Isolation, Characterization and Complementation Analysis of nir B Mutants of Escherichia coli Deficient Only in NADH-dependent Nitrite Reductase Activity

Macdonald, Heather; Pope, Nicolas; Cole, J. A.

doi:10.1099/00221287-131-10-2771

Cited by 19 publications

(15 citation statements)

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“…This gene is located more than 10 min away from cysJIH on the chromosomal map (34) and is not tightly regulated as part of the cysteine regulon (27,28). In E. coli, cysG is closely linked to nirB, the gene for another siroheme-containing enzyme, nitrite reductase (18). The DNA sequences of cysG and the upstream nirB and nirC genes have been determined for E. coli (5a; GenBank sequence ECONIRBC).…”

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confidence: 99%

High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor

Siegel

Kredich

1991

J Bacteriol

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The flavoprotein and hemoprotein components of Escherichia coli B NADPH-sulfite reductase are encoded by cysJ and cysl, respectively. Plasmids containing these two genes overexpressed flavoprotein catalytic activity and apohemoprotein by 13-to 35-fold, but NADPH-sulfite reductase holoenzyme activity was increased only 3-fold. Maximum overexpression of holoenzyme activity was achieved by the inclusion in such plasmids of Salmonella typhimurium cysG, which encodes a uroporphyrinogen III methyltransferase required for the synthesis of siroheme, a cofactor for the hemoprotein. Thus, cofactor deficiency, in this case siroheme, can limit overexpression of a cloned enzyme. Catalytically active holoenzyme accounted for 10% of total soluble protein in a host containing cloned cysJ, cysI, and cysG. A 5.3-kb DNA fragment containing S. typhimurium cysG was sequenced, and the open reading frame corresponding to cysG was identified by subcloning and by identifying plasmid-encoded peptides in maxicells. Comparison with the sequence reported for the E. coli cysG region (J. A. Cole, unpublished data; GenBank sequence ECONIRBC) indicates a gene order of nirB-nirC-cysG in the cloned S. typhimurium fragment. In addition, two open reading frames of unknown identity were found immediately downstream of cysG. One of these contains 11 direct repeats of 33 nucleotides each, which correspond to the consensus amino acid sequence Asp-Asp-Val-Thr-Pro-Pro-Asp-Asp-Ser-Gly-Asp.NADPH-sulfite reductase (SiR) of Escherichia coli and Salmonella typhimurium catalyzes the reduction of sulfite to sulfide and is required for synthesis of L-cysteine from inorganic sulfate (8,14). The native enzyme has a subunit structure a8,4, where a8 is a flavoprotein (SiR-FP) containing both flavin adenine dinucleotide and flavin mononucleotide and P is a hemoprotein (SiR-HP) containing an Fe4S4 center and a single molecule of siroheme (24,25,37,39). Electron flow between these cofactors proceeds from NADPH to flavin adenine dinucleotide to flavin mononucleotide in the flavoprotein, then to a closely coupled Fe4S4-siroheme center in the hemoprotein, and finally from siroheme to sulfite (38).The SiR-FP and SiR-HP components of SiR are encoded by cysJ and cysl, respectively. These genes are contiguous and together with cysH, the gene for 3'-phosphoadenosine 5'-phosphosulfate sulfotransferase, comprise an operon with the gene order promoter-cysJ-cysI-cysH (7,17,(26)(27)(28)(29). The cysJIH operon is part of the positively regulated cysteine regulon (15) and requires sulfur limitation, CysB protein, and either O-acetyl-L-serine or N-acetyl-L-serine for expression (10,11,14,28). SiR activity is also dependent on cysG, which encodes a uroporphyrinogen III methyltransferase necessary for the synthesis of siroheme (42). This gene is located more than 10 min away from cysJIH on the chromosomal map (34) and is not tightly regulated as part of the cysteine regulon (27,28). In E. coli, cysG is closely linked to nirB, the gene for another siroheme-containing enzyme, nitrite redu...

