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. 1993 Feb;175(4):1153–1164. doi: 10.1128/jb.175.4.1153-1164.1993

Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor.

D W Martin 1, B W Holloway 1, V Deretic 1
PMCID: PMC193032  PMID: 8432708

Abstract

Overproduction of the exopolysaccharide alginate by Pseudomonas aeruginosa results in mucoid colony morphology and is an important virulence determinant expressed by this organism in cystic fibrosis. Mucoidy is transcriptionally regulated by signal transduction systems and histone-like elements. One point of convergence of regulatory elements controlling mucoidy is the algD promoter. A newly described genetic locus required for algD transcription was characterized in this study. This DNA region, cloned from a nonmucoid PAO strain, was initially isolated on the basis of its ability to suppress mucoidy when present on a plasmid. The suppressing activity was observed in several mucoid PAO derivatives, including strain PAO568, in which the mapped muc-2 mutation is responsible for its mucoid phenotype, and in close to 40% of cystic fibrosis strains tested. Protein expression studies detected two polypeptides with apparent molecular masses of 27.5 and 20 kDa encoded by the region required for the suppression activity. The gene encoding the polypeptide with an apparent molecular mass of 27.5 kDa, termed algU, was further characterized. A functional chromosomal copy of algU was found to be necessary for the expression of mucoidy. Insertional inactivation of algU on the chromosome of the mucoid strain PAO568 abrogated alginate production and algD transcription. DNA sequence analysis revealed sequence similarity of the predicted algU gene product with sigma H (Spo0H), a sigma factor involved in the control of sporulation and competence in Bacillus spp. Physical mapping revealed that algU resided on the same SpeI fragment (F) as did the pruAB locus, known to be tightly linked with genetic determinants (muc) which can confer mucoidy in genetic crosses. When the chromosomal algU copy was tagged with a Tcr cassette (algU::Tcr), a tight genetic linkage of algU with pruAB was demonstrated by F116L-mediated generalized transduction. Moreover, algU::Tcr derivatives of PAO568 (originally carrying the muc-2 marker) lost the ability to transfer mucoidy in genetic crosses. These results suggest that algU, a regulator of algD transcription showing sequence similarity to an alternative sigma factor, and the genes immediately downstream of algU may be associated with a locus participating in the differentiation into the mucoid phenotype.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen L. N., Hanson R. S. Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol. J Bacteriol. 1985 Mar;161(3):955–962. doi: 10.1128/jb.161.3.955-962.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anwar H., Strap J. L., Costerton J. W. Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antimicrob Agents Chemother. 1992 Jul;36(7):1347–1351. doi: 10.1128/aac.36.7.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barry C. E., 3rd, Hayes S. F., Hackstadt T. Nucleoid condensation in Escherichia coli that express a chlamydial histone homolog. Science. 1992 Apr 17;256(5055):377–379. doi: 10.1126/science.256.5055.377. [DOI] [PubMed] [Google Scholar]
  4. Carter H. L., 3rd, Moran C. P., Jr New RNA polymerase sigma factor under spo0 control in Bacillus subtilis. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9438–9442. doi: 10.1073/pnas.83.24.9438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chater K. F., Bruton C. J., Plaskitt K. A., Buttner M. J., Méndez C., Helmann J. D. The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B. subtilis. Cell. 1989 Oct 6;59(1):133–143. doi: 10.1016/0092-8674(89)90876-3. [DOI] [PubMed] [Google Scholar]
  6. Costerton J. W., Cheng K. J., Geesey G. G., Ladd T. I., Nickel J. C., Dasgupta M., Marrie T. J. Bacterial biofilms in nature and disease. Annu Rev Microbiol. 1987;41:435–464. doi: 10.1146/annurev.mi.41.100187.002251. [DOI] [PubMed] [Google Scholar]
  7. Costerton J. W., Lam J., Lam K., Chan R. The role of the microcolony mode of growth in the pathogenesis of Pseudomonas aeruginosa infections. Rev Infect Dis. 1983 Nov-Dec;5 (Suppl 5):S867–S873. doi: 10.1093/clinids/5.supplement_5.s867. [DOI] [PubMed] [Google Scholar]
  8. Deretic V., Chandrasekharappa S., Gill J. F., Chatterjee D. K., Chakrabarty A. M. A set of cassettes and improved vectors for genetic and biochemical characterization of Pseudomonas genes. Gene. 1987;57(1):61–72. doi: 10.1016/0378-1119(87)90177-6. [DOI] [PubMed] [Google Scholar]
