Biochemical and Genetic Characterization of nirB Mutants of Escherichia coli K12 Pleiotropically Defective in Nitrite and Sulphite Reduction

Ja, Cole; Bm, Newman; White, Peter D

doi:10.1099/00221287-120-2-475

Cited by 20 publications

(14 citation statements)

References 16 publications

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“…It was previously established that in enteric bacteria, the siroheme cofactor (the product of the CysG activity) is also required for nitrite reductase activity. As a consequence, the cysG mutants could not grow with nitrate as the sole nitrogen source (Nir Ϫ ) (2). To test the ability of the CTNUX8 strain of R. etli to grow in minimal medium with various nitrogen sources, a modified RMM medium with thiosulfate substituted for sulfate was prepared.…”

Section: Isolation Of Cysmentioning

confidence: 99%

A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation

et al. 1997

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By its inability to grow on sulfate as the sole sulfur source, a mutant strain (CTNUX8) of Rhizobium etli carrying Tn5 was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a cysG (siroheme synthetase)-homologous gene. By RNase protection assays, it was established that the cysG-like gene had a basal level of expression in thiosulfate-or cysteine-grown cells, which was induced when sulfate or methionine was used. Unlike its wild-type parent (strain CE3), the mutant strain, CTNUX8, was also unable to grow on nitrate as the sole nitrogen source and was unable to induce a high level of nitrite reductase. Despite its pleiotropic phenotype, strain CTNUX8 was able to induce pink, effective (N 2 -fixing) nodules on the roots of Phaseolus vulgaris plants. However, mixed inoculation experiments showed that strain CTNUX8 is significantly different from the wild type in its ability to nodulate. Our data support the notion that sulfate (or sulfite) is the sulfur source of R. etli in the rhizosphere, while cysteine, methionine, or glutathione is supplied by the root cells to bacteria growing inside the plant.The soil bacterium Rhizobium etli has the ability to induce the formation of nitrogen-fixing nodules on the root of the common bean (Phaseolus vulgaris). From the point of view of the bacterial partner, the development of these new plant organs is a multistage process involving bacterial multiplication in the rhizosphere, the recognition and infection of the root hairs, bacterial growth inside a network of infection threads, and the release of bacteria from the threads into the cytoplasm of host cells. Within the infected plant cells, the bacteria differentiate into nondividing bacteroids which are equivalent to plant cell organelles able to reduce atmospheric dinitrogen (28,30).In poor soils, where Rhizobium-legume symbiosis takes place, and prior to the onset of N 2 fixation, the host plant should provide nutrients (e.g., carbon, nitrogen, and sulfur sources) to support the growth of the developing nodules. It has recently been proposed (6) that bacterial nutrients are delivered by the plant only at the tip (and not at the base) of the infection threads, where bacterial growth was observed. It has also been speculated (19) that provision of nutrients from seed storage reserves may serve as a mechanism for regulating both the bacterial growth inside the infection threads and the bacteroid differentiation inside the invaded plant cells. However, the kind of nutrients used by Rhizobium bacteria (as carbon, nitrogen, or sulfur sources) during the early steps of the symbiotic interaction remain undefined. Furthermore, since both root metabolism and nodule metabolism are different for different legumes (19), the bacterial nutrient(s) might be specific for each symbiotic relationship.Considering the compounds commonly used by bacteria as a sulfur source, inorganic sulfate is particularly interesting, since its assimilation represents the pathway by which inorganic sulfur is fixed into an organic l...

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Section: Isolation Of Cysmentioning

confidence: 99%

A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation

et al. 1997

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“…The hemoprotein subunit contains a novel heme group, called siroheme, in its catalytic center. Siroheme is also present in nitrite reductase, an anaerobically expressed enzyme that catalyzes the six-electron transfer reduction of nitrite to ammonia (6)(7)(8). The cysG gene, in which mutations lead to defective nitrite and biosynthetic sulfite reduction, is essential for siroheme biosynthesis (12).…”

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confidence: 99%

Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium

Huang

Barrett

1990

J Bacteriol

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“…All previous cysG mutants were isolated as cysteine auxotrophs and were thus defective in synthesis of siroheme (required for sulfite and nitrite reductases) (10,11,16,24,26). Previously, a set of 30 such Salmonella cysG mutants were found to be defective for cobalamin synthesis (B 12 Ϫ ) (16).…”

Section: Isolation Of Cysg Mutants With a Cysmentioning

confidence: 99%

“…The auxotrophy of cysG mutants is due to their failure to produce siroheme, the cofactor of the CysIJ enzyme (11,24,27,28). Since siroheme is also required for nitrite reductase (NirB), cysG mutants are also defective in reduction of nitrite (Nir Ϫ ) (11). After the discovery of B 12 synthesis in S. typhimurium (18), it was found that cysG mutants fail to make cobalamin (i.e., are B 12 Ϫ ) (16).…”

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confidence: 99%

“…These mutants, as well as cysIJ mutants were shown to be defective in reduction of sulfite to sulfide (14), which is required for cysteine biosynthesis. The auxotrophy of cysG mutants is due to their failure to produce siroheme, the cofactor of the CysIJ enzyme (11,24,27,28). Since siroheme is also required for nitrite reductase (NirB), cysG mutants are also defective in reduction of nitrite (Nir Ϫ ) (11).…”

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confidence: 99%

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Evidence that the CysG protein catalyzes the first reaction specific to B12 synthesis in Salmonella typhimurium, insertion of cobalt

Fazzio

Roth

1996

J Bacteriol

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The cysG gene of Salmonella typhimurium is involved in synthesis of both cobalamin (B 12 ) and siroheme (a cofactor required for SO 3 2؊ and NO 2 2؊ reductases). The failure to reduce SO 3 2؊ leads to cysteine auxotrophy, for which the enzyme is named. Although Escherichia coli does not synthesize B 12 de novo, it possesses a very similar CysG enzyme which has been shown to catalyze two methylations (uroporphyrinogen III to precorrin-2), ring oxidation (precorrin-2 to factor II), and iron insertion (factor II to siroheme). In S. typhimurium, precorrin-2 is a precursor of both siroheme and B 12 . All previously known Salmonella cysG mutants are defective in the synthesis of both siroheme and cobalamin. We describe two new classes of cysG mutants that cannot synthesize B 12 but still make siroheme. For class I mutants, exogenous cobalt corrects the B 12 defect but inhibits ability to make siroheme; B 12 synthesis is inhibited by added iron. Class II mutants are unaffected by exogenous cobalt, but their B 12 defect is corrected by derepression of the B 12 biosynthetic genes (cob). We propose that all mutants are defective in insertion of cobalt into factor II and that the Salmonella CysG enzyme normally catalyzes this insertion-the first reaction dedicated to cobalamin synthesis. Although E. coli does not make B 12 , its CysG enzyme has been shown in vitro to insert cobalt into factor II and may have evolved to support B 12 synthesis in some ancestor common to Salmonella species and E. coli.

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Biochemical and Genetic Characterization of nirB Mutants of Escherichia coli K12 Pleiotropically Defective in Nitrite and Sulphite Reduction

Cited by 20 publications

References 16 publications

A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation

A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation

Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium

Evidence that the CysG protein catalyzes the first reaction specific to B12 synthesis in Salmonella typhimurium, insertion of cobalt

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