Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12

Giordano, Gérard; Saracino, Laurence; Grillet, Lydia

doi:10.1016/0304-4165(85)90035-2

Cited by 12 publications

(6 citation statements)

References 24 publications

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“…The results of the present study indicate that perchlorate reduction is tightly regulated at a genetic level and that the expression of the enzymes associated with the reduction of perchlorate is controlled by the presence of both oxygen and nitrate. Nitrate, chlorate, and perchlorate [(per)chlorate] are structurally analogous to each other and may potentially be incorporated into the same enzyme active site, as is evidenced by the fact that chlorate can be used as a substrate by various nitrate reductases (3,16,33,44 (11), and the fact that D. agitata can simultaneously reduce both perchlorate and nitrate would suggest that nitrate can alternatively be utilized as an analogous substrate by the (per)chlorate reductase enzymes. In support of this, the perchlorate reductase purified from the ClRB strain GR-1 could alternatively reduce nitrate as a substrate (21).…”

Section: Resultsmentioning

confidence: 99%

Environmental Factors That Control Microbial Perchlorate Reduction

Chaudhuri

O’Connor

Gustavson

et al. 2002

Appl Environ Microbiol

163

131

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As part of a study to elucidate the environmental parameters that control microbial perchlorate respiration, we investigated the reduction of perchlorate by the dissimilatory perchlorate reducer Dechlorosoma suillum under a diverse set of environmental conditions. Our results demonstrated that perchlorate reduction by D. suillum only occurred under anaerobic conditions in the presence of perchlorate and was dependent on the presence of molybdenum. Perchlorate reduction was dependent on the presence of the enzyme chlorite dismutase, which was induced during metabolism of perchlorate. Anaerobic conditions alone were not enough to induce expression of this enzyme. Dissolved oxygen concentrations less than 2 mg liter ؊1 were enough to inhibit perchlorate reduction by D. suillum. Similarly to oxygen, nitrate also regulated chlorite dismutase expression and repressed perchlorate reduction by D. suillum. Perchlorate-grown cultures of D. suillum preferentially reduced nitrate in media with equimolar amounts of perchlorate and nitrate. In contrast, an extended (40 h) lag phase was observed if a similar nitrate-perchlorate medium was inoculated with a nitrate-grown culture. Perchlorate reduction commenced only when nitrate was completely removed in either of these experiments. In contrast to D. suillum, nitrate had no inhibitory effects on perchlorate reduction by the perchlorate reducer Dechloromonas agitata strain CKB. Nitrate was reduced to nitrite concomitant with perchlorate reduction to chloride. These studies demonstrate that microbial respiration of perchlorate is significantly affected by environmental conditions and perchlorate reduction is directly dependent on bioavailable molybdenum and the presence or absence of competing electron acceptors. A microbial treatment strategy can achieve and maintain perchlorate concentrations below the recommended regulatory level, but only in environments in which the variables described above can be controlled.

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Section: Resultsmentioning

confidence: 99%

Environmental Factors That Control Microbial Perchlorate Reduction

Chaudhuri

O’Connor

Gustavson

et al. 2002

Appl Environ Microbiol

163

131

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“…Furthermore, all pleiotropic chl mutants tested, including chiA, had immunologically detectable PA, although only chlB had detectable PA activity (6). The chlB mutant from which protein PA was isolated is thought to synthesize MPT but not insert it into molybdoenzymes (1,6). These findings suggest that protein PA may be an MPT carrier protein which loses MPT during purification.…”

Section: Discussionmentioning

confidence: 98%

“…Subsequent work has shown that protein PA is synthesized in an inactive form when cells are grown in tungsten (a Mo antagonist) and restored to activity when cells are grown in Mo. Furthermore, all pleiotropic chl mutants tested, including chiA, had immunologically detectable PA, although only chlB had detectable PA activity (6). The chlB mutant from which protein PA was isolated is thought to synthesize MPT but not insert it into molybdoenzymes (1,6).…”

