• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Oct 1995; 177(19): 5670–5679.
PMCID: PMC177379

Multiple promoters and induction by heat shock of the gene encoding the alternative sigma factor AlgU (sigma E) which controls mucoidy in cystic fibrosis isolates of Pseudomonas aeruginosa.

Abstract

Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable with sigma E from Escherichia coli and Salmonella typhimurium. sigma E has been implicated in the expression of systems enhancing bacterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. aeruginosa from multiple promoters (P1 through P5) that fall into three categories: (i) the P1 and P3 promoters, which display strong similarity to the -35 and -10 canonical sequences of sigma E promoters and were found to be absolutely dependent on AlgU; (ii) the P2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tcr cells; and (iii) the transcripts corresponding to P4 and P5, which were not affected by inactivation of algU. Introduction of E. coli rpoE (encoding sigma E) or algU into P. aeruginosa algU::Tcr strains restored P1 and P3 transcription and brought the P2 signal back to the wild-type level. The AlgU-dependent promoters P1 and P3 were inducible by heat shock in wild-type nonmucoid P. aeruginosa PAO1. At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, two-dimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock. Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively, these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AlgU and, by extension, its homologs in other organisms (e.g., sigma E in S. typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions.

Full Text

The Full Text of this article is available as a PDF (736K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anastassiou ED, Mintzas AC, Kounavis C, Dimitracopoulos G. Alginate production by clinical nonmucoid Pseudomonas aeruginosa strains. J Clin Microbiol. 1987 Apr;25(4):656–659. [PMC free article] [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Bukau B. Regulation of the Escherichia coli heat-shock response. Mol Microbiol. 1993 Aug;9(4):671–680. [PubMed]
  • Darzins A, Wang SK, Vanags RI, Chakrabarty AM. Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa. J Bacteriol. 1985 Nov;164(2):516–524. [PMC free article] [PubMed]
  • Deretic V, Dikshit R, Konyecsni WM, Chakrabarty AM, Misra TK. The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, belongs to a class of environmentally responsive genes. J Bacteriol. 1989 Mar;171(3):1278–1283. [PMC free article] [PubMed]
  • Deretic V, Gill JF, Chakrabarty AM. Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa. J Bacteriol. 1987 Jan;169(1):351–358. [PMC free article] [PubMed]
  • Deretic V, Gill JF, Chakrabarty AM. Pseudomonas aeruginosa infection in cystic fibrosis: nucleotide sequence and transcriptional regulation of the algD gene. Nucleic Acids Res. 1987 Jun 11;15(11):4567–4581. [PMC free article] [PubMed]
  • Deretic V, Hibler NS, Holt SC. Immunocytochemical analysis of AlgP (Hp1), a histonelike element participating in control of mucoidy in Pseudomonas aeruginosa. J Bacteriol. 1992 Feb;174(3):824–831. [PMC free article] [PubMed]
  • Deretic V, Konyecsni WM. Control of mucoidy in Pseudomonas aeruginosa: transcriptional regulation of algR and identification of the second regulatory gene, algQ. J Bacteriol. 1989 Jul;171(7):3680–3688. [PMC free article] [PubMed]
  • Deretic V, Schurr MJ, Boucher JC, Martin DW. Conversion of Pseudomonas aeruginosa to mucoidy in cystic fibrosis: environmental stress and regulation of bacterial virulence by alternative sigma factors. J Bacteriol. 1994 May;176(10):2773–2780. [PMC free article] [PubMed]
  • DeVries CA, Ohman DE. Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation. J Bacteriol. 1994 Nov;176(21):6677–6687. [PMC free article] [PubMed]
  • Erickson JW, Gross CA. Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression. Genes Dev. 1989 Sep;3(9):1462–1471. [PubMed]
  • Erickson JW, Vaughn V, Walter WA, Neidhardt FC, Gross CA. Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene. Genes Dev. 1987 Jul;1(5):419–432. [PubMed]
  • Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. [PMC free article] [PubMed]
  • Flachmann R, Kunz N, Seifert J, Gütlich M, Wientjes FJ, Läufer A, Gassen HG. Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB. Eur J Biochem. 1988 Aug 1;175(2):221–228. [PubMed]
  • Fujiwara S, Zielinski NA, Chakrabarty AM. Enhancer-like activity of A1gR1-binding site in alginate gene activation: positional, orientational, and sequence specificity. J Bacteriol. 1993 Sep;175(17):5452–5459. [PMC free article] [PubMed]
  • Fyfe JA, Govan JR. Alginate synthesis in mucoid Pseudomonas aeruginosa: a chromosomal locus involved in control. J Gen Microbiol. 1980 Aug;119(2):443–450. [PubMed]
  • Gilligan PH. Microbiology of airway disease in patients with cystic fibrosis. Clin Microbiol Rev. 1991 Jan;4(1):35–51. [PMC free article] [PubMed]
  • Goldberg JB, Dahnke T. Pseudomonas aeruginosa AlgB, which modulates the expression of alginate, is a member of the NtrC subclass of prokaryotic regulators. Mol Microbiol. 1992 Jan;6(1):59–66. [PubMed]
  • Goldberg JB, Gorman WL, Flynn JL, Ohman DE. A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species. J Bacteriol. 1993 Mar;175(5):1303–1308. [PMC free article] [PubMed]
  • Grossman AD, Erickson JW, Gross CA. The htpR gene product of E. coli is a sigma factor for heat-shock promoters. Cell. 1984 Sep;38(2):383–390. [PubMed]
