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. 1996 Apr;178(7):1850–1857. doi: 10.1128/jb.178.7.1850-1857.1996

RfaH enhances elongation of Escherichia coli hlyCABD mRNA.

J A Leeds 1, R A Welch 1
PMCID: PMC177878  PMID: 8606157

Abstract

Escherichia coli hlyCABD operons encode the polypeptide component (Hly A) of an extracellular cytolytic toxin, as well as proteins required for its acylation (HlyC) and sec-independent secretion (HlyBD). Previous reports suggested that the E. coli protein RfaH is required for wild-type hemolysin expression, either by positively activating hly transcript initiation (M. J. A. Bailey, V. Koronakis, T. Schmoll, and C. Hughes, Mol. Microbiol. 6:1003-1012, 1992) or by promoting proper insertion of hemolysin export machinery in the E. coli outer membrane (C. Wandersman and S. Letoffe, Mol. Microbiol. 7:141-150, 1993). RfaH is also required for wild-type levels of mRNA transcribed from promoter-distal genes in the rfaQ-K, traY-Z, and rplK-rpoC gene clusters, suggesting that RfaH is a transcriptional antiterminator. We tested these models by analyzing the effects of rfaH mutations on hlyCABD mRNA synthesis and decay, HlyA protein levels, and hemolytic activity. The model system included a uropathogenic strain of E. coli harboring hlyCABD on the chromosome and E. coli K-12 transformed with the hlyCABD operon on a recombinant plasmid. Our results suggest that RfaH enhances hlyCABD transcript elongation, consistent with the model of RfaH involvement in transcriptional antitermination in E. coli. We also demonstrated that RfaH increases toxin efficacy. Modulation of hemolysin activity may be an indirect effect of RfaH-dependent E. coli outer membrane chemotype, which is consistent with the model of lipopolysaccharide involvement in hemolytic activity.

