title: Disruption of rcsB by a duplicated sequence in a curli-producing Escherichia coli O157:H7 results in differential gene expression in relation to biofilm formation, stress responses and metabolism.
abstract: Escherichia coli O157:H7 (O157) strain 86-24, linked to a 1986 disease outbreak, displays curli- and biofilm-negative phenotypes that are correlated with the lack of Congo red (CR) binding and formation of white colonies (CR-) on a CR-containing medium. However, on a CR medium this strain produces red isolates (CR+) capable of producing curli fimbriae and biofilms.
  
  
  
  To identify genes controlling differential expression of curli fimbriae and biofilm formation, the RNA-Seq profile of a CR+isolate was compared to the CR-parental isolate. Of the 242 genes expressed differentially in the CR+isolate, 201 genes encoded proteins of known functions while the remaining 41 encoded hypothetical proteins. Among the genes with known functions, 149 were down- and 52 were up-regulated. Some of the upregulated genes were linked to biofilm formation through biosynthesis of curli fimbriae and flagella. The genes encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD, BssR and FlhDC, which modulate biofilm formation, were significantly altered in their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH, htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB-phoR and pst) response pathways, and amino acid metabolism were downregulated in the CR+isolate. Many genes mediating acid resistance and colanic acid biosynthesis, which influence biofilm formation directly or indirectly, were also down-regulated. Comparative genomics of CR+and CR-isolates revealed the presence of a short duplicated sequence in the rcsB gene of the CR+isolate. The alignment of the amino acid sequences of RcsB of the two isolates showed truncation of RcsB in the CR+isolate at the insertion site of the duplicated sequence. Complementation of CR+isolate with rcsB of the CR-parent restored parental phenotypes to the CR+isolate.
  
  
  
  The results of this study indicate that RcsB is a global regulator affecting bacterial survival in growth-restrictive environments through upregulation of genes promoting biofilm formation while downregulating certain metabolic functions. Understanding whether rcsB inactivation enhances persistence and survival of O157 in carrier animals and the environment would be important in developing strategies for controlling this bacterial pathogen in these niches.
authors.affiliation: Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, ARS-USDA, P. O. Box 70, 1920 Dayton Avenue, Ames, IA, 50010, USA. vijay.sharma@ars.usda.gov.
authors.forename: V K
authors.initials: VK
authors.lastname: Sharma
authors.affiliation: Infectious Bacterial Diseases Research Unit, National Animal Disease Center, ARS-USDA, Ames, IA, 50010, USA.
authors.forename: D O
authors.initials: DO
authors.lastname: Bayles
authors.affiliation: Infectious Bacterial Diseases Research Unit, National Animal Disease Center, ARS-USDA, Ames, IA, 50010, USA.
authors.forename: D P
authors.initials: DP
authors.lastname: Alt
authors.affiliation: Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, ARS-USDA, P. O. Box 70, 1920 Dayton Avenue, Ames, IA, 50010, USA.
authors.forename: T
authors.initials: T
authors.lastname: Looft
authors.affiliation: Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, ARS-USDA, P. O. Box 70, 1920 Dayton Avenue, Ames, IA, 50010, USA.
authors.forename: B W
authors.initials: BW
authors.lastname: Brunelle
authors.affiliation: Microscopy Services Unit, National Animal Disease Center, ARS-USDA, Ames, IA, 50010, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Stasko
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001422:Bacterial Adhesion; D001483:Base Sequence; D018441:Biofilms / Q000254:growth & development*; D003224:Congo Red / Q000378:metabolism; D003470:Culture Media; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D015536:Down-Regulation; D019453:Escherichia coli O157 / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D006360:Heat-Shock Proteins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics; D046529:Microscopy, Electron, Transmission; D009997:Osmotic Pressure; D018384:Oxidative Stress; D010641:Phenotype; D011134:Polysaccharides / Q000096:biosynthesis / Q000235:genetics; D012329:RNA, Bacterial / Q000302:isolation & purification; D016415:Sequence Alignment; D013312:Stress, Physiological / Q000235:genetics*; D013315:Stress, Psychological / Q000235:genetics; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D000596:Amino Acids; C491730:CpxP protein, E coli; D003470:Culture Media; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011134:Polysaccharides; D012329:RNA, Bacterial; C489568:RcsB protein, E coli; D014157:Transcription Factors; D003224:Congo Red; C004275:colanic acid
keywords: Biofilms; Curli; EHEC; Stress signaling