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

Abstract

Mutant strain 25-1 of the facultative chemoautotroph Ralstonia eutropha H16 had previously been shown to exhibit an obligately high-CO 2 -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 CO 2 . In the present study, a gene designated can and encoding a ␤-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 MgSO 4 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 CO 2 concentrations. The HCR phenotype of HB1 was overcome by complementation with heterologous CA genes, indicating that growth of the organism at low CO 2 concentrations requires sufficient CA activity rather than the specific function of Can. The metabolic function(s) depending on CA activity remains to be identified.Carbon dioxide and bicarbonate (dissolved inorganic carbon [DIC]) are essential growth factors for bacteria. The metabolic need for DIC is evident in autotrophs utilizing CO 2 as the sole carbon source, but heterotrophs also fix significant amounts of both carbon species. Although sufficient CO 2 is produced during catabolism, deprivation of atmospheric CO 2 leads to growth inhibition or even death of heterotrophs (7,14,22). Pathogenic bacteria seem to be adapted to high DIC concentrations in their host environment, as they usually require 5 to 10% (vol/vol) CO 2 for growth (9,45,60). Furthermore, elevated DIC was found to shorten the lag phase and accelerate growth of bacteria even though the organisms were not generally dependent on high DIC concentrations (46,47,60). This "sparking effect" is most pronounced when cultures are inoculated at low cell densities. The need for DIC is generally attributed to CO 2 fixation in anaplerotic or other biosynthetic reactions. Consequently, the requirement is often satisfied by supplementation of the growth media with metabolites, particularly intermediates of the tricarboxylic acid cycle such as oxaloacetate and 2-oxoglutarate (28). Most high-CO 2 -requiring (HCR) mutants of Escherichia coli and other microorganisms regained the ability to grow at air concentrations of CO 2 (0.035% [vol/vol]) upon provision with appropriate metabolites, but some depended strictly on high CO 2 concentrations (5 to 10% [vol/vol]) (1,10,64). In contrast to their general DIC requirement, many microorganisms are inhibited by very high CO 2 concentrations (ca. 20% [vol/vol] and above), an ef...

Why Is Carbonic Anhydrase Essential to Escherichia coli ?

“…Is this because of the increased CO 2 partial pressure (which also increases the rate of supply of HCO 3 Ϫ ) or because of the lack of O 2 ? Oxygen sensitivity has been reported as a phenotype associated with deletion of the can homolog NCE103 from Saccharomyces cerevisiae (15), and can mutants of R. eutropha can grow in an atmosphere enriched with CO 2 (20). To distinguish between the need to exclude O 2 and a requirement for an increased partial pressure of CO 2 , we tested the ability of EDCM636 to grow anaerobically under H 2 alone and found that it cannot, indicating that it must be the increased partial pressure of CO 2 in the earlier experiment that allowed growth.…”

“…It has been shown that a human ␣-CA can substitute for the R. eutropha Can (20). The ability of ␥-CAs to substitute for bacterial ␤-CAs has not been We introduced the can deletion into this strain (in the presence of azide) to create SM1.…”

“…The Ralstonia eutropha homolog of YadF, Can, has recently been characterized (20) and found to be essential. Unpublished results of these authors (quoted in reference 20) show that Can is able to suppress an E. coli yadF mutant and that YadF can suppress a can mutant in R. eutropha.…”

Lessons from Studies ofSymbiobacteriumthermophilum, a Unique Syntrophic Bacterium

“…A similar requirement of elevated CO 2 concentration for growth is known as a phenotype of mutants for carbonic anhydrase in Ralstonia eutropha 17) and Escherichia coli. 18,19) Carbonic anhydrase (EC 4.2.1.1) is a ubiquitous enzyme that catalyses the conversion between CO 2 and bicarbonate.…”

Carbonic anhydrases in fungi

“…1). Surprisingly, the deletion of a single cas gene does not cause lethality, as it does in yeasts and several prokaryotes (Götz et al, 1999;Klengel et al, 2005;Kusian et al, 2002). From analysis of the phenotypes of Dcas1, Dcas2 and Dcas3 deletion strains, cas2 appears to encode the major CA in S. macrospora, since only the Dcas2 mutant is drastically impaired in vegetative growth.…”