Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1984 Jun;158(3):1115–1121. doi: 10.1128/jb.158.3.1115-1121.1984

Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate.

J B Goldberg, D E Ohman
PMCID: PMC215558  PMID: 6427188

Abstract

Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis commonly produce a capsule-like exopolysaccharide called alginate. The alginate-producing (Alg+) phenotype results in a mucoid colony morphology and is an unstable trait. A mutant of P. aeruginosa FRD (a cystic fibrosis isolate) was obtained which was temperature sensitive for alginate production ( Algts ). At elevated growth temperatures (41 degrees C), no alginate was detected in culture supernatants of the Algts mutant, but yields of alginate increased as the temperature of incubation was reduced. The mutation responsible for the Algts phenotype, alg-50(Ts), has been mapped to a region of the FRD chromosome closely linked to trp-2. The alg-50(Ts) marker did not map near the met-l-linked chromosomal mutations responsible for the instability of the Alg+ phenotype. A broad host range cosmid cloning system based upon derivatives of plasmid RK2 was used to construct a P. aeruginosa clone bank. After transfer of the clone bank to the Algts mutant, hybrid plasmids were obtained which complemented the Algts defect. Deletion mapping of the original 20.3 kilobases of P. aeruginosa DNA cloned showed that a 4.7-kilobase fragment would complement the alg-50(Ts) mutation.

Full text

PDF
1115

Selected References

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

  1. Baltimore R. S., Mitchell M. Immunologic investigations of mucoid strains of Pseudomonas aeruginosa: comparison of susceptibility to opsonic antibody in mucoid and nonmucoid strains. J Infect Dis. 1980 Feb;141(2):238–247. doi: 10.1093/infdis/141.2.238. [DOI] [PubMed] [Google Scholar]
  2. Banerjee P. C., Vanags R. I., Chakrabarty A. M., Maitra P. K. Alginic acid synthesis in Pseudomonas aeruginosa mutants defective in carbohydrate metabolism. J Bacteriol. 1983 Jul;155(1):238–245. doi: 10.1128/jb.155.1.238-245.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Burkardt H. J., Riess G., Pühler A. Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol. 1979 Oct;114(2):341–348. doi: 10.1099/00221287-114-2-341. [DOI] [PubMed] [Google Scholar]
  5. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Di Sant'Agnese P. A., Davis P. B. Research in cystic fibrosis (first of three parts). N Engl J Med. 1976 Aug 26;295(9):481–485. doi: 10.1056/NEJM197608262950905. [DOI] [PubMed] [Google Scholar]
  8. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doggett R. G., Harrison G. M., Carter R. E. Mucoid Pseudomonas aeruginosa in patients with chronic illnesses. Lancet. 1971 Jan 30;1(7692):236–237. doi: 10.1016/s0140-6736(71)90973-1. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Haas D., Holloway B. W. Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics. Mol Gen Genet. 1978 Jan 17;158(3):229–237. doi: 10.1007/BF00267194. [DOI] [PubMed] [Google Scholar]
  16. Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
  17. Linker A., Jones R. S. A new polysaccharide resembling alginic acid isolated from pseudomonads. J Biol Chem. 1966 Aug 25;241(16):3845–3851. [PubMed] [Google Scholar]
  18. 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]
  19. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mian F. A., Jarman T. R., Righelato R. C. Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa. J Bacteriol. 1978 May;134(2):418–422. doi: 10.1128/jb.134.2.418-422.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Ohman D. E., Chakrabarty A. M. Utilization of human respiratory secretions by mucoid Pseudomonas aeruginosa of cystic fibrosis origin. Infect Immun. 1982 Aug;37(2):662–669. doi: 10.1128/iai.37.2.662-669.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pindar D. F., Bucke C. The biosynthesis of alginic acid by Azotobacter vinelandii. Biochem J. 1975 Dec;152(3):617–622. doi: 10.1042/bj1520617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pugashetti B. K., Vadas L., Prihar H. S., Feingold D. S. GDPmannose dehydrogenase and biosynthesis of alginate-like polysaccharide in a mucoid strain of Pseudomonas aeruginosa. J Bacteriol. 1983 Feb;153(2):1107–1110. doi: 10.1128/jb.153.2.1107-1110.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. di Sant'Agnese P. A., Davis P. B. Research in cystic fibrosis (second of three parts). N Engl J Med. 1976 Sep 2;295(10):534–541. doi: 10.1056/NEJM197609022951005. [DOI] [PubMed] [Google Scholar]
  29. di Sant'Agnese P. A., Davis P. B. Research in cystic fibrosis (third of three parts). N Engl J Med. 1976 Sep 9;295(11):597–602. doi: 10.1056/NEJM197609092951105. [DOI] [PubMed] [Google Scholar]

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

RESOURCES