Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangement

The NarL and NarP proteins are homologous response regulators of Escherichia coli that control the expression of several operons in response to nitrate and nitrite. A consensus heptameric NarL DNA-binding sequence has been identified, and previous observations suggest that the NarP protein has a similar sequence specificity. However, some operons are regulated by NarL alone, whereas others are controlled by both NarL and NarP. In this study, DNase I footprinting experiments with the fdnG , nirB and nrfA control regions revealed that NarP only binds to heptamer sequences organized as an inverted repeat with a 2 bp spacing (7–2–7 sites). The NarL protein also binds to these 7–2–7 sites but, unlike NarP, also recognizes heptamers in other arrangements. These results provide an explanation for the regulation of some operons by NarL alone and for the different effects of NarL and NarP at common target operons, such as fdnG and nrfA . To investigate this differential DNA binding further, derivatives of the nrfA control region were constructed in which the spacing of the 7–2–7 heptamers was increased (7– n –7 constructs). Increasing the spacing to four or more basepairs abolished NarP binding and significantly reduced NarL binding. The NarL protein also had a reduced binding affinity for heptamers adjacent to the 7– n –7 heptamer pair, suggesting a decrease in cooperative interactions. In conclusion, we propose that 7–2–7 sites are preferred by both NarL and NarP. NarL can also recognize other binding site arrangements, an ability that appears to be lacking in 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.

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).     

Integration host factor is required for positive regulation of the tdc operon of Escherichia coli.

A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.