Characterization of the Escherichia coli K-12 ydhYVWXUT operon: regulation by FNR, NarL and NarP

• Published in: Microbiology (Society for General Microbiology) 2008-02, Vol.154 (2), p.608-618
• Main Authors: Partridge, Jonathan D, Browning, Douglas F, Xu, Meng, Newnham, Louise J, Scott, Colin, Roberts, Ruth E, Poole, Robert K, Green, Jeffrey
• Format: Article
• Language: English
• Subjects: Bacteria, Anaerobic - growth & development; Bacteriology; Base Sequence; Binding Sites; Biological and medical sciences; DNA Footprinting; DNA-Binding Proteins - genetics; Down-Regulation; Electrophoretic Mobility Shift Assay; Escherichia coli; Escherichia coli K12 - genetics; Escherichia coli K12 - growth & development; Escherichia coli Proteins - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genetics; Iron-Sulfur Proteins - genetics; Microbiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrates - metabolism; Nitrites - metabolism; Oligonucleotide Array Sequence Analysis; Operon; Oxidoreductases - genetics; Regulatory Sequences, Nucleic Acid; Repressor Proteins - genetics; Sequence Analysis; Transcription Initiation Site; Transcription, Genetic
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2007/012146-0

1 Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK 2 The School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK Correspondence Jeffrey Green jeff.green{at}sheffield.ac.uk 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 (P ydhY ) 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–P ydhY 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 . Abbreviations: MBP, maltose-binding protein; RACE, random amplification of cDNA ends