title: HlyX, the FNR homologue of Actinobacillus pleuropneumoniae, is a [4Fe-4S]-containing oxygen-responsive transcription regulator that anaerobically activates FNR-dependent class I promoters via an enhanced AR1 contact.
abstract: The hlyX gene of the pig pathogen Actinobacillus pleuropneumoniae encodes HlyX, a homologue of FNR, the anaerobic transcription regulator of Escherichia coli. The hlyX gone complements the anaerobic respiratory deficiencies of E. coli fnr mutants but also induces the expression of an otherwise latent haemolysin. Therefore, FNR and HlyX have distinct but overlapping regulons. The hlyX gene has been overexpressed as a gst::hlyX fusion and the HlyX protein purified. Similar to FNR, HlyX can acquire a [4Fe-4S] cluster, which promotes binding to the FNR box (Kd of 20-30 nM) under anaerobic conditions. Expression of hlyX in E. coli induced the anaerobic production of at least five polypeptides, including the yfiD gene product, which were not induced by fnr. Analysis of the yfiD promoter region revealed the presence of two FNR boxes situated at -61.5 and -114.5. Consistent with this observation, expression from the semi-synthetic Class I promoter FF + 20pmelR was efficiently activated by HlyX but not by FNR. The weaker level of FNR-mediated activation of Class I promoters suggests that there is a poorer activating contact (activating region 1 (AR1) equivalent) between FNR and RNA polymerase at these promoters and that HlyX possesses an additional or improved AR1. The AR1 of HlyX is partially characterized by a surface-exposed region around amino acid A187, which confers the altered specificity and provides an explanation for the existence of distinct but overlapping HlyX and FNR regulons.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, UK. jeff,green@sheffield.ac.uk
authors.forename: J
authors.initials: J
authors.lastname: Green
authors.forename: M L
authors.initials: ML
authors.lastname: Baldwin
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D018085:Regulon; D013552:Swine; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; C087924:HlyX protein, Actinobacillus pleuropneumoniae; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors