Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Jul;170(7):3228–3236. doi: 10.1128/jb.170.7.3228-3236.1988

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 L Flynn 1, D E Ohman 1
PMCID: PMC211274  PMID: 2838462

Abstract

Pseudomonas aeruginosa can convert to a mucoid colony morphology by a genetic mechanism called alginate conversion; this results in the production of copious amounts of the exopolysaccharide alginate. The mucoid phenotype of P. aeruginosa is commonly associated with its ability to cause chronic pulmonary tract infections in patients with cystic fibrosis. In this study we isolated the cis-acting locus involved in alginate conversion, called algS, from both mucoid and nonmucoid isogenic strains. We then examined the role of algS in the control of algT, a trans-active gene required for alginate production in P. aeruginosa. We used a new cosmid cloning vector, called pEMR2, that permitted both the cloning of large DNA fragments and their subsequent gene replacement in P. aeruginosa. To verify the predicted properties of this vector, we isolated and tested a pEMR2 hisI+ clone. Using cloned algS-containing DNA and a method for gene replacement, we constructed isogenic strains of P. aeruginosa that had Tn501 adjacent to algS on the chromosome. Two pEMR2 clone banks containing genomic fragments from isogenic algS(On) (exhibiting the alginate production phenotype) and algS(Off) (exhibiting the non-alginate production phenotype) strains were constructed, and Tn501 served as an adjacent marker to select for clones containing the respective algS allele. The pEMR2 algS(On) and pEMR2 algS(Off) clones were shown to contain the indicated algS allele by gene replacement with the chromosome of strains that carried the opposite allele. To test whether algS controls the expression of the adjacent algT gene, we constructed a pLAFR1 algS(Off)T clone and showed it to be unable to complement an algT::Tn501 mutation in trans. In contrast, a pLAFR1 algS(On)T clone did complement algT::Tn501 in trans. Thus, algS appears to control the activation of algT expression, bringing about alginate conversion.

Full text

PDF
3228

Selected References

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

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. Brown N. L., Choi C. L., Grinsted J., Richmond M. H., Whitehead P. R. Nucleotide sequences at the ends of the mercury resistance transposon, Tn501. Nucleic Acids Res. 1980 May 10;8(9):1933–1945. doi: 10.1093/nar/8.9.1933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [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. Darzins A., Wang S. K., Vanags R. I., Chakrabarty A. M. Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa. J Bacteriol. 1985 Nov;164(2):516–524. doi: 10.1128/jb.164.2.516-524.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Evans L. R., Linker A. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacteriol. 1973 Nov;116(2):915–924. doi: 10.1128/jb.116.2.915-924.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Goldberg J. B., Ohman D. E. Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate. J Bacteriol. 1987 Apr;169(4):1593–1602. doi: 10.1128/jb.169.4.1593-1602.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Govan J. R., Fyfe J. A., McMillan C. The instability of mucoid Pseudomonas aeruginosa: fluctuation test and improved stability of the mucoid form in shaken culture. J Gen Microbiol. 1979 Jan;110(1):229–232. doi: 10.1099/00221287-110-1-229. [DOI] [PubMed] [Google Scholar]
  15. Govan J. R., Fyfe J. A. Mucoid Pseudomonas aeruginosa and cystic fibrosis: resistance of the mucoid from to carbenicillin, flucloxacillin and tobramycin and the isolation of mucoid variants in vitro. J Antimicrob Chemother. 1978 May;4(3):233–240. doi: 10.1093/jac/4.3.233. [DOI] [PubMed] [Google Scholar]
  16. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  17. Holloway B. W., Krishnapillai V., Morgan A. F. Chromosomal genetics of Pseudomonas. Microbiol Rev. 1979 Mar;43(1):73–102. doi: 10.1128/mr.43.1.73-102.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Holloway B. W., Rossiter H., Burgess D., Dodge J. Aeruginocin tolerant mutants of Pseudomonas aeruginosa. Genet Res. 1973 Dec;22(3):239–253. doi: 10.1017/s0016672300013069. [DOI] [PubMed] [Google Scholar]
  19. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Martin D. R. Mucoid variation in Pseudomonas aeruginosa induced by the action of phage. J Med Microbiol. 1973 Feb;6(1):111–118. doi: 10.1099/00222615-6-1-111. [DOI] [PubMed] [Google Scholar]
  23. Miller R. V., Rubero V. J. Mucoid conversion by phages of Pseudomonas aeruginosa strains from patients with cystic fibrosis. J Clin Microbiol. 1984 May;19(5):717–719. doi: 10.1128/jcm.19.5.717-719.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. O'Hoy K., Krishnapillai V. Recalibration of the Pseudomonas aeruginosa strain PAO chromosome map in time units using high-frequency-of-recombination donors. Genetics. 1987 Apr;115(4):611–618. doi: 10.1093/genetics/115.4.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ohman D. E., Chakrabarty A. M. Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun. 1981 Jul;33(1):142–148. doi: 10.1128/iai.33.1.142-148.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ohman D. E. Molecular genetics of exopolysaccharide production by mucoid Pseudomonas aeruginosa. Eur J Clin Microbiol. 1986 Feb;5(1):6–10. doi: 10.1007/BF02013452. [DOI] [PubMed] [Google Scholar]
  27. Ohman D. E., West M. A., Flynn J. L., Goldberg J. B. Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production. J Bacteriol. 1985 Jun;162(3):1068–1074. doi: 10.1128/jb.162.3.1068-1074.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Royle P. L., Matsumoto H., Holloway B. W. Genetic circularity of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol. 1981 Jan;145(1):145–155. doi: 10.1128/jb.145.1.145-155.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schwarzmann S., Boring J. R. Antiphagocytic Effect of Slime from a Mucoid Strain of Pseudomonas aeruginosa. Infect Immun. 1971 Jun;3(6):762–767. doi: 10.1128/iai.3.6.762-767.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Selvaraj G., Fong Y. C., Iyer V. N. A portable DNA sequence carrying the cohesive site (cos) of bacteriophage lambda and the mob (mobilization) region of the broad-host-range plasmid RK2: a module for the construction of new cosmids. Gene. 1984 Dec;32(1-2):235–241. doi: 10.1016/0378-1119(84)90051-9. [DOI] [PubMed] [Google Scholar]
  31. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  32. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  33. Williams R. J., Govan J. R. Pyocine typing of mucoid strains of Pseudomonas aeruginosa isolated from children with cystic fibrosis. J Med Microbiol. 1973 Aug;6(3):409–412. doi: 10.1099/00222615-6-3-409. [DOI] [PubMed] [Google Scholar]
  34. Wilson R., Roberts D., Cole P. Effect of bacterial products on human ciliary function in vitro. Thorax. 1985 Feb;40(2):125–131. doi: 10.1136/thx.40.2.125. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES