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. 1989 Sep;171(9):5071–5078. doi: 10.1128/jb.171.9.5071-5078.1989

Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.

R A Siddiqui 1, K Benthin 1, H G Schlegel 1
PMCID: PMC210319  PMID: 2549012

Abstract

The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.

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  1. Barkay T., Fouts D. L., Olson B. H. Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities. Appl Environ Microbiol. 1985 Mar;49(3):686–692. doi: 10.1128/aem.49.3.686-692.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bender C. L., Cooksey D. A. Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato. J Bacteriol. 1987 Feb;169(2):470–474. doi: 10.1128/jb.169.2.470-474.1987. [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. Cooksey D. A. Characterization of a Copper Resistance Plasmid Conserved in Copper-Resistant Strains of Pseudomonas syringae pv. tomato. Appl Environ Microbiol. 1987 Feb;53(2):454–456. doi: 10.1128/aem.53.2.454-456.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. El Solh N., Ehrlich S. D. A small cadmium resistance plasmid isolated from Staphylococcus aureus. Plasmid. 1982 Jan;7(1):77–84. doi: 10.1016/0147-619x(82)90029-4. [DOI] [PubMed] [Google Scholar]
  6. Foster T. J. Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol Rev. 1983 Sep;47(3):361–409. doi: 10.1128/mr.47.3.361-409.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Friedrich B., Friedrich C. G., Meyer M., Schlegel H. G. Expression of hydrogenase in Alcaligenes spp. is altered by interspecific plasmid exchange. J Bacteriol. 1984 Apr;158(1):331–333. doi: 10.1128/jb.158.1.331-333.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Friedrich B., Hogrefe C., Schlegel H. G. Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus. J Bacteriol. 1981 Jul;147(1):198–205. doi: 10.1128/jb.147.1.198-205.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Friedrich B., Meyer M., Schlegel H. G. Transfer and expression of the herbicide-degrading plasmid pJP4 in aerobic autotrophic bacteria. Arch Microbiol. 1983 Feb;134(2):92–97. doi: 10.1007/BF00407938. [DOI] [PubMed] [Google Scholar]
  10. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  11. Hogrefe C., Römermann D., Friedrich B. Alcaligenes eutrophus hydrogenase genes (Hox). J Bacteriol. 1984 Apr;158(1):43–48. doi: 10.1128/jb.158.1.43-48.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jobling M. G., Ritchie D. A. Genetic and physical analysis of plasmid genes expressing inducible resistance of tellurite in Escherichia coli. Mol Gen Genet. 1987 Jun;208(1-2):288–293. doi: 10.1007/BF00330455. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  16. Kuhn M., Jendrossek D., Fründ C., Steinbüchel A., Schlegel H. G. Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene. J Bacteriol. 1988 Feb;170(2):685–692. doi: 10.1128/jb.170.2.685-692.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mergeay M., Houba C., Gerits J. Extrachromosomal inheritance controlling resistance to cadmium, cobalt, copper and zinc ions: evidence from curing in a Pseudomonas [proceedings]. Arch Int Physiol Biochim. 1978 May;86(2):440–442. [PubMed] [Google Scholar]
  18. Mergeay M., Nies D., Schlegel H. G., Gerits J., Charles P., Van Gijsegem F. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacteriol. 1985 Apr;162(1):328–334. doi: 10.1128/jb.162.1.328-334.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mobley H. L., Silver S., Porter F. D., Rosen B. P. Homology among arsenate resistance determinants of R factors in Escherichia coli. Antimicrob Agents Chemother. 1984 Feb;25(2):157–161. doi: 10.1128/aac.25.2.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nies A., Nies D. H., Silver S. Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus. J Bacteriol. 1989 Sep;171(9):5065–5070. doi: 10.1128/jb.171.9.5065-5070.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nies D. H., Silver S. Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus. J Bacteriol. 1989 Feb;171(2):896–900. doi: 10.1128/jb.171.2.896-900.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nies D., Mergeay M., Friedrich B., Schlegel H. G. Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34. J Bacteriol. 1987 Oct;169(10):4865–4868. doi: 10.1128/jb.169.10.4865-4868.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rouch D., Camakaris J., Lee B. T., Luke R. K. Inducible plasmid-mediated copper resistance in Escherichia coli. J Gen Microbiol. 1985 Apr;131(4):939–943. doi: 10.1099/00221287-131-4-939. [DOI] [PubMed] [Google Scholar]
  24. Schubert P., Steinbüchel A., Schlegel H. G. Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J Bacteriol. 1988 Dec;170(12):5837–5847. doi: 10.1128/jb.170.12.5837-5847.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sensfuss C., Reh M., Schlegel H. G. No correlation exists between the conjugative transfer of the autotrophic character and that of plasmids in Nocardia opaca strains. J Gen Microbiol. 1986 Apr;132(4):997–1007. doi: 10.1099/00221287-132-4-997. [DOI] [PubMed] [Google Scholar]
  26. Siddiqui R. A., Schlegel H. G., Meyer M. Inducible and constitutive expression of pMOL28-encoded nickel resistance in Alcaligenes eutrophus N9A. J Bacteriol. 1988 Sep;170(9):4188–4193. doi: 10.1128/jb.170.9.4188-4193.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Silver S., Nucifora G., Chu L., Misra T. K. Bacterial resistance ATPases: primary pumps for exporting toxic cations and anions. Trends Biochem Sci. 1989 Feb;14(2):76–80. doi: 10.1016/0968-0004(89)90048-0. [DOI] [PubMed] [Google Scholar]
  28. Simon R. High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol Gen Genet. 1984;196(3):413–420. doi: 10.1007/BF00436188. [DOI] [PubMed] [Google Scholar]
  29. Slater S. C., Voige W. H., Dennis D. E. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway. J Bacteriol. 1988 Oct;170(10):4431–4436. doi: 10.1128/jb.170.10.4431-4436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smith D. H. R factors mediate resistance to mercury, nickel, and cobalt. Science. 1967 May 26;156(3778):1114–1116. doi: 10.1126/science.156.3778.1114. [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. Summers A. O. Organization, expression, and evolution of genes for mercury resistance. Annu Rev Microbiol. 1986;40:607–634. doi: 10.1146/annurev.mi.40.100186.003135. [DOI] [PubMed] [Google Scholar]
  33. Tynecka Z., Gos Z., Zajac J. Energy-dependent efflux of cadmium coded by a plasmid resistance determinant in Staphylococcus aureus. J Bacteriol. 1981 Aug;147(2):313–319. doi: 10.1128/jb.147.2.313-319.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Weiss A. A., Murphy S. D., Silver S. Mercury and organomercurial resistances determined by plasmids in Staphylococcus aureus. J Bacteriol. 1977 Oct;132(1):197–208. doi: 10.1128/jb.132.1.197-208.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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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