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Selected References

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  1. Bailey M. J., Koronakis V., Schmoll T., Hughes C. Escherichia coli HlyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes. Mol Microbiol. 1992 Apr;6(8):1003–1012. doi: 10.1111/j.1365-2958.1992.tb02166.x. [DOI] [PubMed] [Google Scholar]
  2. Beutin L., Achtman M. Two Escherichia coli chromosomal cistrons, sfrA and sfrB, which are needed for expression of F factor tra functions. J Bacteriol. 1979 Sep;139(3):730–737. doi: 10.1128/jb.139.3.730-737.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beutin L., Manning P. A., Achtman M., Willetts N. sfrA and sfrB products of Escherichia coli K-12 are transcriptional control factors. J Bacteriol. 1981 Feb;145(2):840–844. doi: 10.1128/jb.145.2.840-844.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brazas R., Davie E., Farewell A., Rothfield L. I. Transcriptional organization of the rfaGBIJ locus of Salmonella typhimurium. J Bacteriol. 1991 Oct;173(19):6168–6173. doi: 10.1128/jb.173.19.6168-6173.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Choe M., Reznikoff W. S. Anaerobically expressed Escherichia coli genes identified by operon fusion techniques. J Bacteriol. 1991 Oct;173(19):6139–6146. doi: 10.1128/jb.173.19.6139-6146.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Creeger E. S., Schulte T., Rothfield L. I. Regulation of membrane glycosyltransferases by the sfrB and rfaH genes of Escherichia coli and Salmonella typhimurium. J Biol Chem. 1984 Mar 10;259(5):3064–3069. [PubMed] [Google Scholar]
  7. Cross M. A., Koronakis V., Stanley P. L., Hughes C. HlyB-dependent secretion of hemolysin by uropathogenic Escherichia coli requires conserved sequences flanking the chromosomal hly determinant. J Bacteriol. 1990 Mar;172(3):1217–1224. doi: 10.1128/jb.172.3.1217-1224.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Emory S. A., Belasco J. G. The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency. J Bacteriol. 1990 Aug;172(8):4472–4481. doi: 10.1128/jb.172.8.4472-4481.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Felmlee T., Pellett S., Lee E. Y., Welch R. A. Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide. J Bacteriol. 1985 Jul;163(1):88–93. doi: 10.1128/jb.163.1.88-93.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Felmlee T., Pellett S., Welch R. A. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol. 1985 Jul;163(1):94–105. doi: 10.1128/jb.163.1.94-105.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gaffney D., Skurray R., Willetts N. Regulation of the F conjugation genes studied by hybridization and tra-lacZ fusion. J Mol Biol. 1983 Jul 25;168(1):103–122. doi: 10.1016/s0022-2836(83)80325-8. [DOI] [PubMed] [Google Scholar]
  12. Hardie K. R., Issartel J. P., Koronakis E., Hughes C., Koronakis V. In vitro activation of Escherichia coli prohaemolysin to the mature membrane-targeted toxin requires HlyC and a low molecular-weight cytosolic polypeptide. Mol Microbiol. 1991 Jul;5(7):1669–1679. doi: 10.1111/j.1365-2958.1991.tb01914.x. [DOI] [PubMed] [Google Scholar]
  13. Hobbs M., Reeves P. R. The JUMPstart sequence: a 39 bp element common to several polysaccharide gene clusters. Mol Microbiol. 1994 Jun;12(5):855–856. doi: 10.1111/j.1365-2958.1994.tb01071.x. [DOI] [PubMed] [Google Scholar]
  14. Karls R., Schulz V., Jovanovich S. B., Flynn S., Pak A., Reznikoff W. S. Pseudorevertants of a lac promoter mutation reveal overlapping nascent promoters. Nucleic Acids Res. 1989 May 25;17(10):3927–3949. doi: 10.1093/nar/17.10.3927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knapp S., Then I., Wels W., Michel G., Tschäpe H., Hacker J., Goebel W. Analysis of the flanking regions from different haemolysin determinants of Escherichia coli. Mol Gen Genet. 1985;200(3):385–392. doi: 10.1007/BF00425721. [DOI] [PubMed] [Google Scholar]
  16. Koronakis V., Hughes C. Identification of the promoters directing in vivo expression of hemolysin genes in Proteus vulgaris and Escherichia coli. Mol Gen Genet. 1988 Jul;213(1):99–104. doi: 10.1007/BF00333404. [DOI] [PubMed] [Google Scholar]
  17. Müller D., Hughes C., Goebel W. Relationship between plasmid and chromosomal hemolysin determinants of Escherichia coli. J Bacteriol. 1983 Feb;153(2):846–851. doi: 10.1128/jb.153.2.846-851.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nicaud J. M., Mackman N., Gray L., Holland I. B. Characterisation of HlyC and mechanism of activation and secretion of haemolysin from E. coli 2001. FEBS Lett. 1985 Aug 5;187(2):339–344. doi: 10.1016/0014-5793(85)81272-2. [DOI] [PubMed] [Google Scholar]
  19. Pradel E., Schnaitman C. A. Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12. J Bacteriol. 1991 Oct;173(20):6428–6431. doi: 10.1128/jb.173.20.6428-6431.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ralling G., Linn T. Evidence that Rho and NusA are involved in termination in the rplL-rpoB intercistronic region. J Bacteriol. 1987 May;169(5):2277–2280. doi: 10.1128/jb.169.5.2277-2280.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rehemtulla A., Kadam S. K., Sanderson K. E. Cloning and analysis of the sfrB (sex factor repression) gene of Escherichia coli K-12. J Bacteriol. 1986 May;166(2):651–657. doi: 10.1128/jb.166.2.651-657.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roncero C., Casadaban M. J. Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus. J Bacteriol. 1992 May;174(10):3250–3260. doi: 10.1128/jb.174.10.3250-3260.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stanley P. L., Diaz P., Bailey M. J., Gygi D., Juarez A., Hughes C. Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly. Mol Microbiol. 1993 Nov;10(4):781–787. doi: 10.1111/j.1365-2958.1993.tb00948.x. [DOI] [PubMed] [Google Scholar]
  25. Stevens M. P., Hänfling P., Jann B., Jann K., Roberts I. S. Regulation of Escherichia coli K5 capsular polysaccharide expression: evidence for involvement of RfaH in the expression of group II capsules. FEMS Microbiol Lett. 1994 Nov 15;124(1):93–98. doi: 10.1111/j.1574-6968.1994.tb07267.x. [DOI] [PubMed] [Google Scholar]
  26. Vogel M., Hess J., Then I., Juarez A., Goebel W. Characterization of a sequence (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli. Mol Gen Genet. 1988 Apr;212(1):76–84. doi: 10.1007/BF00322447. [DOI] [PubMed] [Google Scholar]
  27. Wagner W., Vogel M., Goebel W. Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol. 1983 Apr;154(1):200–210. doi: 10.1128/jb.154.1.200-210.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wandersman C., Delepelaire P. TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4776–4780. doi: 10.1073/pnas.87.12.4776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wandersman C., Létoffé S. Involvement of lipopolysaccharide in the secretion of Escherichia coli alpha-haemolysin and Erwinia chrysanthemi proteases. Mol Microbiol. 1993 Jan;7(1):141–150. doi: 10.1111/j.1365-2958.1993.tb01105.x. [DOI] [PubMed] [Google Scholar]
  30. Welch R. A., Hull R., Falkow S. Molecular cloning and physical characterization of a chromosomal hemolysin from Escherichia coli. Infect Immun. 1983 Oct;42(1):178–186. doi: 10.1128/iai.42.1.178-186.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Welch R. A., Pellett S. Transcriptional organization of the Escherichia coli hemolysin genes. J Bacteriol. 1988 Apr;170(4):1622–1630. doi: 10.1128/jb.170.4.1622-1630.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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