Characterization of the Escherichia coli K-12 ydhYVWXUT operon: regulation by FNR, NarL and NarP.
In Escherichia coli K-12 the expression of many genes is controlled by the oxygen-responsive transcription factor FNR and the nitrate- and nitrite-responsive two-component systems NarXL and NarPQ. Here, the ydhY gene is shown to be the first gene of a six-gene operon (ydhYVWXUT) that encodes proteins predicted to be components of an oxidoreductase. Mapping the ydhY-T transcript start and site-directed mutagenesis confirmed that the ydhY-T genes are transcribed from an FNR-dependent class II promoter and showed that the FNR site is centred at -42.5. In the presence of nitrate or nitrite, NarXL and NarPQ repressed ydhY-T expression. Analysis of the DNA sequence of the ydhY promoter region (PydhY) revealed the presence of four heptameric sequences similar to NarL/P binding sites centred at -42, -16, +6 and +15. The latter heptamers are arranged as a 7-2-7 inverted repeat, which is required for recognition by NarP. Accordingly, NarP protected the 7-2-7 region in DNase I footprints, and mutation of either heptamer +6 or heptamer +15 impaired nitrite-mediated repression, whereas mutation of heptamer -42 and heptamer -16 did not affect the response to nitrite. The NarL protein also protected the 7-2-7 region, but in contrast to NarP, the NarL footprint extended further upstream to encompass the -16 heptamer. The extended NarL footprint was consistent with the presence of multiple NarL-PydhY complexes in gel retardation assays. Mutation of heptamer -42, which is located within the FNR binding site, or heptamer +6 (but not heptamers -16 or +15) impaired nitrate-mediated repression. Thus, although the region of the ydhY-T promoter containing the -16 and +15 heptamers was recognized by NarL in vitro, mutation of these heptamers did not affect NarL-mediated repression in vivo.
Partridge
Jonathan D
JD
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK.
Browning
Douglas F
DF
The School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK.
Xu
Meng
M
The School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK.
Newnham
Louise J
LJ
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK.
Scott
Colin
C
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK.
Roberts
Ruth E
RE
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK.
Poole
Robert K
RK
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK.
Green
Jeffrey
J
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK.
D001421:Bacteria, Anaerobic / Q000254:growth & development; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics*; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D048168:Escherichia coli K12 / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009566:Nitrates / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics; D017421:Sequence Analysis; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D009566:Nitrates; D009573:Nitrites; D012097:Repressor Proteins; C081466:narP protein, E coli; C069780:NarL protein, E coli; D010088:Oxidoreductases