The antiterminator RfaH is required for the expression of LPS, capsule, haemolysin, exotoxin, haemin uptake receptor and F pilus. As these structures are critical for bacterial virulence, loss of RfaH usually leads to attenuation. Here, we inactivated the gene of O:3 to study its role in this enteropathogen. RNA sequencing of the WT and Δ strain transcriptomes revealed that RfaH acted as a highly specific regulator that enhanced the transcription of the operons involved in biosynthesis of LPS O-antigen and outer core (OC), but did not affect the expression of enterobacterial common antigen. Interestingly, the transcriptome of the Δ strain was very similar to that of an O-antigen-negative mutant. This indicated that some of the changes seen in the Δ strain, such as the genes involved in outer membrane homeostasis or in the stress-response-associated Cpx pathway, were actually due to indirect responses via the loss of O-antigen. The decreased amount of LPS on the Δ strain cell surface resulted in an attenuated stress response, and lower resistance to compounds such as SDS and polymyxin B. However, the Δ strain was significantly more resistant to complement-mediated killing by normal human serum. Taken together, our results revealed a novel role of RfaH acting as a highly specific regulator of O-antigen and OC of LPS in O:3. It may be speculated that RfaH might have an role in controlling tissue-specific expression of bacterial surface oligo/polysaccharides.


Article metrics loading...

Loading full text...

Full text loading...



