Anaerobically expressed Escherichia coli genes identified by operon fusion techniques

Choe, Muhyeon; Reznikoff, William S.

doi:10.1128/jb.173.19.6139-6146.1991

Cited by 61 publications

(47 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A more complex scenario is presented by the regulation of two more recently identified genes, termed aeg-46.5 and aeg-93 (4). Nitrate induction of aeg-46.5 is enhanced in a narL mutant (4) and is mediated by NarP (21). Regulation of aeg-93 is more complex than that of aeg-46.5, since nitrite is an effective activator and it appears that NarL represses expression in the presence of nitrate but activates it in the presence of nitrite, while NarP is an activator with both nitrate and nitrite.…”

Section: Discussionmentioning

confidence: 96%

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP

Kaiser¹,

Sawers²

1995

J Bacteriol

View full text Add to dashboard Cite

The pfl operon is expressed at high levels anaerobically. Growth of Escherichia coli in the presence of nitrate or nitrite led to a 45% decrease in expression when cells were cultivated in rich medium. Nitrate repression, however, was significantly enhanced (sevenfold) when the cells were cultured in minimal medium. Regulation of pfl expression by nitrate was dependent on the NarL, NarP, NarQ, and NarX proteins but independent of FNR, ArcA, and integration host factor, which are additional regulators of pfl expression. Strains unable to synthesize any one of the NarL, NarP, NarQ, or NarX proteins, but retaining the capacity to synthesize the remaining three, exhibited essentially normal nitrate regulation. In contrast, narL narP and narX narQ double null mutants were devoid of nitrate regulation when cultured in rich medium but they retained some nitrate repression (1.3-fold) when grown in minimal medium. By using lacZ fusions, it was possible to localize the DNA sequences required to mediate nitrate repression to the pfl promoter-regulatory region. DNase I footprinting studies identified five potential binding sites for the wild-type NarL protein in the pfl promoter-regulatory region. Specific footprints were obtained only when NarL was phosphorylated with acetyl phosphate before the binding reaction was performed. Each of the protected regions contained at least one heptamer sequence which has been deduced from mutagenesis studies to be essential for NarL binding (K. Tyson, A. Bell, J. Cole, and S. Busby, Mol. Microbiol. 7:151-157, 1993).Nitrate respiration is an efficient means of energy generation for Escherichia coli. The use of this electron acceptor naturally requires that a particular complement of enzymes is synthesized. Included in this set of enzymes is a respiratory pathway comprising a formate dehydrogenase termed FDH-N (encoded by the fdnGHI operon) and a nitrate reductase (encoded by narGHJI). The coordinate synthesis of these enzymes occurs only anaerobically and when nitrate is available (33). The presence of nitrate also reduces or prevents the expression of a number of genes, e.g., the frdABCD operon encoding fumarate reductase and the dmsABC operon encoding dimethylsulfoxide reductase, whose products either are not required during nitrate respiration or are required but in much reduced amounts. A complex system of proteins that is involved in sensing nitrate in the environment and transmitting its presence to the level of gene expression has been discovered (33). The proteins include two sensor-regulator pairs termed NarX-NarL and NarQ-NarP of which NarX and NarQ are membrane-associated histidine kinases and NarL and NarP are transcription regulators (3,7,14,20,21,28,35,39). The NarQ and NarX proteins are not only capable of sensing nitrate but they are also responsive to nitrite. Mutational studies have demonstrated that either NarX or NarQ is capable of modulating the DNA-binding activity of the NarL and NarP proteins in response to nitrate and nitrite (for a detailed review, see reference ...

show abstract

Section: Discussionmentioning

confidence: 96%

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP

Kaiser¹,

Sawers²

1995

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…A molybdopterin enzyme(s) is, therefore, implicated as the nitrosating activity and also as the source of a mutagenic methylating agent in E. coli under the growth conditions used. E. coli has at least three nitrate reductases, A, P, and Z, that all require the molybdopterin cofactor (10,17). The moa mutation may, therefore, have been particularly effective because it eliminates all three nitrate reductase activities, whereas narG and fnr mutations which did not affect spontaneous mutagenesis in ada ogt eliminate only nitrate reductase A.…”