  9. Deretic V., Dikshit R., Konyecsni W. M., Chakrabarty A. M., Misra T. K. 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. doi: 10.1128/jb.171.3.1278-1283.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deretic V., Gill J. F., Chakrabarty A. M. Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa. J Bacteriol. 1987 Jan;169(1):351–358. doi: 10.1128/jb.169.1.351-358.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deretic V., Govan J. R., Konyecsni W. M., Martin D. W. Mucoid Pseudomonas aeruginosa in cystic fibrosis: mutations in the muc loci affect transcription of the algR and algD genes in response to environmental stimuli. Mol Microbiol. 1990 Feb;4(2):189–196. doi: 10.1111/j.1365-2958.1990.tb00586.x. [DOI] [PubMed] [Google Scholar]
  12. Deretic V., Hibler N. S., Holt S. C. Immunocytochemical analysis of AlgP (Hp1), a histonelike element participating in control of mucoidy in Pseudomonas aeruginosa. J Bacteriol. 1992 Feb;174(3):824–831. doi: 10.1128/jb.174.3.824-831.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Deretic V., Konyecsni W. M. A procaryotic regulatory factor with a histone H1-like carboxy-terminal domain: clonal variation of repeats within algP, a gene involved in regulation of mucoidy in Pseudomonas aeruginosa. J Bacteriol. 1990 Oct;172(10):5544–5554. doi: 10.1128/jb.172.10.5544-5554.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Deretic V., Konyecsni W. M. 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. doi: 10.1128/jb.171.7.3680-3688.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Deretic V., Leveau J. H., Mohr C. D., Hibler N. S. In vitro phosphorylation of AlgR, a regulator of mucoidy in Pseudomonas aeruginosa, by a histidine protein kinase and effects of small phospho-donor molecules. Mol Microbiol. 1992 Oct;6(19):2761–2767. doi: 10.1111/j.1365-2958.1992.tb01455.x. [DOI] [PubMed] [Google Scholar]
  16. Deretic V., Mohr C. D., Martin D. W. Mucoid Pseudomonas aeruginosa in cystic fibrosis: signal transduction and histone-like elements in the regulation of bacterial virulence. Mol Microbiol. 1991 Jul;5(7):1577–1583. doi: 10.1111/j.1365-2958.1991.tb01903.x. [DOI] [PubMed] [Google Scholar]
  17. Dubnau D. The regulation of genetic competence in Bacillus subtilis. Mol Microbiol. 1991 Jan;5(1):11–18. doi: 10.1111/j.1365-2958.1991.tb01820.x. [DOI] [PubMed] [Google Scholar]
  18. Dubnau E., Weir J., Nair G., Carter L., 3rd, Moran C., Jr, Smith I. Bacillus sporulation gene spo0H codes for sigma 30 (sigma H). J Bacteriol. 1988 Mar;170(3):1054–1062. doi: 10.1128/jb.170.3.1054-1062.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Figurski D. H., Helinski D. R. 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. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Flynn J. L., Ohman D. E. Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype. J Bacteriol. 1988 Apr;170(4):1452–1460. doi: 10.1128/jb.170.4.1452-1460.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fyfe J. A., Govan J. R. Alginate synthesis in mucoid Pseudomonas aeruginosa: a chromosomal locus involved in control. J Gen Microbiol. 1980 Aug;119(2):443–450. doi: 10.1099/00221287-119-2-443. [DOI] [PubMed] [Google Scholar]
  22. Goldberg J. B., Won J., Ohman D. E. Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa. J Gen Microbiol. 1990 May;136(5):789–796. doi: 10.1099/00221287-136-5-789. [DOI] [PubMed] [Google Scholar]
  23. Govan J. R., Harris G. S. Pseudomonas aeruginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis. Microbiol Sci. 1986 Oct;3(10):302–308. [PubMed] [Google Scholar]
  24. HOLLOWAY B. W. Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol. 1955 Dec;13(3):572–581. doi: 10.1099/00221287-13-3-572. [DOI] [PubMed] [Google Scholar]
  25. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  26. Hindahl M. S., Frank D. W., Hamood A., Iglewski B. H. Characterization of a gene that regulates toxin A synthesis in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Jun 24;16(12):5699–5699. doi: 10.1093/nar/16.12.5699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ishimoto K. S., Lory S. Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1954–1957. doi: 10.1073/pnas.86.6.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kato J., Chakrabarty A. M. 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. doi: 10.1073/pnas.88.5.1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kato J., Misra T. K., Chakrabarty A. M. AlgR3, a protein resembling eukaryotic histone H1, regulates alginate synthesis in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2887–2891. doi: 10.1073/pnas.87.8.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kimbara K., Chakrabarty A. M. Control of alginate synthesis in Pseudomonas aeruginosa: regulation of the algR1 gene. Biochem Biophys Res Commun. 1989 Oct 31;164(2):601–608. doi: 10.1016/0006-291x(89)91502-7. [DOI] [PubMed] [Google Scholar]
  31. Knutson C. A., Jeanes A. A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem. 1968 Sep;24(3):470–481. doi: 10.1016/0003-2697(68)90154-1. [DOI] [PubMed] [Google Scholar]
  32. Konyecsni W. M., Deretic V. Broad-host-range plasmid and M13 bacteriophage-derived vectors for promoter analysis in Escherichia coli and Pseudomonas aeruginosa. Gene. 1988 Dec 30;74(2):375–386. doi: 10.1016/0378-1119(88)90171-0. [DOI] [PubMed] [Google Scholar]
