title: Sigma factor N, liaison to an ntrC and rpoS dependent regulatory pathway controlling acid resistance and the LEE in enterohemorrhagic Escherichia coli.
abstract: Enterohemorrhagic Escherichia coli (EHEC) is dependent on acid resistance for gastric passage and low oral infectious dose, and the locus of enterocyte effacement (LEE) for intestinal colonization. Mutation of rpoN, encoding sigma factor N (σ(N)), dramatically alters the growth-phase dependent regulation of both acid resistance and the LEE. This study reports on the determinants of σ(N)-directed acid resistance and LEE expression, and the underlying mechanism attributable to this phenotype. Glutamate-dependent acid resistance (GDAR) in TW14359ΔrpoN correlated with increased expression of the gadX-gadW regulatory circuit during exponential growth, whereas upregulation of arginine-dependent acid resistance (ADAR) genes adiA and adiC in TW14359ΔrpoN did not confer acid resistance by the ADAR mechanism. LEE regulatory (ler), structural (espA and cesT) and effector (tir) genes were downregulated in TW14359ΔrpoN, and mutation of rpoS encoding sigma factor 38 (σ(S)) in TW14359ΔrpoN restored acid resistance and LEE genes to WT levels. Stability, but not the absolute level, of σ(S) was increased in TW14359ΔrpoN; however, increased stability was not solely attributable to the GDAR and LEE expression phenotype. Complementation of TW14359ΔrpoN with a σ(N) allele that binds RNA polymerase (RNAP) but not DNA, did not restore WT levels of σ(S) stability, gadE, ler or GDAR, indicating a dependence on transcription from a σ(N) promoter(s) and not RNAP competition for the phenotype. Among a library of σ(N) enhancer binding protein mutants, only TW14359ΔntrC, inactivated for nitrogen regulatory protein NtrC, phenocopied TW14359ΔrpoN for σ(S) stability, GDAR and ler expression. The results of this study suggest that during exponential growth, NtrC-σ(N) regulate GDAR and LEE expression through downregulation of σ(S) at the post-translational level; likely by altering σ(S) stability or activity. The regulatory interplay between NtrC, other EBPs, and σ(N)-σ(S), represents a mechanism by which EHEC can coordinate GDAR, LEE expression and other cellular functions, with nitrogen availability and physiologic stimuli.
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology (CMMB), University of South Florida, Tampa, Florida, United States of America.
authors.forename: Avishek
authors.initials: A
authors.lastname: Mitra
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Fay
authors.forename: Jason K
authors.initials: JK
authors.lastname: Morgan
authors.forename: Khoury W
authors.initials: KW
authors.lastname: Vendura
authors.forename: Salvatore L
authors.initials: SL
authors.lastname: Versaggi
authors.forename: James T
authors.initials: JT
authors.lastname: Riordan
mesh_terms: D000143:Acids; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism; D017920:Antiporters / Q000235:genetics / Q000378:metabolism; D051777:AraC Transcription Factor / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D020895:Enterocytes / Q000382:microbiology; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54 / Q000235:genetics* / Q000378:metabolism; D012808:Sigma Factor / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D000143:Acids; C476528:AdiC protein, E coli; D026905:Amino Acid Transport Systems; D017920:Antiporters; D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C474648:GadW protein, E coli; C446316:GadX protein, E coli; D051922:PII Nitrogen Regulatory Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C497876:glnG protein, E coli; C497751:rpoN protein, E coli; C069567:sigma factor KatF protein, Bacteria; D018698:Glutamic Acid; D001120:Arginine; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D002262:Carboxy-Lyases; C019722:arginine decarboxylase; D009584:Nitrogen