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. 1994 Jun;176(11):3375–3382. doi: 10.1128/jb.176.11.3375-3382.1994

Gene cluster controlling conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa: functional analysis in a heterologous host and role in the instability of mucoidy.

M J Schurr 1, D W Martin 1, M H Mudd 1, V Deretic 1
PMCID: PMC205510  PMID: 8195094

Abstract

Conversion to mucoidy, caused by the overproduction of the exopolysaccharide alginate in laboratory and cystic fibrosis strains of Pseudomonas aeruginosa, can occur via frameshift or nonsense mutations in the second gene of the algU mucA mucB cluster. The first gene of the cluster, algU, encodes a putative alternative sigma factor required for algD transcription. The algD gene encodes a critical alginate biosynthetic enzyme and is invariably activated in mucoid P. aeruginosa cells. To investigate the function of the genes controlling conversion to mucoidy, the wild-type algU mucA mucB cluster from the standard genetic strain PAO1 was used to reconstitute algD transcription in Escherichia coli. Transcription of an algD-lacZ chromosomal fusion in E. coli was detected upon introduction of plasmid-borne algU mucA mucB. Moreover, insertional inactivation of either mucA or mucB resulted in further stimulation of transcriptional activity from the algD promoter. This activation was dependent on algU, since a double algU mucA mutation abrogated transcription of algD. These experiments suggest that the phenotypic manifestations of muc mutations, i.e., increased algD expression and mucoid phenotype, depend on the presence of an active algU gene and that this regulator and the factors encoded by the downstream genes interact. Further support for these conclusions came from the investigations of the mechanism of reversion to nonmucoidy in P. aeruginosa, a phenomenon frequently referred to as the instability of mucoid phenotype. Spontaneous nonmucoid derivatives of the mucoid strain PAO578 carrying the mucA22 mutation were examined for the presence of alterations within the algU mucA mucB locus. Point mutations which inactivated algU were detected in some, but not all, nonmucoid revertants. No reversion of the original mucA22 mutation (a deletion of one C) was observed in any of the investigated strains. This observation suggests that the process of conversion to nonmucoidy ban be explained, at least partially, by second-site suppressor mutations and that a fraction of such mutations occurs in algU.

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Selected References

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  1. 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]
  2. Benson A. K., Haldenwang W. G. Bacillus subtilis sigma B is regulated by a binding protein (RsbW) that blocks its association with core RNA polymerase. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2330–2334. doi: 10.1073/pnas.90.6.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. Deretic V., Martin D. W., Schurr M. J., Mudd M. H., Hibler N. S., Curcic R., Boucher J. C. Conversion to mucoidy in Pseudomonas aeruginosa. Biotechnology (N Y) 1993 Oct;11(10):1133–1136. doi: 10.1038/nbt1093-1133. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Deretic V., Schurr M. J., Boucher J. C., Martin D. W. 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. doi: 10.1128/jb.176.10.2773-2780.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Duncan L., Losick R. SpoIIAB is an anti-sigma factor that binds to and inhibits transcription by regulatory protein sigma F from Bacillus subtilis. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2325–2329. doi: 10.1073/pnas.90.6.2325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gilligan P. H. Microbiology of airway disease in patients with cystic fibrosis. Clin Microbiol Rev. 1991 Jan;4(1):35–51. doi: 10.1128/cmr.4.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goldberg J. B., Gorman W. L., Flynn J. L., Ohman D. E. A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species. J Bacteriol. 1993 Mar;175(5):1303–1308. doi: 10.1128/jb.175.5.1303-1308.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hughes K. T., Gillen K. L., Semon M. J., Karlinsey J. E. Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator. Science. 1993 Nov 19;262(5137):1277–1280. doi: 10.1126/science.8235660. [DOI] [PubMed] [Google Scholar]
  17. Innes R. W., Bent A. F., Kunkel B. N., Bisgrove S. R., Staskawicz B. J. Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes. J Bacteriol. 1993 Aug;175(15):4859–4869. doi: 10.1128/jb.175.15.4859-4869.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. Learn D. B., Brestel E. P., Seetharama S. Hypochlorite scavenging by Pseudomonas aeruginosa alginate. Infect Immun. 1987 Aug;55(8):1813–1818. doi: 10.1128/iai.55.8.1813-1818.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Mai G. T., Seow W. K., Pier G. B., McCormack J. G., Thong Y. H. Suppression of lymphocyte and neutrophil functions by Pseudomonas aeruginosa mucoid exopolysaccharide (alginate): reversal by physicochemical, alginase, and specific monoclonal antibody treatments. Infect Immun. 1993 Feb;61(2):559–564. doi: 10.1128/iai.61.2.559-564.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Martin D. W., Holloway B. W., 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. doi: 10.1128/jb.175.4.1153-1164.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Martin D. W., Schurr M. J., Mudd M. H., 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. doi: 10.1111/j.1365-2958.1993.tb01711.x. [DOI] [PubMed] [Google Scholar]
  27. Martin D. W., Schurr M. J., Mudd M. H., Govan J. R., Holloway B. W., 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. doi: 10.1073/pnas.90.18.8377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McGowan S. J., Gorham H. C., Hodgson D. A. Light-induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region. Mol Microbiol. 1993 Nov;10(4):713–735. doi: 10.1111/j.1365-2958.1993.tb00943.x. [DOI] [PubMed] [Google Scholar]
  29. Min K. T., Hilditch C. M., Diederich B., Errington J., Yudkin M. D. Sigma F, the first compartment-specific transcription factor of B. subtilis, is regulated by an anti-sigma factor that is also a protein kinase. Cell. 1993 Aug 27;74(4):735–742. doi: 10.1016/0092-8674(93)90520-z. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Mohr C. D., Deretic V. In vitro interactions of the histone-like protein IHF with the algD promoter, a critical site for control of mucoidy in Pseudomonas aeruginosa. Biochem Biophys Res Commun. 1992 Dec 15;189(2):837–844. doi: 10.1016/0006-291x(92)92279-7. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Ohnishi K., Kutsukake K., Suzuki H., Lino T. A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an antisigma factor inhibits the activity of the flagellum-specific sigma factor, sigma F. Mol Microbiol. 1992 Nov;6(21):3149–3157. doi: 10.1111/j.1365-2958.1992.tb01771.x. [DOI] [PubMed] [Google Scholar]
  35. Pier G. B., Small G. J., Warren H. B. Protection against mucoid Pseudomonas aeruginosa in rodent models of endobronchial infections. Science. 1990 Aug 3;249(4968):537–540. doi: 10.1126/science.2116663. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Schurr M. J., Martin D. W., Mudd M. H., Hibler N. S., Boucher J. C., 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] [Google Scholar]
  38. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  39. Simpson J. A., Smith S. E., Dean R. T. Scavenging by alginate of free radicals released by macrophages. Free Radic Biol Med. 1989;6(4):347–353. doi: 10.1016/0891-5849(89)90078-6. [DOI] [PubMed] [Google Scholar]
  40. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Xiao Y., Heu S., Yi J., Lu Y., Hutcheson S. W. Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss61 hrp and hrmA genes. J Bacteriol. 1994 Feb;176(4):1025–1036. doi: 10.1128/jb.176.4.1025-1036.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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