  1. Al-Hendy A. (1992). Lipopolysaccharide of Yersinia enterocolitica: molecular genetics and role in virulence PhD thesisTurku, FinlandUniversity of Turku [Google Scholar]
  2. Al-Hendy A., Toivanen P., Skurnik M. (1991a). Expression cloning of Yersinia enterocolitica O:3 rfb gene cluster in Escherichia coli K12. Microb Pathog 10, 4759 [View Article][PubMed] [Google Scholar]
  3. Al-Hendy A., Toivanen P., Skurnik M. (1991b). Rapid method for isolation and staining of bacterial lipopolysaccharide. Microbiol Immunol 35, 331333 [View Article][PubMed] [Google Scholar]
  4. Al-Hendy A., Toivanen P., Skurnik M. (1992). Lipopolysaccharide O side chain of Yersinia enterocolitica O:3 is an essential virulence factor in an orally infected murine model. Infect Immun 60, 870875[PubMed] [Google Scholar]
  5. Bailey M. J. A., Hughes C., Koronakis V. (1997). RfaH and the ops element, components of a novel system controlling bacterial transcription elongation. Mol Microbiol 26, 845851 [View Article][PubMed] [Google Scholar]
  6. Bailey M. J., Hughes C., Koronakis V. (2000). In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element. Mol Gen Genet 262, 10521059 [View Article][PubMed] [Google Scholar]
  7. Bengoechea J. A., Pinta E., Salminen T., Oertelt C., Holst O., Radziejewska-Lebrecht J., Piotrowska-Seget Z., Venho R., Skurnik M. (2002). Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8. J Bacteriol 184, 42774287 [View Article][PubMed] [Google Scholar]
  8. Bengoechea J. A., Najdenski H., Skurnik M. (2004). Lipopolysaccharide O antigen status of Yersinia enterocolitica O:8 is essential for virulence and absence of O antigen affects the expression of other Yersinia virulence factors. Mol Microbiol 52, 451469 [View Article][PubMed] [Google Scholar]
  9. Bertani G. (2004). Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriol 186, 595600 [View Article][PubMed] [Google Scholar]
  10. Biedzka-Sarek M., Venho R., Skurnik M. (2005). Role of YadA, Ail, and lipopolysaccharide in serum resistance of Yersinia enterocolitica serotype O:3. Infect Immun 73, 22322244 [View Article][PubMed] [Google Scholar]
  11. Brill W. J., Magasanik B. (1969). Genetic and metabolic control of histidase and urocanase in Salmonella typhimurium, strain 15-59. J Biol Chem 244, 53925402[PubMed] [Google Scholar]
  12. Burmann B. M., Schweimer K., Luo X., Wahl M. C., Stitt B. L., Gottesman M. E., Rösch P. (2010). A NusE : NusG complex links transcription and translation. Science 328, 501504 [View Article][PubMed] [Google Scholar]
  13. Burmann B. M., Knauer S. H., Sevostyanova A., Schweimer K., Mooney R. A., Landick R., Artsimovitch I., Rösch P. (2012). An α helix to β barrel domain switch transforms the transcription factor RfaH into a translation factor. Cell 150, 291303 [View Article][PubMed] [Google Scholar]
  14. Carter H. D., Svetlov V., Artsimovitch I. (2004). Highly divergent RfaH orthologs from pathogenic proteobacteria can substitute for Escherichia coli RfaH both in vivo and in vitro . J Bacteriol 186, 28292840 [View Article][PubMed] [Google Scholar]
  15. Cimini D., De Rosa M., Carlino E., Ruggiero A., Schiraldi C. (2013). Homologous overexpression of RfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide. Microb Cell Fact 12, 46 [View Article][PubMed] [Google Scholar]
  16. Demarre G., Guérout A. M., Matsumoto-Mashimo C., Rowe-Magnus D. A., Marlière P., Mazel D. (2005). A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPalpha) conjugative machineries and their cognate Escherichia coli host strains. Res Microbiol 156, 245255 [View Article][PubMed] [Google Scholar]
  17. Hitchcock P. J., Brown T. M. (1983). Morphological heterogeneity amongst Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 154, 269277[PubMed] [Google Scholar]
  18. Hunke S., Keller R., Müller V. S. (2012). Signal integration by the Cpx-envelope stress system. FEMS Microbiol Lett 326, 1222 [View Article][PubMed] [Google Scholar]
  19. Kapatral V., Campbell J. W., Minnich S. A., Thomson N. R., Matsumura P., Prüss B. M. (2004). Gene array analysis of Yersinia enterocolitica FlhD and FlhC: regulation of enzymes affecting synthesis and degradation of carbamoylphosphate. Microbiology 150, 22892300 [View Article][PubMed] [Google Scholar]
  20. Korpela M. T., Kurittu J. S., Karvinen J. T., Karp M. T. (1998). A recombinant Escherichia coli sensor strain for the detection of tetracyclines. Anal Chem 70, 44574462 [View Article][PubMed] [Google Scholar]
  21. Krauss J. H., Weckesser J., Mayer H. (1988). Electrophoretic analysis of lipopolysaccharides of purple nonsulfur bacteria. Int J Syst Bacteriol 38, 157163 [View Article] [Google Scholar]
  22. Langmead B., Trapnell C., Pop M., Salzberg S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 [View Article][PubMed] [Google Scholar]
  23. Lindberg A. A., Hellerqvist C. -G. (1980). Rough mutants of Salmonella typhimurium: immunochemical and structural analysis of lipopolysaccharides from rfaH mutants. J Gen Microbiol 116, 2532[PubMed] [Google Scholar]
  24. Meier-Dieter U., Acker G., Mayer H. (1989). Detection of enterobacterial common antigen on bacterial cell surfaces by colony-immunoblotting: effect of its linkage to lipopolysaccharide. FEMS Microbiol Lett 59, 215219 [View Article][PubMed] [Google Scholar]
  25. Michiels T., Cornelis G. R. (1991). Secretion of hybrid proteins by the Yersinia Yop export system. J Bacteriol 173, 16771685[PubMed] [Google Scholar]
  26. Moores A., Chipper-Keating S., Sun L., McVicker G., Wales L., Gashi K., Blomfield I. C. (2014). RfaH suppresses small RNA MicA inhibition of fimB expression in Escherichia coli K-12. J Bacteriol 196, 148156 [View Article][PubMed] [Google Scholar]
  27. Morales V. M., Bäckman A., Bagdasarian M. (1991). A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97, 3947 [View Article][PubMed] [Google Scholar]
  28. Muszyński A., Rabsztyn K., Knapska K., Duda K. A., Duda-Grychtoł K., Kasperkiewicz K., Radziejewska-Lebrecht J., Holst O., Skurnik M. (2013). Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O:3. Microbiology 159, 17821793 [View Article][PubMed] [Google Scholar]
