Abstract
Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide “A” of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an “A” residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene.
Highlights
Carbonic anhydrase (CA) (EC 4.2.1.1) catalyzes the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and plays an important role in various biochemical and physiological processes in prokaryotic and eukaryotic organisms [1, 2]
The transformants were grown in minimal medium containing 1% (w/v) glucose and total RNA was isolated from cells harvested at the mid-exponential growth phase. cDNAs for the bca-cat and the gca-cat transcriptional fusions were synthesized, tailed with poly-(A), amplified and cloned into the pJET1.2/blunt cloning vector
CO2 is in chemical equilibrium with HCO3, carbonic acid and carbonate
Summary
Carbonic anhydrase (CA) (EC 4.2.1.1) catalyzes the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and plays an important role in various biochemical and physiological processes in prokaryotic and eukaryotic organisms [1, 2]. CAs are ubiquitously found in eukarya, bacteria and archaea domains of life [2, 3, 4] and five genetically distinct CA families are known to date, namely the α-, β-, γ-, δ-, and z-CAs [5]. The β and γ are the ancient classes of CAs, predominantly found in prokaryotes and their presence in species of archaea and bacteria indicate their fundamental role in prokaryotic biology [6, 7, 8]. PLOS ONE | DOI:10.1371/journal.pone.0154382 April 27, 2016
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