Cloning, expression and some properties of α-carbonic anhydrase from Helicobacter pylori

Chirică, Laura C.; Elleby, Björn; Lindskog, Sven

doi:10.1016/s0167-4838(00)00204-1

Cited by 63 publications

(35 citation statements)

References 27 publications

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“…In Neisseria gonorrhoeae and Helicobacter pylori, putative ␤-and ␥-CA genes were identified (51,62). The ␣-CAs of these organisms have been characterized recently (12,27). The cyanobacterium Synechococcus sp.…”

Section: Resultsmentioning

confidence: 99%

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

2002

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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...

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Section: Resultsmentioning

confidence: 99%

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

2002

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“…The carbonic anhydrase of the human gastric pathogen H. pylori has been studied in some detail because of its possible involvement in acid acclimation that allows colonization and thence virulence (7,8,22,37,44). These studies have resulted in a large amount of biochemical and physiological data concerning the specific roles of H. pylori carbonic anhydrase but have left analysis of the transcriptional regulation of the carbonic anhydrase gene almost untouched.…”

Section: Discussionmentioning

confidence: 99%

The HP0165-HP0166 Two-Component System (ArsRS) Regulates Acid-Induced Expression of HP1186 α-Carbonic Anhydrase in Helicobacter pylori by Activating the pH-Dependent Promoter

et al. 2007

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The periplasmic ␣-carbonic anhydrase of Helicobacter pylori is essential for buffering the periplasm at acidic pH. This enzyme is an integral component of the acid acclimation response that allows this neutralophile to colonize the stomach. Transcription of the HP1186 ␣-carbonic anhydrase gene is upregulated in response to low environmental pH. A binding site for the HP0166 response regulator (ArsR) has been identified in the promoter region of the HP1186 gene. To investigate the mechanism that regulates the expression of HP1186 in response to low pH and the role of the HP0165-HP0166 two-component system (ArsRS) in this acidinducible regulation, Northern blot analysis was performed with RNAs isolated from two different wild-type H. pylori strains (26695 and 43504) and mutants with HP0165 histidine kinase (ArsS) deletions, after exposure to either neutral pH or low pH (pH 4.5). ArsS-dependent upregulation of HP1186 ␣-carbonic anhydrase in response to low pH was found in both strains. Western blot analysis of H. pylori membrane proteins confirmed the regulatory role of ArsS in HP1186 expression in response to low pH. Analysis of the HP1186 promoter region revealed two possible transcription start points (TSP 1 and TSP 2 ) located 43 and 11 bp 5 of the ATG start codon, respectively, suggesting that there are two promoters transcribing the HP1186 gene. Quantitative primer extension analysis showed that the promoter from TSP 1 (43 bp 5 of the ATG start codon) is a pH-dependent promoter and is regulated by ArsRS in combating environmental acidity, whereas the promoter from TSP 2 may be responsible for control of the basal transcription of HP1186 ␣-carbonic anhydrase.

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“…The Mr. Gene Company, specialized in gene synthesis, designed the synthetic Sulfurihydrogenibium sp. gene encoding for the α carbonic anhydrase lacking the peptide signal (first 20 amino acid of the peptide sequence) 18 and containing a NdeI and XhoI site at the 5′ and 3′ end of the SspCa gene, respectively. The resulting plasmid was amplified into E. coli DH5 α cells.…”

Section: Gene Identification Construct Preparation Protein Expressimentioning

confidence: 99%

Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase fromSulfurihydrogenibium yellowstonense YO3AOP1

Capasso

Luca

Carginale

et al. 2012

Journal of Enzyme Inhibition and Medicinal Chemistry

122

109

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Cloning, expression and some properties of α-carbonic anhydrase from Helicobacter pylori

Cited by 63 publications

References 27 publications

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

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

The HP0165-HP0166 Two-Component System (ArsRS) Regulates Acid-Induced Expression of HP1186 α-Carbonic Anhydrase in Helicobacter pylori by Activating the pH-Dependent Promoter

Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase fromSulfurihydrogenibium yellowstonense YO3AOP1

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Cloning, expression and some properties of α-carbonic anhydrase from Helicobacter pylori

Cited by 63 publications

References 27 publications

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

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

The HP0165-HP0166 Two-Component System (ArsRS) Regulates Acid-Induced Expression of HP1186 α-Carbonic Anhydrase in Helicobacter pylori by Activating the pH-Dependent Promoter

Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase fromSulfurihydrogenibium yellowstonense YO3AOP1

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

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