  • Helmann JD, Chamberlin MJ. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. [PubMed]
  • HOLLOWAY BW. Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol. 1955 Dec;13(3):572–581. [PubMed]
  • Johnson K, Charles I, Dougan G, Pickard D, O'Gaora P, Costa G, Ali T, Miller I, Hormaeche C. The role of a stress-response protein in Salmonella typhimurium virulence. Mol Microbiol. 1991 Feb;5(2):401–407. [PubMed]
  • Kato J, Chakrabarty AM. Purification of the regulatory protein AlgR1 and its binding in the far upstream region of the algD promoter in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1760–1764. [PMC free article] [PubMed]
  • Knutson CA, Jeanes A. A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem. 1968 Sep;24(3):470–481. [PubMed]
  • Konyecsni WM, Deretic V. DNA sequence and expression analysis of algP and algQ, components of the multigene system transcriptionally regulating mucoidy in Pseudomonas aeruginosa: algP contains multiple direct repeats. J Bacteriol. 1990 May;172(5):2511–2520. [PMC free article] [PubMed]
  • Lipinska B, Fayet O, Baird L, Georgopoulos C. Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures. J Bacteriol. 1989 Mar;171(3):1574–1584. [PMC free article] [PubMed]
  • Lipinska B, Sharma S, Georgopoulos C. Sequence analysis and regulation of the htrA gene of Escherichia coli: a sigma 32-independent mechanism of heat-inducible transcription. Nucleic Acids Res. 1988 Nov 11;16(21):10053–10067. [PMC free article] [PubMed]
  • Lonetto MA, Brown KL, Rudd KE, Buttner MJ. Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7573–7577. [PMC free article] [PubMed]
  • Lonetto M, Gribskov M, Gross CA. The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol. 1992 Jun;174(12):3843–3849. [PMC free article] [PubMed]
  • Martin DW, Holloway BW, Deretic V. Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor. J Bacteriol. 1993 Feb;175(4):1153–1164. [PMC free article] [PubMed]
  • Martin DW, Schurr MJ, Mudd MH, Deretic V. Differentiation of Pseudomonas aeruginosa into the alginate-producing form: inactivation of mucB causes conversion to mucoidy. Mol Microbiol. 1993 Aug;9(3):497–506. [PubMed]
  • Martin DW, Schurr MJ, Mudd MH, Govan JR, Holloway BW, Deretic V. Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8377–8381. [PMC free article] [PubMed]
  • Martin DW, Schurr MJ, Yu H, Deretic V. Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to sigma E and stress response. J Bacteriol. 1994 Nov;176(21):6688–6696. [PMC free article] [PubMed]
  • May TB, Shinabarger D, Maharaj R, Kato J, Chu L, DeVault JD, Roychoudhury S, Zielinski NA, Berry A, Rothmel RK, et al. Alginate synthesis by Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients. Clin Microbiol Rev. 1991 Apr;4(2):191–206. [PMC free article] [PubMed]
  • Mecsas J, Rouviere PE, Erickson JW, Donohue TJ, Gross CA. The activity of sigma E, an Escherichia coli heat-inducible sigma-factor, is modulated by expression of outer membrane proteins. Genes Dev. 1993 Dec;7(12B):2618–2628. [PubMed]
  • Mecsas J, Welch R, Erickson JW, Gross CA. Identification and characterization of an outer membrane protein, OmpX, in Escherichia coli that is homologous to a family of outer membrane proteins including Ail of Yersinia enterocolitica. J Bacteriol. 1995 Feb;177(3):799–804. [PMC free article] [PubMed]
  • Mohr CD, Hibler NS, Deretic V. AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site. J Bacteriol. 1991 Aug;173(16):5136–5143. [PMC free article] [PubMed]
  • Mohr CD, Sonsteby SK, Deretic V. The Pseudomonas aeruginosa homologs of hemC and hemD are linked to the gene encoding the regulator of mucoidy AlgR. Mol Gen Genet. 1994 Jan;242(2):177–184. [PubMed]
  • Nasu S, Wicks FD, Gholson RK. L-Aspartate oxidase, a newly discovered enzyme of Escherichia coli, is the B protein of quinolinate synthetase. J Biol Chem. 1982 Jan 25;257(2):626–632. [PubMed]
  • O'Farrell PZ, Goodman HM, O'Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. [PubMed]
  • Pier GB, Matthews WJ, Jr, Eardley DD. Immunochemical characterization of the mucoid exopolysaccharide of Pseudomonas aeruginosa. J Infect Dis. 1983 Mar;147(3):494–503. [PubMed]
  • Schurr MJ, Martin DW, Mudd MH, Deretic V. Gene cluster controlling conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa: functional analysis in a heterologous host and role in the instability of mucoidy. J Bacteriol. 1994 Jun;176(11):3375–3382. [PMC free article] [PubMed]
  • Schurr MJ, Martin DW, Mudd MH, Hibler NS, Boucher JC, Deretic V. The algD promoter: regulation of alginate production by Pseudomonas aeruginosa in cystic fibrosis. Cell Mol Biol Res. 1993;39(4):371–376. [PubMed]
  • Tam JP. Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5409–5413. [PMC free article] [PubMed]
  • Totten PA, Lara JC, Lory S. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J Bacteriol. 1990 Jan;172(1):389–396. [PMC free article] [PubMed]
  • Wang QP, Kaguni JM. A novel sigma factor is involved in expression of the rpoH gene of Escherichia coli. J Bacteriol. 1989 Aug;171(8):4248–4253. [PMC free article] [PubMed]
  • Wozniak DJ, Ohman DE. Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription. J Bacteriol. 1991 Feb;173(4):1406–1413. [PMC free article] [PubMed]
  • Yu H, Schurr MJ, Deretic V. Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E. coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P. aeruginosa. J Bacteriol. 1995 Jun;177(11):3259–3268. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...