobium buryatense 5GB1C . Under prolonged O2-starvation in a closed vial, this methanotroph increases the amount of acetate excreted about 10-fold, but the formate, lactate, and succinate excreted do not respond to this culture condition. In bioreactor cultures, the amount of each excreted product is similar across a range of growth rates and limiting substrates, including O2-limitation. A set of mutants were generated in genes predicted to be involved in generating or regulating excretion of these compounds and tested for growth defects, and changes in excretion products. The phenotypes and associated metabolic flux modeling suggested that in M. buryatense 5GB1C, formate and acetate are excreted in response to redox imbalance, and the resulting metabolic state represents a combination of fermentation and respiration metabolism. Overall design: RNA-seq analysis of gene expression of M. buryatense 5GB1 grown in continuous bioreactor culture. Several conditions were used to isolate the effects of O2 limitation on 5GB1 metabolism: O2 limitation at slow growth, O2 limitation with fast growth, CH4 limitation with fast growth, and a terminal oxidase knockout mutant. Each condition had two biological replicates, and between 1 and 4 technical replicates.