Engineering Ability to Utilize Glucose and Ethanol in Azospirillum brasilense Sp7 for better Colonization of Rice Roots

Azospirillum brasilense Sp7 is a plant growth promoting, rhizobacterium known to colonize the roots of several C4 and C3 grasses and cereals including rice (1). It preferentially utilizes C4-dicarboxylates for nitrogen fixation and growth, but shows very limited ability to utilize carbohydrates and alcohols as carbon source (1). Rice root exudates are rich in glucose. Under oxygen limiting submerged conditions, rice roots accumulate ethanol (2).

When alcohol is supplemented with glycerol or fructose, A. brasilense grows well, and both alcohol and glycerol/fructose are utilized simultaneously. Cloning of glucose-phosphotransferase operon alongwith glucose-6P-dehydrogenase gene from Azospirillum lipoferum and their overexpression conferred the ability in A. brasilense Sp7 to utilize glucose as sole carbon source. Proteome of A. brasilense Sp7 grown with glycerol/fructose alongwith 1.5% ethanol showed an upregulation of NAD-dependent aldehyde dehydrogenase (AdhA), which was absent in glycerol/fructose minimal medium or in malate minimal medium supplemented with 1.5% ethanol. Inactivation of adhA in A. brasilense led to the failure to utilize alcohol, and only fructose or glycerol was utilized. A. brasilense genome encodes two PQQ-dependent alcohol dehydrogenases (ExaA and ExaA1): ExaA plays major role in ethanol catabolism when cultures are grown in fructose or glycerol minimal medium, ExaA1 facilitates ethanol utilization in cultures grown with glycerol as carbon source. Single- and double-knockouts of exaA and exaA1 failed to utilize alcohol in the medium containing alcohol and fructose/glycerol. Expression of exaA and adhA in A. brasilense enabled alcohol utilization in presence of malate suggesting that exaA and adhA are needed for alcohol utilization. Expression of both ExaA and ExaA1 in A. brasilense is negatively regulated by RpoH2 sigma factor. However, a two-component system (ExaS and ExaR) regulates exaA expression positively.

References.

1. V. S, Dubey. A. P, Gupta. A, Singh. S, Singh. B. N, and Tripathi. A. K. (2017). Regulation of glycerol-induced quinoprotein alcohol dehydrogenase by σ54 and a LuxR-type regulator in Azospirillum brasilense Sp7. J. Bacteriol. 199:e00035-17.
2. Krause A, Bischoff B, Miché L, Battistoni F and Reinhold-Hurek B. (2011). Exploring the function of alcohol dehydrogenases during the endophytic life of Azoarcus Sp. strain BH72. Mol Plant Microbe Interact. 24:1325-1332.