  33. Konyecsni W. M., 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. doi: 10.1128/jb.172.5.2511-2520.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Krieg D. P., Helmke R. J., German V. F., Mangos J. A. Resistance of mucoid Pseudomonas aeruginosa to nonopsonic phagocytosis by alveolar macrophages in vitro. Infect Immun. 1988 Dec;56(12):3173–3179. doi: 10.1128/iai.56.12.3173-3179.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Krishnapillai V. A novel transducing phage. Its role in recognition of a possible new host-controlled modification system in Pseudomonas aeruginosa. Mol Gen Genet. 1972;114(2):134–143. doi: 10.1007/BF00332784. [DOI] [PubMed] [Google Scholar]
  36. Lonetto M., Gribskov M., Gross C. A. The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol. 1992 Jun;174(12):3843–3849. doi: 10.1128/jb.174.12.3843-3849.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Losick R., Youngman P., Piggot P. J. Genetics of endospore formation in Bacillus subtilis. Annu Rev Genet. 1986;20:625–669. doi: 10.1146/annurev.ge.20.120186.003205. [DOI] [PubMed] [Google Scholar]
  38. MacGeorge J., Korolik V., Morgan A. F., Asche V., Holloway B. W. Transfer of a chromosomal locus responsible for mucoid colony morphology in Pseudomonas aeruginosa isolated from cystic fibrosis patients to P. aeruginosa PAO. J Med Microbiol. 1986 Jun;21(4):331–336. doi: 10.1099/00222615-21-4-331. [DOI] [PubMed] [Google Scholar]
  39. Meile L., Soldati L., Leisinger T. Regulation of proline catabolism in Pseudomonas aeruginosa PAO. Arch Microbiol. 1982 Aug;132(2):189–193. doi: 10.1007/BF00508729. [DOI] [PubMed] [Google Scholar]
  40. Merrick M. J., Gibbins J. R. The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. Nucleic Acids Res. 1985 Nov 11;13(21):7607–7620. doi: 10.1093/nar/13.21.7607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Miller J. F., Mekalanos J. J., Falkow S. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science. 1989 Feb 17;243(4893):916–922. doi: 10.1126/science.2537530. [DOI] [PubMed] [Google Scholar]
  42. Mohr C. D., Deretic V. Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa. J Bacteriol. 1990 Nov;172(11):6252–6260. doi: 10.1128/jb.172.11.6252-6260.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mohr C. D., Hibler N. S., 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. doi: 10.1128/jb.173.16.5136-5143.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mohr C. D., Leveau J. H., Krieg D. P., Hibler N. S., Deretic V. AlgR-binding sites within the algD promoter make up a set of inverted repeats separated by a large intervening segment of DNA. J Bacteriol. 1992 Oct;174(20):6624–6633. doi: 10.1128/jb.174.20.6624-6633.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mohr C. D., Martin D. W., Konyecsni W. M., Govan J. R., Lory S., Deretic V. Role of the far-upstream sites of the algD promoter and the algR and rpoN genes in environmental modulation of mucoidy in Pseudomonas aeruginosa. J Bacteriol. 1990 Nov;172(11):6576–6580. doi: 10.1128/jb.172.11.6576-6580.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Predich M., Nair G., Smith I. Bacillus subtilis early sporulation genes kinA, spo0F, and spo0A are transcribed by the RNA polymerase containing sigma H. J Bacteriol. 1992 May;174(9):2771–2778. doi: 10.1128/jb.174.9.2771-2778.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rappuoli R., Aricó B., Scarlato V. Thermoregulation and reversible differentiation in Bordetella: a model for pathogenic bacteria. Mol Microbiol. 1992 Aug;6(15):2209–2211. doi: 10.1111/j.1365-2958.1992.tb01395.x. [DOI] [PubMed] [Google Scholar]
  49. Ratnaningsih E., Dharmsthiti S., Krishnapillai V., Morgan A., Sinclair M., Holloway B. W. A combined physical and genetic map of Pseudomonas aeruginosa PAO. J Gen Microbiol. 1990 Dec;136(12):2351–2357. doi: 10.1099/00221287-136-12-2351. [DOI] [PubMed] [Google Scholar]
  50. Sadoff H. L. Encystment and germination in Azotobacter vinelandii. Bacteriol Rev. 1975 Dec;39(4):516–539. doi: 10.1128/br.39.4.516-539.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shortridge V. D., Pato M. L., Vasil A. I., Vasil M. L. Physical mapping of virulence-associated genes in Pseudomonas aeruginosa by transverse alternating-field electrophoresis. Infect Immun. 1991 Oct;59(10):3596–3603. doi: 10.1128/iai.59.10.3596-3603.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tatti K. M., Carter H. L., 3rd, Moir A., Moran C. P., Jr Sigma H-directed transcription of citG in Bacillus subtilis. J Bacteriol. 1989 Nov;171(11):5928–5932. doi: 10.1128/jb.171.11.5928-5932.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. West S. E., Iglewski B. H. Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Oct 11;16(19):9323–9335. doi: 10.1093/nar/16.19.9323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wozniak D. J., Ohman D. E. 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. doi: 10.1128/jb.173.4.1406-1413.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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