Section: Discussionmentioning

confidence: 99%

Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli

Taverna¹,

Sedgwick²

1996

J Bacteriol

144

113

View full text Add to dashboard Cite

Escherichia coli ada ogt mutants, which are totally deficient in O 6 -methylguanine-DNA methyltransferases, have an increased spontaneous mutation rate. This phenotype is particularly evident in starving cells and suggests the generation of an endogenous DNA alkylating agent under this growth condition. We have found that in wild-type cells, the level of the inducible Ada protein is 20-fold higher in stationary-phase and starving cells than in rapidly growing cells, thus enhancing the defense of these cells against DNA damage. The increased level of Ada in stationary cells is dependent on RpoS, a stationary-phase-specific sigma subunit of RNA polymerase. We have also identified a potential source of the mutagenic agent. Nitrosation of amides and related compounds can generate directly acting methylating agents and can be catalyzed by bacterial enzymes. E. coli moa mutants, which are defective in the synthesis of a molybdopterin cofactor required by several reductases, are deficient in nitrosation activity. It is reported here that a moa mutant shows reduced generation of a mutagenic methylating agent from methylamine (or methylurea) and nitrite added to agar plates. Moreover, a moa mutation eliminates much of the spontaneous mutagenesis in ada ogt mutants. These observations indicate that the major endogenous mutagen is not S-adenosylmethionine but arises by bacterially catalyzed nitrosation.Only a few types of cancer have been attributed to environmental agents. Other carcinogens of primary concern may be transient but highly reactive metabolites that arise during normal metabolism (24, 36). Indeed, several metabolites of amino acids, sugars, oxygen, and lipids are known to be mutagenic. It is, therefore, important to identify metabolites that damage DNA in vivo and result in spontaneous mutagenesis in the absence of adequate DNA repair. For example, reactive oxygen species generated during aerobic metabolism damage DNA bases and sugar residues. Specific oxygen radical detoxification and DNA repair mechanisms prevent or remove this damage (13,14). The increase in the rate of spontaneous mutagenesis in bacteria and yeast mutants that are specifically deficient in the repair of aberrantly methylated DNA bases indicates that methylation of DNA also occurs endogenously (16,34,52,53). Rodent cells may be similarly affected (2).The major mutagenic adduct induced in DNA by methylating agents is O 6 -methylguanine (O 6 -meG). This modified base mispairs with thymine during DNA replication, resulting in GC-to-AT transition mutations. In wild-type cells, O 6 -meG is repaired by ubiquitous O 6 -meG-DNA methyltransferases that directly transfer the methyl group from the modified base to one of their own cysteine residues. As a result of this process, the methyltransferases are inactivated (25). Escherichia coli has two O 6 -meG-DNA methyltransferases, a constitutive Ogt protein and an inducible Ada protein. Each exponentially growing cell contains only two to four molecules of Ada protein and 10-fold more Ogt protein (32,3...

show abstract

“…In E. coli, pyruvate formate-lyase (PFL), the central enzyme of anaerobic glucose metabolism (Sawers & Bock, 1988), the formate hydrogen lyase complex, which converts the formate formed as a result of the breakdown of pyruvate by the PFL to CO, and H, (Choe & Reznikoff, 1991), and the fermentative alcohol dehydrogenase involved in ethanol generation are repressed by nitrate (Clark, 1989). It would seem that B. licbeniformis, like E. cob, has developed adaptive responses that are controlled by hierarchical regulatory mechanisms which ensure that the most energetically favourable metabolic mode is adopted in a specific environment (Gunsalus, 1992).…”

Section: Isolation Of Mutants Deficient In Nitrate Respirationmentioning

confidence: 99%

Anaerobic metabolism in Bacillus licheniformis NCIB 6346

et al. 1995

View full text Add to dashboard Cite

The products of anaerobic metabolism of glucose and its derivatives sorbitol, gluconate and glucuronate by Bacillus lichenifomis have been determined by proton NMR. Glucose was fermented through mixed-acid fermentation pathways t o acetate, 2,3-butanediol, ethanol, formate, lactate, succinate and pyruvate. However, the bacterium was incapable of fermenting the three glucose derivatives. When B. lichenifomis cells were incubated anaerobically with glucose in the presence of nitrate, the reduced products and formate did not appear and acetate was formed as the major metabolite. Growth and formation of acetate was also observed when B. lichenifomis cells were incubated anaerobically with each of the three glucose derivatives, in the presence of nitrate. A formate-nitrate oxidolreductase system was induced under anaerobic conditions, with increased activities when nitrate was added t o the anaerobic growth medium. However no activity was detected when cells were grown in the presence of molecular oxygen. Formate-nitrate oxidol reductase activity was absent in chlorate-resistant mutants isolated spontaneously or following Tn917 insertional mutagenesis. The spontaneous mutants fermented glucose in the presence of nitrate suggesting that they were incapable of nitrate respiration, due to a deficiency in one or more components of the formate-nitrate oxido-reductase system. Two insertional mutants exhibited elevated pgalactosidase activity when grown in the presence of nitrate.

show abstract

Anaerobically expressed Escherichia coli genes identified by operon fusion techniques

Cited by 61 publications

References 28 publications

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP

Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli

Anaerobic metabolism in Bacillus licheniformis NCIB 6346

Contact Info

Product

Resources

About