Isolation of Genes Required for Hydrogenase Synthesis in Escherichia coli

Chaudhuri, Arijit; Krasna, Alvin I.

doi:10.1099/00221287-133-12-3289

Cited by 13 publications

(34 citation statements)

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“…Hydrogenase-positive C. vinosum and P. vulgaris were grown as previously described (Gitlitz & Krasna, 1975;Schengrund & Krasna, 1969) and the DNA isolated from these cells was used to prepare genomic libraries (Chaudhuri & Krasna, 1987. The wild-type hydrogenase-positive E. coli strain was K 12W6 met bio (ATCC 25019).…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme activities of interest were measured at 25 "C in washed cell suspensions. Hydrogenase activity was assayed by the deuterium exchange method and reduction of viologen dyes; formate hydrogenlyase (FHL) activity was determined by manometric measurements of evolution of H2 from formate as described previously (Chaudhuri & Krasna, 1987).…”

Section: Methodsmentioning

confidence: 99%

“…Bacteria were grown in complex or minimal medium containing appropriate antibiotics when required for selection as described previously (Chaudhuri & Krasna, 1987).…”

Section: Methodsmentioning

confidence: 99%

“…The hydC and hydD genes, which map at minute 77, are also required for synthesis of all three isoenzymes ( Mandrand-Berthelot, 1986). Mutations in the hydC and hydE genes can be overcome by growing cells in the presence of high concentrations of nickel (Waugh & Boxer, 1986;Wu & Mandrand-Berthelot, 1986;Chaudhuri & Krasna, 1987). The role of the hydA, hydB and hydF genes is not known.…”

Section: Introductionmentioning

confidence: 99%

“…Four of these genes, hydA, hydB, hydE and hydF, have been mapped near minute 59 of the E. coli genome map and DNA fragments have been isolated which complemented the individual mutations Waugh & Boxer, 1986;Chaudhuri & Krasna, 1987;Sankar & Shanmugam, 1988a, b). Strains with mutations in these genes are devoid of all hydrogenase activities (reduction of viologen dyes with H2, exchange reaction, growth on fumarate plus H2, evolution of H2 from formate) and lack the three hydrogenase isoenzymes Sawers et al, 1985;Waugh & Boxer, 1986).…”

Section: Introductionmentioning

confidence: 99%

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Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

Chaudhuri

Krasna

1990

Journal of General Microbiology

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DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4-7 kb from C. vinosum and 2.3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosurn were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Bacteria were grown in complex or minimal medium containing appropriate antibiotics when required for selection as described previously (Chaudhuri & Krasna, 1987).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

Chaudhuri

Krasna

1990

Journal of General Microbiology

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Mutations in trans which affect the anaerobic expression of a formate dehydrogenase (fdhF) structural gene

Schlensog¹,

Birkmann²,

Böck³

1989

Arch. Microbiol.

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An operon fusion was constructed in which the chloramphenicol acetyltransferase gene (cat) is under the transcriptional control of the anaerobically-activated formate dehydrogenase (fdhF) gene promoter. It was used as a screening system for mutations in trans which prevent the formate-dependent anaerobic induction of fdhF gene expression. Five classes of mutants were identified. The defect in class I mutants was complemented by a plasmid (pBA11) or subclones thereof, which harbor genes of the Escherichia coli 58 min hyd (hydrogenase) gene cluster. They may comprise regulatory gene mutants. The phenotype of class II mutants was reversed by supplementing the medium with 100 microM MoO4(2-); WO4(2-) could substitute for MoO4(2-) in restoring anaerobic induction by formate. Similarly, class III mutants were phenotypically suppressed by inclusion of 500 microM Ni2+ in the medium; these mutants were shown to carry a defective fnr gene. The mutant of class IV had a defect in a formate dehydrogenase structural gene and that of class V was unable to grow under fermentative conditions while maintaining the capability to grow anaerobically in the presence of electron acceptors.

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The hydrogenases and formate dehydrogenases ofEscherichia coli

Sawers¹

1994

Antonie van Leeuwenhoek

224

205

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Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.

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Isolation of Genes Required for Hydrogenase Synthesis in Escherichia coli

Cited by 13 publications

References 28 publications

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris

Mutations in trans which affect the anaerobic expression of a formate dehydrogenase (fdhF) structural gene

The hydrogenases and formate dehydrogenases ofEscherichia coli

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