  29. Nagy G., Dobrindt U., Schneider G., Khan A. S., Hacker J., Emödy L. (2002). Loss of regulatory protein RfaH attenuates virulence of uropathogenic Escherichia coli . Infect Immun 70, 44064413 [View Article][PubMed] [Google Scholar]
  30. Nagy G., Danino V., Dobrindt U., Pallen M., Chaudhuri R., Emödy L., Hinton J. C., Hacker J. (2006). Down-regulation of key virulence factors makes the Salmonella enterica serovar Typhimurium rfaH mutant a promising live-attenuated vaccine candidate. Infect Immun 74, 59145925 [View Article][PubMed] [Google Scholar]
  31. NandyMazumdar M., Artsimovitch I. (2015). Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins – shifting shapes and paradigms. Bioessays 37, 324334 [View Article][PubMed] [Google Scholar]
  32. Pajunen M. I., Kiljunen S. J., Söderholm M. -E. L., Skurnik M. (2001). Complete genomic sequence of the lytic bacteriophage φYeO3-12 of Yersinia enterocolitica serotype O:3. J Bacteriol 183, 19281937 [View Article][PubMed] [Google Scholar]
  33. Parada J. L., Magasanik B. (1975). Expression of the hut operons of Salmonella typhimurium in Klebsiella aerogenes and in Escherichia coli . J Bacteriol 124, 12631268[PubMed] [Google Scholar]
  34. Pekkola-Heino K., Viljanen M. K., Ståhlberg T. H., Granfors K., Toivanen A. (1987). Monoclonal antibodies reacting selectively with core and O-polysaccharide of Yersinia enterocolitica O:3 lipopolysaccharide. Acta Pathol Microbiol Immunol Scand [C] 95, 2734[PubMed] [Google Scholar]
  35. Pinta E., Duda K. A., Hanuszkiewicz A., Salminen T. A., Bengoechea J. A., Hyytiäinen H., Lindner B., Radziejewska-Lebrecht J., Holst O., Skurnik M. (2010). Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core. J Biol Chem 285, 2833328342 [View Article][PubMed] [Google Scholar]
  36. Pinta E., Li Z., Batzilla J., Pajunen M., Kasanen T., Rabsztyn K., Rakin A., Skurnik M. (2012). Identification of three oligo-/polysaccharide-specific ligases in Yersinia enterocolitica . Mol Microbiol 83, 125136 [View Article][PubMed] [Google Scholar]
  37. Reinés M., Llobet E., Llompart C. M., Moranta D., Pérez-Gutiérrez C., Bengoechea J. A. (2012). Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides. J Bacteriol 194, 31733188 [View Article][PubMed] [Google Scholar]
  38. Robinson M. D., McCarthy D. J., Smyth G. K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139140 [View Article][PubMed] [Google Scholar]
  39. Skurnik M. (1984). Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis . J Appl Bacteriol 56, 355363 [View Article][PubMed] [Google Scholar]
  40. Skurnik M. (1995). Role of YadA in Yersinia-enterocolitica-induced reactive arthritis: a hypothesis. Trends Microbiol 3, 318319 [View Article][PubMed] [Google Scholar]
  41. Skurnik M., Bengoechea J. A. (2003). The biosynthesis and biological role of lipopolysaccharide O-antigens of pathogenic Yersiniae . Carbohydr Res 338, 25212529 [View Article][PubMed] [Google Scholar]
  42. Skurnik M., Venho R., Toivanen P., al-Hendy A. (1995). A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis. Mol Microbiol 17, 575594 [View Article][PubMed] [Google Scholar]
  43. Skurnik M., Venho R., Bengoechea J. -A., Moriyón I. (1999). The lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3 is required for virulence and plays a role in outer membrane integrity. Mol Microbiol 31, 14431462 [View Article][PubMed] [Google Scholar]
  44. Skurnik M., Hyytiäinen H. J., Happonen L. J., Kiljunen S., Datta N., Mattinen L., Williamson K., Kristo P., Szeliga M., other authors. (2012). Characterization of the genome, proteome, and structure of yersiniophage φR1-37. J Virol 86, 1262512642 [View Article][PubMed] [Google Scholar]
  45. Trapnell C., Hendrickson D. G., Sauvageau M., Goff L., Rinn J. L., Pachter L. (2013). Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31, 4653 [View Article][PubMed] [Google Scholar]
  46. Uliczka F., Pisano F., Schaake J., Stolz T., Rohde M., Fruth A., Strauch E., Skurnik M., Batzilla J., other authors. (2011). Unique cell adhesion and invasion properties of Yersinia enterocolitica O:3, the most frequent cause of human Yersiniosis. PLoS Pathog 7, e1002117.doi:10.1371/journal.ppat. 7, e1002117 [View Article][PubMed] [Google Scholar]
  47. Varjosalo M., Keskitalo S., Van Drogen A., Nurkkala H., Vichalkovski A., Aebersold R., Gstaiger M. (2013). The protein interaction landscape of the human CMGC kinase group. Cell Rep 3, 13061320 [View Article][PubMed] [Google Scholar]
  48. Vogt S. L., Raivio T. L. (2012). Just scratching the surface: an expanding view of the Cpx envelope stress response. FEMS Microbiol Lett 326, 211 [View Article][PubMed] [Google Scholar]
  49. Yethon J. A., Vinogradov E., Perry M. B., Whitfield C. (2000). Mutation of the lipopolysaccharide core glycosyltransferase encoded by waaG destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation. J Bacteriol 182, 56205623 [View Article][PubMed] [Google Scholar]
  50. Zhang L., Skurnik M. (1994). Isolation of an R M+ mutant of Yersinia enterocolitica serotype O:8 and its application in construction of rough mutants utilizing mini-Tn5 derivatives and lipopolysaccharide-specific phage. J Bacteriol 176, 17561760[PubMed] [Google Scholar]
  51. Zhang L., Al-Hendy A., Toivanen P., Skurnik M. (1993). Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-l-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems. Mol Microbiol 9, 309321 [View Article][PubMed] [Google Scholar]
  52. Zhang L., Radziejewska-Lebrecht J., Krajewska-Pietrasik D., Toivanen P., Skurnik M. (1997). Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8. Mol Microbiol 23, 6376 [View Article][PubMed] [Google Scholar]
  53. Zhang G., Meredith T. C., Kahne D. (2013). On the essentiality of lipopolysaccharide to Gram-negative bacteria. Curr Opin Microbiol 16, 779785 [View Article][PubMed] [Google Scholar]

Data & Media loading...


Supplementary Data


Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error