A periplasmic, alpha-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

Intact cells of the purple non-sulfur bacterium Rhodopseudomonas palustris growing anaerobically, but not aerobically, contain carbonic anhydrase (CA) activity. The native enzyme was purified >2000-fold to apparent homogeneity and found to be a dimer with an estimated molecular mass of 54 kDa and a subunit molecular mass of 27 kDa. The CA gene (acaP) was cloned and its sequence revealed that it was homologous to alpha-type CAs. The upstream region of acaP was fused to the lacZ gene and beta-galactosidase activity was measured under different growth conditions. Acetazolamide inhibited purified CA with an IC(50) in the range of 10(-8) M, and in the culture media concentrations as low as 30 microM inhibited phototrophic growth under anaerobic, light conditions when bicarbonate was used. An acaP::KAN:(r) mutant strain was constructed by insertion of a kanamycin-resistance cassette and showed a growth pattern similar to wild-type cells grown in the presence of CA inhibitor. CO(2) gas supplied as an inorganic carbon source reversed the effect of mutation or acetazolamide. CA activity measurements, fusion and Western blot experiments confirmed that CA is expressed under different anaerobic conditions independently of bicarbonate or CO(2) and that there is no expression under aerobic conditions.