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confidence: 99%

High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor

Siegel

Kredich

1991

J Bacteriol

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“…Mutants defective in the cysG gene are unable to grow without cysteine either aerobically or anaerobically because the cysG product is essential to convert uroporphyrinogen 111 into sirohaem. As the NADH-dependent nitrite reductase is the most active enzyme for reducing nitrite to ammonia in E. coli, cysG mutants are also defective in nitrite reduction [2].The cysG gene is located extremely close to nirB, the structural gene for the nitrite reductase apoprotein, but is separated from it by three open reading frames which, in the preceding paper, we have designated nirD, nirE and nirC [3]. The third of these open reading frames, nirC, encodes a 28.5-kDa polypeptide with regions of amino acid sequence similar to cytochrome oxidase polypeptide 1 [3].…”

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Transcriptional control of the cysG gene of Escherichia coli K‐12 during aerobic and anaerobic growth

Peakman

Busby

Cole

1990

European Journal of Biochemistry

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The 74-min region of the Escherichiu coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH-dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functiona1,fnr gene and were shown by S1 mapping experiments to be due to transcriptional readthrough from the Fnr-dependent nirB promoter.No promoter activity was associated with DNA fragments between the BumHI site located within the Nterminal coding region of the nirB gene and the cysG promoter located at the C-terminus of nirC.Sirohaem is one of the prosthetic groups of both the NADH-dependent nitrite reductase and the NADPH-dependent sulphite reductase of Escherichiu coli [I]. Sulphite reductase is an essential enzyme in the synthesis of cysteine from sulphate. Mutants defective in the cysG gene are unable to grow without cysteine either aerobically or anaerobically because the cysG product is essential to convert uroporphyrinogen 111 into sirohaem. As the NADH-dependent nitrite reductase is the most active enzyme for reducing nitrite to ammonia in E. coli, cysG mutants are also defective in nitrite reduction [2].The cysG gene is located extremely close to nirB, the structural gene for the nitrite reductase apoprotein, but is separated from it by three open reading frames which, in the preceding paper, we have designated nirD, nirE and nirC [3]. The third of these open reading frames, nirC, encodes a 28.5-kDa polypeptide with regions of amino acid sequence similar to cytochrome oxidase polypeptide 1 [3].The nirB gene is not expressed during aerobic growth. Transcription of nirB during anaerobic growth is dependent upon a functional Fnr protein and is further induced by nitrite (4, 51. As nirB and cysG are transcribed in the same direction (from nirB towards cysG), the aims of the experiments deCorrespondence to J. A. Cole,

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“…The nirB gene, the structural gene for an NADH-dependent nitrite reductase, maps very close to the cysG gene (25) Complementation data, using a chromosomal duplication, suggest that all cys mutations which map to min 72 affect one gene. These data suggest that all cysG mutations are blocked in the synthesis of DSC and that no other enzyme of the siroheme synthetic pathway is encoded at the cysG locus.…”

Section: Resultsmentioning

confidence: 99%

“…Transcription of the cysG gene was induced in response to nitrite when cells were grown under anaerobic conditions. Molecular analysis of the cysG gene in E. coli suggests that the gene is in an operon with the nirB gene, which encodes the structural gene for the NADH-dependent nitrite reductase (25,31,32). Sequence analysis of the cysG gene suggests that this is also the case in S. typhimurium (41).…”

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Genetic structure and regulation of the cysG gene in Salmonella typhimurium

Goldman

Roth

1993

J Bacteriol

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Siroheme, a cofactor of both sulfite and nitrite reductase in Sabnonella typhimurium, requires the cysG gene for its synthesis. Three steps are required to synthesize siroheme from uroporphyrinogen m, the last common intermediate in the heme and siroheme pathways. All previously characterized cysG mutants were shown to be defective for the synthesis of cobalamin (B12), which shares a common precursor with siroheme. Since few cysG auxotrophs had been previously analyzed and since there is no evidence of siroheme mutants outside of the cysG region, we sought to expand the analysis of the region by isolating more mutations and studying the transcriptional regulation of the cysG gene using IacZ fusions. We isolated and analyzed 66 cysG auxotrophs. All were defective for both siroheme and cobalamin synthesis. Five exceptional mutants were partially defective for the synthesis of both and appear to be leaky. Complementation tests with tandem duplications suggest that the mutations causing the Cys auxotrophy affect only one cistron. The cysG gene is transcribed in a clockwise direction; this was demonstrated by a method that permits determining the orientation of two genes of unknown orientation provided their relative map order is known. The cysG gene was not part of the cysteine regulon, but had a substantial basal level of expression which was induced fivefold when cells were grown anaerobically on nitrite. Finally, we used Mud-generated duplications to genetically determine the organization of the cysG and nirB genes.

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Isolation, Characterization and Complementation Analysis of nir B Mutants of Escherichia coli Deficient Only in NADH-dependent Nitrite Reductase Activity

Cited by 19 publications

References 19 publications

High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor

High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor

Transcriptional control of the cysG gene of Escherichia coli K‐12 during aerobic and anaerobic growth

Genetic structure and regulation of the cysG gene in Salmonella typhimurium

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