Section: Discussionmentioning

confidence: 99%

In vitro system for molybdopterin biosynthesis

Johnson¹,

Rajagopalan²

1987

J Bacteriol

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A high-Mr fraction present in chl+ and chLIA strains of Escherichia coli synthesizes molybdopterin (MPT) from the low-Mr fraction of several MPT-deficient mutants. Using this in vitro complementation as an assay, we have partially characteirzed the high-Mr fraction as a protein, termed MPT converting factor, of Mr 45,000, distinguishable from the Mo cofactor carrier protein of similar Mr by its absolute requirement for the low-Mr fraction of a non-chLIA mutant in the nit-i reconstitution assay. MPT converting factor was rapidly inactivated in the absence of a reduced sulfhydryl compound. Anaerobic incubation of MPT Previous experiments have shown that the pleiotropic mutants Escherichia coli chlAl (12) and Neurospora crassa nit-i (13) lack molybdopterin (MPT), the organic moiety of the molybdenum cofactor. It has also been demonstrated that a high-molecular-weight fraction in E. coli chlAl can generate authentic MPT when incubated with a lowmolecular-weight fraction from N. crassa nit-i (12). In this paper we present a partial characterization of the highmolecular-weight factor in chlAl extracts and discuss the nature of the compounds supplied by the nit-i lowmolecular-weight fraction. Our experiments were designed to test the hypothesis that the chlAl factor is an enzyme in the MPT biosynthetic pathway and that reconstitution of nit-i apo-nitrate reductase occurs by the action of the converting factor on MPT precursor(s) in nit-i to generate true MPT. MATERIALS AND METHODSMaterials and methods (including terminology) were as described in the accompanying paper (12), with the following additions.Chemicals. Protein molecular weight standards for sizeexclusion high-pressure liquid chromatography (HPLC) were from U.S. Biochemical Corp. N-Bromobimane was from Calbiochem. Neopterin was from B. Schirks. Matrex Red gel was from Amicon Corp. Preswollen microgranular DE-52 cellulose was from Whatman, Inc. All other biochemicals were from Sigma Chemical Co. All inorganic reagents were reagent grade. Pterin-6-carlboxylic-7-sulfonic acid was synthesized from deoxyurothione as described previously (10). Sulfite oxidase was purified from chicken liver and quantitated as described previously (11).Bacterial strains. All strains are described in the accompanying paper (12), except that MJ351 (chlAI) was constructed by P1 MJ345 (gal' chlAI) crossed with spontaneous * Corresponding author.2-deoxygalactose-resistant MJ17 (gal chl+) to yield the Gal' phenotype. HPLC. For size-exclusion HPLC, a Kratos TSK3000SW column was used with the equipment described in the accompanying paper (12). Crude extracts were filtered through a Gelman Metricel 0.2-,um filter before injection. Reverse-phase (C18) HPLC was as described in the accompanying paper (12).Enzyme assays. MPT converting factor activity is defined as the ability to reconstitute nitrate reductase in crude nit-] extract, but not in the G-25-excluded fraction of nit-i extract. MPT converting factor in chlAI extract was assayed by incubating the extract with nit-i crude extract in t...

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“…Molybdenum is required for the activity of a number of these gene products. The presence of molybdate or tungstate is essential for the accumulation of the subunits of nitrate reductase (8,30); for the accumulation of active Mo cofactor in both wild-type and chlD cells (1,21); for the activity of the PA protein, which is the chiA gene product (9); and also for the increase in expression of the chlI gene in response to nitrate (23 Strains with the chlD-lac fusion combined with an additional chl or nar mutation show that the regulation of chlD by molybdenum is not mediated by any of the chl or nar genes we tested. The presence of high concentrations of molybdate always resulted in greatly reduced gene expression, even in the absence of the chl or nar gene products.…”

Section: Discussionmentioning

confidence: 99%

Molybdenum-sensitive transcriptional regulation of the chlD locus of Escherichia coli

Miller¹,

Scott²,

Amy³

1987

J Bacteriol

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The chiD gene in Escherichja coli is required for the incorporation and utilization of molybdenum when the cells are grown with low concentrations of molybdate. We constructed chiD-lac operon fusions and measured expression of the fusion, Mo cofactor, and nitrate reductase activities under a variety of growth conditions. The chlD-lac fusion was highly expressed when cells were grown with less than 10 nm molybdate. Increasing concentrations of molybdate caused loss of activity, with less than 5% of the activity remaining at 500 nM molybdate; when tungstate replaced molybdate, it bad an identical affect on chiD expression. Expression of chiD-lac was increased in cells grown with nitrate. Strains with chiD-lac plus an additional mutation in a chl or nar gene were constructed to test whether the regulation of chiD-lac required the concerted action of gene products involved with Mo cofactor or nitrate reductase synthesis. Mutations in narL prevented the increase in activity in response' to nitrate; mutations in chM, narC, or narI resulted in partial constitutive expression of the chiD-lac fusion: the fusion was regulated by molybdate, but it no longer required the presence of nitrate for maximal activity. Mutations in chMA, chE, or chiG which affect Mo cofactor metabolism, did not affect the expression of chiD-lac.

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Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12

Cited by 12 publications

References 24 publications

Environmental Factors That Control Microbial Perchlorate Reduction

Environmental Factors That Control Microbial Perchlorate Reduction

In vitro system for molybdopterin biosynthesis

Molybdenum-sensitive transcriptional regulation of the chlD locus of Escherichia coli

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