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. 1997 Nov;63(11):4292–4297. doi: 10.1128/aem.63.11.4292-4297.1997

cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8.

R Holtwick 1, F Meinhardt 1, H Keweloh 1
PMCID: PMC168749  PMID: 9361416

Abstract

Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.

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Selected References

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  1. Banerjee R. V., Johnston N. L., Sobeski J. K., Datta P., Matthews R. G. Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain. J Biol Chem. 1989 Aug 15;264(23):13888–13895. [PubMed] [Google Scholar]
  2. 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]
  3. Cai X. Y., Maxon M. E., Redfield B., Glass R., Brot N., Weissbach H. Methionine synthesis in Escherichia coli: effect of the MetR protein on metE and metH expression. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4407–4411. doi: 10.1073/pnas.86.12.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Diefenbach R., Keweloh H. Synthesis of trans unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids. Arch Microbiol. 1994;162(1-2):120–125. doi: 10.1007/s002030050112. [DOI] [PubMed] [Google Scholar]
  5. Guckert J. B., Hood M. A., White D. C. Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio and proportions of cyclopropyl fatty acids. Appl Environ Microbiol. 1986 Oct;52(4):794–801. doi: 10.1128/aem.52.4.794-801.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Heipieper H. J., Diefenbach R., Keweloh H. Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity. Appl Environ Microbiol. 1992 Jun;58(6):1847–1852. doi: 10.1128/aem.58.6.1847-1852.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Heipieper H. J., de Bont J. A. Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes. Appl Environ Microbiol. 1994 Dec;60(12):4440–4444. doi: 10.1128/aem.60.12.4440-4444.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Keweloh H., Heipieper H. J. Trans unsaturated fatty acids in bacteria. Lipids. 1996 Feb;31(2):129–137. doi: 10.1007/BF02522611. [DOI] [PubMed] [Google Scholar]
  9. Loffeld B., Keweloh H. cis/trans isomerization of unsaturated fatty acids as possible control mechanism of membrane fluidity in Pseudomonas putida P8. Lipids. 1996 Aug;31(8):811–815. doi: 10.1007/BF02522976. [DOI] [PubMed] [Google Scholar]
  10. Makula R. A. Phospholipid composition of methane-utilizing bacteria. J Bacteriol. 1978 Jun;134(3):771–777. doi: 10.1128/jb.134.3.771-777.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Morita N., Shibahara A., Yamamoto K., Shinkai K., Kajimoto G., Okuyama H. Evidence for cis-trans isomerization of a double bond in the fatty acids of the psychrophilic bacterium Vibrio sp. strain ABE-1. J Bacteriol. 1993 Feb;175(3):916–918. doi: 10.1128/jb.175.3.916-918.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Müller-Newen G., Janssen U., Stoffel W. Enoyl-CoA hydratase and isomerase form a superfamily with a common active-site glutamate residue. Eur J Biochem. 1995 Feb 15;228(1):68–73. doi: 10.1111/j.1432-1033.1995.tb20230.x. [DOI] [PubMed] [Google Scholar]
  14. Müller K. D., Husmann H., Nalik H. P. A new and rapid method for the assay of bacterial fatty acids using high resolution capillary gas chromatography and trimethylsulfonium hydroxide. Zentralbl Bakteriol. 1990 Nov;274(2):174–182. doi: 10.1016/s0934-8840(11)80100-3. [DOI] [PubMed] [Google Scholar]
  15. Okuyama H., Enari D., Shibahara A., Yamamoto K., Morita N. Identification of activities that catalyze the cis-trans isomerization of the double bond of a mono-unsaturated fatty acid in Pseudomonas sp. strain E-3. Arch Microbiol. 1996 Jun;165(6):415–417. doi: 10.1007/s002030050346. [DOI] [PubMed] [Google Scholar]
  16. Okuyama H., Sasaki S., Higashi S., Murata N. A trans-unsaturated fatty acid in a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol. 1990 Jun;172(6):3515–3518. doi: 10.1128/jb.172.6.3515-3518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rehm B. H., Hancock R. E. Membrane topology of the outer membrane protein OprH from Pseudomonas aeruginosa: PCR-mediated site-directed insertion and deletion mutagenesis. J Bacteriol. 1996 Jun;178(11):3346–3349. doi: 10.1128/jb.178.11.3346-3349.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rosenberg S. M., Stahl M. M., Kobayashi I., Stahl F. W. Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage. Gene. 1985;38(1-3):165–175. doi: 10.1016/0378-1119(85)90215-x. [DOI] [PubMed] [Google Scholar]
  20. Taghavi S., van der Lelie D., Mergeay M. Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments. Appl Environ Microbiol. 1994 Oct;60(10):3585–3591. doi: 10.1128/aem.60.10.3585-3591.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Urbanowski M. L., Stauffer G. V. The control region of the metH gene of Salmonella typhimurium LT2: an atypical met promoter. Gene. 1988 Dec 15;73(1):193–200. doi: 10.1016/0378-1119(88)90325-3. [DOI] [PubMed] [Google Scholar]
  22. Weber F. J., Isken S., de Bont J. A. Cis/trans isomerization of fatty acids as a defence mechanism of Pseudomonas putida strains to toxic concentrations of toluene. Microbiology. 1994 Aug;140(Pt 8):2013–2017. doi: 10.1099/13500872-140-8-2013. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. de Lorenzo V., Timmis K. N. Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol. 1994;235:386–405. doi: 10.1016/0076-6879(94)35157-0. [DOI] [PubMed] [Google Scholar]

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