Open AccessRegulation of expression and biochemical characterization of a β‐class carbonic anhydrase from the plant growth‐promoting rhizobacterium, Azospirillum brasilense Sp7
Author(s) -
Kaur Simarjot,
Mishra Mukti Nath,
Tripathi Anil K.
Publication year - 2009
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2009.01736.x
Subject(s) - azospirillum brasilense , homotetramer , carbonic anhydrase , biology , biochemistry , escherichia coli , enzyme , microbiology and biotechnology , lac operon , bacteria , gene , microbial inoculant , genetics , protein subunit
Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO 2 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% CO 2 enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO 2 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 CO 2 from the ambient air and the requirement of CO 2 hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.