Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1981 Mar;145(3):1448–1451. doi: 10.1128/jb.145.3.1448-1451.1981

Versatile cloning vector for Pseudomonas aeruginosa.

D O Wood, M F Hollinger, M B Tindol
PMCID: PMC217156  PMID: 6259134

Abstract

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.

Full text

PDF
1448

Images in this article

Selected References

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

  1. Bachmann B. J., Low K. B. Linkage map of Escherichia coli K-12, edition 6. Microbiol Rev. 1980 Mar;44(1):1–56. doi: 10.1128/mr.44.1.1-56.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [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. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gautier F., Bonewald R. The use of plasmid R1162 and derivatives for gene cloning in the methanol-utilizing Pseudomonas AM1. Mol Gen Genet. 1980;178(2):375–380. doi: 10.1007/BF00270487. [DOI] [PubMed] [Google Scholar]
  6. Guerry P., van Embden J., Falkow S. Molecular nature of two nonconjugative plasmids carrying drug resistance genes. J Bacteriol. 1974 Feb;117(2):619–630. doi: 10.1128/jb.117.2.619-630.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hedges R. W., Jacob A. E., Crawford I. P. Wide ranging plasmid bearing the Pseudomonas aeruginosa tryptophan synthase genes. Nature. 1977 May 19;267(5608):283–284. doi: 10.1038/267283a0. [DOI] [PubMed] [Google Scholar]
  8. Holloway B. W. Isolation and characterization of an R' plasmid in Pseudomonas aeruginosa. J Bacteriol. 1978 Mar;133(3):1078–1082. doi: 10.1128/jb.133.3.1078-1082.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Markowitz S. M., Macrina F. L., Phibbs P. V., Jr R-factor inheritance and plasmid content in mucoid Pseudomonas aeruginosa. Infect Immun. 1978 Nov;22(2):530–539. doi: 10.1128/iai.22.2.530-539.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mercer A. A., Loutit J. S. Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions. J Bacteriol. 1979 Oct;140(1):37–42. doi: 10.1128/jb.140.1.37-42.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Meyers J. A., Sanchez D., Elwell L. P., Falkow S. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J Bacteriol. 1976 Sep;127(3):1529–1537. doi: 10.1128/jb.127.3.1529-1537.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nagahari K., Sakaguchi K. RSF1010 plasmid as a potentially useful vector in Pseudomonas species. J Bacteriol. 1978 Mar;133(3):1527–1529. doi: 10.1128/jb.133.3.1527-1529.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]

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

RESOURCES