Co-metabolism of ethanol in Azospirillum brasilense Sp7 is mediated by fructose and glycerol and regulated negatively by an alternative sigma factor RpoH2.

Azospirillum brasilense is a plant growth promoting rhizobacterium which is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can co-metabolize ethanol in media having fructose or glycerol as carbon source, and contribute to its growth. In minimal medium containing fructose or glycerol as carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense. But, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense led to the loss of the AldA protein and ethanol utilization ability in fructose or glycerol supplemented media. Furthermore, ethanol inhibited the growth of the aldA::km mutant. The exaA::km mutant also lost the ability to utilize ethanol in fructose supplemented medium. But, in glycerol supplemented media, it utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced only by glycerol, not by fructose. Unlike exaA, expression of aldA and exaA1 were not dependent on σ54. Instead, they were negatively regulated by RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as carbon source without or with malate overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating co-metabolism of ethanol by inducing the expression of ethanol oxidizing enzymes and of RpoH2 in repressing them. IMPORTANCE This study has unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol is used as primary growth substrate. It opens the possibility of studying the regulation of expression of ethanol oxidation pathway for generating high yielding strains, which can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions.