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. 1989 Jul;171(7):3680–3688. doi: 10.1128/jb.171.7.3680-3688.1989

Control of mucoidy in Pseudomonas aeruginosa: transcriptional regulation of algR and identification of the second regulatory gene, algQ.

V Deretic 1, W M Konyecsni 1
PMCID: PMC210111  PMID: 2544550

Abstract

A new alginate regulatory gene, algQ, was identified in a chromosomal region which, when tandemly amplified, induces mucoidy in Pseudomonas aeruginosa. The algQ gene was found closely linked to the previously identified algR gene. Both algQ and algR were required for transcription of the key alginate biosynthetic gene, algD. In addition, expression of the algR gene was studied. The algR promoter was mapped by S1 nuclease and reverse transcription and found to be activated in mucoid cells. However, even in nonmucoid cells, transcription of algR was detectable at an approximately 50-fold-lower level, as opposed to the algD promoter, which was silent in the nonmucoid background. Transcription of both promoters was studied by using algR- and algD-specific oligonucleotides and total cellular RNA from fresh cystic fibrosis isolates of mucoid P. aeruginosa and their nonmucoid revertants. Identical patterns of activity were found in all strains: in mucoid cells, both algR and algD were activated. This finding indicated that common mechanisms were involved in the regulation of alginate gene expression. However, when the algR gene was cloned behind the tac promoter on a broad-host-range-controlled expression vector, induction of transcription with isopropropyl-beta-D-thiogalactopyranoside (IPTG) caused the appearance of a nonmucoid phenotype in previously mucoid cells. This effect was transient, since removal of the inducer (IPTG) made cells mucoid again. Since the algR gene product is homologous to transcriptional regulators from a class of environmentally responsive systems (known to have a second, sensory component), the algQ gene could be a candidate for the sensory component of the alginate system.

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Selected References

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  1. Bradford M. M. 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. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Buck M. Deletion analysis of the Klebsiella pneumoniae nitrogenase promoter: importance of spacing between conserved sequences around positions -12 and -24 for activation by the nifA and ntrC (glnG) products. J Bacteriol. 1986 May;166(2):545–551. doi: 10.1128/jb.166.2.545-551.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chandler P. M., Krishnapillai V. Isolation and properties of recombination-deficient mutants of Pseudomonas aeruginosa. Mutat Res. 1974 Apr;23(1):15–23. doi: 10.1016/0027-5107(74)90155-9. [DOI] [PubMed] [Google Scholar]
  4. Dale R. M., McClure B. A., Houchins J. P. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. Plasmid. 1985 Jan;13(1):31–40. doi: 10.1016/0147-619x(85)90053-8. [DOI] [PubMed] [Google Scholar]
  5. Darzins A., Chakrabarty A. M. Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J Bacteriol. 1984 Jul;159(1):9–18. doi: 10.1128/jb.159.1.9-18.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Deretic V., Gill J. F., Chakrabarty A. M. 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. doi: 10.1093/nar/15.11.4567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deretic V., Tomasek P., Darzins A., Chakrabarty A. M. Gene amplification induces mucoid phenotype in rec-2 Pseudomonas aeruginosa exposed to kanamycin. J Bacteriol. 1986 Feb;165(2):510–516. doi: 10.1128/jb.165.2.510-516.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dixon R. The xylABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli. Mol Gen Genet. 1986 Apr;203(1):129–136. doi: 10.1007/BF00330393. [DOI] [PubMed] [Google Scholar]
  12. Duchêne M., Schweizer A., Lottspeich F., Krauss G., Marget M., Vogel K., von Specht B. U., Domdey H. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J Bacteriol. 1988 Jan;170(1):155–162. doi: 10.1128/jb.170.1.155-162.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Flynn J. L., Ohman D. E. Use of a gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT. J Bacteriol. 1988 Jul;170(7):3228–3236. doi: 10.1128/jb.170.7.3228-3236.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. George R. H. Pseudomonas infections in cystic fibrosis. Arch Dis Child. 1987 May;62(5):438–439. doi: 10.1136/adc.62.5.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gill J. F., Deretic V., Chakrabarty A. M. Overproduction and assay of Pseudomonas aeruginosa phosphomannose isomerase. J Bacteriol. 1986 Aug;167(2):611–615. doi: 10.1128/jb.167.2.611-615.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goldberg J. B., Ohman D. E. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol. 1984 Jun;158(3):1115–1121. doi: 10.1128/jb.158.3.1115-1121.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gussin G. N., Ronson C. W., Ausubel F. M. Regulation of nitrogen fixation genes. Annu Rev Genet. 1986;20:567–591. doi: 10.1146/annurev.ge.20.120186.003031. [DOI] [PubMed] [Google Scholar]
  21. Haas D., Holloway B. W. R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol Gen Genet. 1976 Mar 30;144(3):243–251. doi: 10.1007/BF00341722. [DOI] [PubMed] [Google Scholar]
  22. Hess J. F., Oosawa K., Kaplan N., Simon M. I. Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell. 1988 Apr 8;53(1):79–87. doi: 10.1016/0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  23. Inouye S., Nakazawa A., Nakazawa T. Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product. Gene. 1986;44(2-3):235–242. doi: 10.1016/0378-1119(86)90187-3. [DOI] [PubMed] [Google Scholar]
  24. Itoh Y., Soldati L., Stalon V., Falmagne P., Terawaki Y., Leisinger T., Haas D. Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene. J Bacteriol. 1988 Jun;170(6):2725–2734. doi: 10.1128/jb.170.6.2725-2734.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johnson K., Parker M. L., Lory S. Nucleotide sequence and transcriptional initiation site of two Pseudomonas aeruginosa pilin genes. J Biol Chem. 1986 Nov 25;261(33):15703–15708. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Minton N. P., Clarke L. E. Identification of the promoter of the Pseudomonas gene coding for carboxypeptidase G2. J Mol Appl Genet. 1985;3(1):26–35. [PubMed] [Google Scholar]
  30. Misra T. K. DNA sequencing: a new strategy to create ordered deletions, modified M13 vector, and improved reaction conditions for sequencing by dideoxy chain termination method. Methods Enzymol. 1987;155:119–139. doi: 10.1016/0076-6879(87)55012-1. [DOI] [PubMed] [Google Scholar]
  31. Mizusawa S., Nishimura S., Seela F. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 1986 Feb 11;14(3):1319–1324. doi: 10.1093/nar/14.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ninfa A. J., Magasanik B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5909–5913. doi: 10.1073/pnas.83.16.5909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ninfa A. J., Ninfa E. G., Lupas A. N., Stock A., Magasanik B., Stock J. Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5492–5496. doi: 10.1073/pnas.85.15.5492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ronson C. W., Nixon B. T., Ausubel F. M. Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell. 1987 Jun 5;49(5):579–581. doi: 10.1016/0092-8674(87)90530-7. [DOI] [PubMed] [Google Scholar]
  35. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stachel S. E., Zambryski P. C. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell. 1986 Aug 1;46(3):325–333. doi: 10.1016/0092-8674(86)90653-7. [DOI] [PubMed] [Google Scholar]
  37. Stibitz S., Weiss A. A., Falkow S. Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir. J Bacteriol. 1988 Jul;170(7):2904–2913. doi: 10.1128/jb.170.7.2904-2913.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Weiss A. A., Falkow S. Genetic analysis of phase change in Bordetella pertussis. Infect Immun. 1984 Jan;43(1):263–269. doi: 10.1128/iai.43.1.263-269.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wozniak D. J., Cram D. C., Daniels C. J., Galloway D. R. Nucleotide sequence and characterization of toxR: a gene involved in exotoxin A regulation in Pseudomonas aeruginosa. Nucleic Acids Res. 1987 Mar 11;15(5):2123–2135. doi: 10.1093/nar/15.5.2123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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