Regulation of expression and biochemical characterization of a β-class carbonic anhydrase from the plant growth-promoting rhizobacterium,Azospirillum brasilense Sp7

Abstract

Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO2 to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to β-class carbonic anhydrases (CAs). Biochemical characteristics of the β-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower β-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO2 enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO2 levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, β-CA in A. brasilense seems to be required for scavenging CO2 from the ambient air and the requirement of CO2 hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.