Cometabolism of Ethanol in Azospirillum brasilense Sp7 Is Mediated by Fructose and Glycerol and Regulated Negatively by an Alternative Sigma Factor RpoH2

Abstract

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 σ 54 . 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.

Aldehyde Dehydrogenase Diversity in Azospirillum Genomes

“…Ethanol degradation is mediated by pyruvate decarboxylases and ALDH2. These findings provide valuable insights into the metabolic capabilities of Azospirillum, and may have implications for future research in this area [22].…”

“…Given the promiscuity observed among phylogenetically related ALDH families, it is plausible that the COG1012 and MSR-1-like families may be able to metabolize substrates to those metabolized by the DhaS and ALDH2 families, although their efficiency may vary, as is the case with AldA, AldB, and AldC of P. syringae DC3000 [58,86]. It is worth noting that aldA from A. brasilense Sp7 belongs to the ALDH2 family and has been shown to convert aldehyde into acetate [22]. It is known that some ALDH families are promiscuous in their substrate specificity [94], which suggests that the ALDH2, COG1012, DhaS, and MSR1-like families could be involved in the metabolism of aldehyde, acetaldehyde, and indole acetaldehyde, with different yields for each substrate type.…”

“…To identify proteins containing the ALDH domain, we performed local alignments against the protein collection on the BLAST platform [21], using the aldA gene from Pseudomonas syringae DC3000 as the query sequence [22]. We set an E-value threshold of 0.05 and stored all hits in FASTA files for classification, alignment, and phylogenetic analysis [23].…”

“…Additionally, our analyses revealed a high identity (90.9% ± 6.2%) among the members of this family. In the bacterium A. brasilense Sp7, experimental evidence has shown that the protein aldehyde dehydrogenase WP_035679551.1 participates in the conversion of acetaldehyde into acetic acid [22]. Notably, this protein has 97% aldehyde dehydrogenase from the bacterium A. brasilense Yu62 [60], which participates in the conversion of indole acetaldehyde into indole acetic acid.…”

Shelf Life of Biofilm Producing PGPR Liquid Inoculants in Different Enriched Media Composition

“…In a minimal medium containing fructose or glycerol as a carbon source, ethanol supplementation increases the production of dehydrogenated alcohols and aldehyde dehydrogenases. Ethanol is used as a secondary carbon source when fructose and glycerol are the primary growth substrates (Singh et al, 2021). Evaluation of pH in liquid-based biofertilizers showed variations among different carrier materials.…”