The Occurrence and Genetics of Some CO2 Mutants in Streptomyces coelicolor

Vivian, Alan; Charles, H. P.

doi:10.1099/00221287-61-2-263

Cited by 5 publications

(4 citation statements)

References 7 publications

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“…Like the original HCR mutant 25-1, can deletion mutant HB1 failed to grow (10) and Streptomyces coelicolor (64) without assigning them to a specific metabolic/genetic defect. The obligate HCR phenotype of mutant HB1 excludes a specific involvement of Can in autotrophic metabolism of R. eutropha.…”

Section: Resultsmentioning

confidence: 96%

“…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.…”

mentioning

confidence: 99%

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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: 96%

mentioning

confidence: 99%

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

2002

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“…The occurrence of mutants of various microorganisms auxotrophic for carbon dioxide has been reviewed by Charles and Broadbent (15) and Charles and Roberts (16). In most cases the mutants respond to particular alternative nutrients, sometimes already explicable in terms of known biosynthetic pathways, and most of the carbon dioxide mutants of S. coelicolor isolated by Vivian and Charles (101) were of this type. However, one mutant, defining a locus cdxA, appeared to have a carbon dioxide requirement for which no alternative nutrient was found.…”

Section: Carbon Dioxide Mutantsmentioning

confidence: 99%

Advances in Streptomyces coelicolor genetics.

Hopwood¹,

Chater²,

Dowding³

et al. 1973

Bacteriological Reviews

128

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“…Significantly, the S. coelicolor sequence included the signature motif for a GntR family transcriptional regulator. The location of cosmid 6D7 is close to the locus of pdxA, the first pdx gene isolated and initially mapped on the chromosome of S. coelicolor A3(2) by genetic procedures (Vivian & Charles, 1970 ;D. A. Hopwood, personal communication).…”

Section: Location Of An Orf1 Homologue In S Coelicolormentioning

confidence: 99%

The pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces venezuelae ISP5230: functional characterization The GenBank accession number for the sequence reported in this paper is AF286159.

Magarvey

et al. 2001

Microbiology

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The pdx-4 mutation in Streptomyces venezuelae ISP5230 confers a growth requirement for pyridoxal (pdx) and is a marker for the genetically mapped cluster of genes associated with chloramphenicol biosynthesis. A gene regulating salvage synthesis of vitamin B6 cofactors in S. venezuelae was cloned by transforming a pdx-4 mutant host with the plasmid vector pDQ101 carrying a library of wild-type genomic DNA fragments, and by selecting for complementation of the host's pdx requirement. However, the corresponding replicative plasmid could not be isolated. Southern hybridizations and transduction analysis indicated that the complementing plasmid had integrated into the chromosome ; after excision by a second crossover, the plasmid failed to propagate. To avoid loss of the recombinant vector, a pdxdependent Streptomyces lividans mutant, KAA1, with a phenotype matching that of S. venezuelae pdx-4, was isolated for use as the cloning host. Introduction of pIJ702 carrying an S. venezuelae genomic library into S. lividans KAA1, and selection of prototrophic transformants, led to the isolation of a stable recombinant vector containing a 25 kb S. venezuelae DNA fragment that complemented requirements for pdx in both S. venezuelae and S. lividans mutants. Sequence analysis of the cloned DNA located an intact ORF with a deduced amino acid sequence that, in its central and C-terminal regions resembled type-I aminotransferases. The N-terminal region of the cloned DNA fragment aligned closely with distinctive helix-turn-helix motifs found near the N termini of GntR family transcriptional regulators. The overall deduced amino acid sequence of the cloned DNA showed 73 % end-to-end identity to a putative GntR-type regulator cloned in cosmid 6D7 from the Streptomyces coelicolor A3(2) genome. This location is close to that of pdxA, the first pdx marker in S. coelicolor A3(2) identified and mapped genetically in Sir David Hopwood's laboratory. The S. venezuelae gene and S. coelicolor pdxA are postulated to be homologues regulating vitamin B6 coenzyme synthesis from pdx.

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The Occurrence and Genetics of Some CO2 Mutants in Streptomyces coelicolor

Cited by 5 publications

References 7 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

Advances in Streptomyces coelicolor genetics.

The pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces venezuelae ISP5230: functional characterization The GenBank accession number for the sequence reported in this paper is AF286159.

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The Occurrence and Genetics of Some CO2 Mutants in Streptomyces coelicolor

Cited by 5 publications

References 7 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

Advances in Streptomyces coelicolor genetics.

The pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces venezuelae ISP5230: functional characterization The GenBank accession number for the sequence reported in this paper is AF286159.

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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