Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.
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 beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-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 beta-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, beta-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.
Kaur
Simarjot
S
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India.
Mishra
Mukti Nath
MN
Tripathi
Anil K
AK
D000332:Aerobiosis; D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D015806:Azospirillum brasilense / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D002245:Carbon Dioxide / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D001616:beta-Galactosidase; D002256:Carbonic Anhydrases