Mutational analysis of nitrate regulatory gene narL in Escherichia coli K-12.
The narL gene product, NarL, is the nitrate-responsive regulator of anaerobic respiratory gene expression. We used genetic analysis of narL mutants to better understand the mechanism of NarL-mediated gene regulation. We selected and analyzed seven nitrate-independent narL mutants. Each of three independent, strongly constitutive mutants had changes of Val-88 to Ala. The other four mutants were weakly constitutive. The narL505(V88A) allele was largely dominant to narL+, while narX+ had a negative influence on its constitutive phenotype, suggesting that NarX may play a negative role in nitrate regulation. We also constructed two narL mutations that are analogous to previously characterized constitutive degU alleles. The first, narL503(H15L), was a recessive null allele. The second, narL504(D110K), functioned essentially as wild type but was dependent on narX+ for full activity. We changed Asp-59 of NarL, which corresponds to the site of phosphorylation of other response regulators, to Asn. This change, narL502(D59N), was a recessive null allele, which is consistent with the hypothesis that NarL requires phosphorylation for activation. Finally, we tested the requirement for molybdate on regulation in a narL505(V88A) strain. Although narL505(V88A) conferred some nitrate-independent expression of fdnGHI (encoding formate dehydrogenase-N) in limiting molybdate, it required excess molybdate for full induction both in the absence and in the presence of nitrate. This finding suggests that narL505(V88A) did not confer molybdate-independent expression of fdnGHI.
Egan
S M
SM
Section of Microbiology, Cornell University, Ithaca, New York 14853-8101.
Stewart
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V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005838:Genotype; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009566:Nitrates / Q000378:metabolism*; D015345:Oligonucleotide Probes; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009566:Nitrates; D015345:Oligonucleotide Probes; C069780:NarL protein, E coli