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

Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizu, Soraku, Kyoto 619-0292, Japan¹

Author for correspondence: Hideaki Yukawa. Tel: +81 774 75 2308. Fax: +81 774 75 2321. e-mail: yukawa{at}rite.or.jp

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 -type CAs. The upstream region of acaP was fused to the lacZ gene and ß-galactosidase activity was measured under different growth conditions. Acetazolamide inhibited purified CA with an IC₅₀ in the range of 10^-8 M, and in the culture media concentrations as low as 30 µM 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. CO₂ 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 CO₂ and that there is no expression under aerobic conditions.

Keywords: carbonic anhydrase, inorganic carbon uptake, Rhodopseudomonas palustris, periplasmic enzyme

Abbreviations: AZ, acetazolamide; CA, carbonic anhydrase; C_i, inorganic carbon; PNSB, purple non-sulfur bacterium; RubisCo, ribulose-bisphosphate carboxylase-oxygenase

^a Present address: Biological Research Center, Hungarian Academy of Sciences, DNA-Chip Laboratory, Szeged, PO Box 521, H-6701, Hungary.

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