Carbonic Anhydrase Is Essential for Growth of Ralstonia eutropha at Ambient CO 2 Concentrations

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Abstract
Mutant strain 25-1 of the facultative chemoautotroph Ralstonia eutropha H16 had previously been shown to exhibit an obligately high-CO2-requiring (HCR) phenotype. Although the requirement varied with the carbon and energy sources utilized, none of these conditions allowed growth at the air concentration of CO2. In the present study, a gene designated can and encoding a β-carbonic anhydrase (CA) was identified as the site altered in strain 25-1. The mutation caused a replacement of the highly conserved glycine residue 98 by aspartate in Can. A can deletion introduced into wild-type strain H16 generated mutant HB1, which showed the same HCR phenotype as mutant 25-1. Overexpression of can in Escherichia coli and mass spectrometric determination of CA activity demonstrated that can encodes a functional CA. The enzyme is inhibited by ethoxyzolamide and requires 40 mM MgSO4 for maximal activity. Low but significant CA activities were detected in wild-type H16 but not in mutant HB1, strongly suggesting that the CA activity of Can is essential for growth of the wild type in the presence of low CO2 concentrations. The HCR phenotype of HB1 was overcome by complementation with heterologous CA genes, indicating that growth of the organism at low CO2 concentrations requires sufficient CA activity rather than the specific function of Can. The metabolic function(s) depending on CA activity remains to be identified.