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. 1992 Jul;174(13):4410–4415. doi: 10.1128/jb.174.13.4410-4415.1992

Fusion-mediated transfer of plasmids into Spiroplasma floricola cells.

M Salman 1, M Tarshis 1, S Rottem 1
PMCID: PMC206226  PMID: 1624433

Abstract

We have developed and characterized a system for the transfer of plasmids encapsulated in large unilamellar vesicles (LUV) into Spiroplasma floricola BNR1 cells. The approach is based on the ability of S. floricola-derived LUV to fuse with S. floricola cells. The fusion was continuously monitored by an assay for lipid mixing based on the dequenching of the fluorescent probe octadecylrhodamine B (R18) that was incorporated into LUV at self-quenching concentrations. The fusion was also evaluated by fluorescence-activated cell sorter measurements and by sucrose density gradient analysis. LUV-cell fusion occurred only in the presence of low concentrations (5%) of polyethylene glycol (polyethylene glycol 8000) and depended on temperature, the LUV/cell ratio, and divalent cations in the incubation medium. Throughout the fusion process, spiroplasma cells remained intact and viable. Under optimal fusion conditions, the plasmid pACYC, encapsulated in LUV by reversed-phase evaporation, was transferred into live S. floricola cells and expressed chloramphenicol acetyltransferase activity. The expression was transient with maximal chloramphenicol acetyltransferase activity observed after 6 h of incubation of the transfected cells.

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Selected References

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  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Barroso G., Labarère J. Chromosomal gene transfer in Spiroplasma citri. Science. 1988 Aug 19;241(4868):959–961. doi: 10.1126/science.3261453. [DOI] [PubMed] [Google Scholar]
  3. Citovsky V., Rottem S., Nussbaum O., Laster Y., Rott R., Loyter A. Animal viruses are able to fuse with prokaryotic cells. Fusion between Sendai or influenza virions and Mycoplasma. J Biol Chem. 1988 Jan 5;263(1):461–467. [PubMed] [Google Scholar]
  4. Düzgüneş N. Membrane fusion. Subcell Biochem. 1985;11:195–286. doi: 10.1007/978-1-4899-1698-3_5. [DOI] [PubMed] [Google Scholar]
  5. Furness G., Cerone A. M. Preparation of competent single-cell suspensions of Mycoplasma hominis tets and Mycoplasma salivarium tets for genetic transformation to tetracycline resistance by DNA extracted from Mycoplamsa hominis tetr. J Infect Dis. 1979 Apr;139(4):444–451. doi: 10.1093/infdis/139.4.444. [DOI] [PubMed] [Google Scholar]
  6. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goulay J. L., Darbord J. C., Desvignes A. Etude comparative de la transformation et de la fusion induite par les polyéthyléleneglycols chez Acholeplasma laidlawii B. Can J Microbiol. 1984 Jan;30(1):40–44. [PubMed] [Google Scholar]
  8. Gross Z., Rottem S. Lipid interconversions in aging Mycoplasma capricolum cultures. J Bacteriol. 1986 Sep;167(3):986–991. doi: 10.1128/jb.167.3.986-991.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Heath T. D. Interaction of liposomes with cells. Methods Enzymol. 1987;149:135–143. doi: 10.1016/0076-6879(87)49051-4. [DOI] [PubMed] [Google Scholar]
  10. Hoekstra D., de Boer T., Klappe K., Wilschut J. Fluorescence method for measuring the kinetics of fusion between biological membranes. Biochemistry. 1984 Nov 20;23(24):5675–5681. doi: 10.1021/bi00319a002. [DOI] [PubMed] [Google Scholar]
  11. Jolly D. J., Yee J. K., Friedmann T. High-efficiency gene transfer into cells. Methods Enzymol. 1987;149:10–25. doi: 10.1016/0076-6879(87)49040-x. [DOI] [PubMed] [Google Scholar]
  12. Lucy J. A., Ahkong Q. F. Membrane fusion. Fusogenic agents and osmotic forces. Subcell Biochem. 1989;14:189–228. [PubMed] [Google Scholar]
  13. Nahas N., Fibach E., Giloh H., Gatt S. Use of the fluorescence activated cell sorter for studying uptake of fluorescent fatty acids into cultured cells. Biochim Biophys Acta. 1987 Jan 13;917(1):86–91. doi: 10.1016/0005-2760(87)90287-6. [DOI] [PubMed] [Google Scholar]
  14. Nicolau C., Rottem S. Expression of a beta-lactamase activity in Mycoplasma capricolum transfected with the liposome-encapsulated E.coli pBR 322 plasmid. Biochem Biophys Res Commun. 1982 Oct 15;108(3):982–986. doi: 10.1016/0006-291x(82)92096-4. [DOI] [PubMed] [Google Scholar]
  15. Razin S. Molecular biology and genetics of mycoplasmas (Mollicutes). Microbiol Rev. 1985 Dec;49(4):419–455. doi: 10.1128/mr.49.4.419-455.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Roberts M. C., Kenny G. E. Conjugal transfer of transposon Tn916 from Streptococcus faecalis to Mycoplasma hominis. J Bacteriol. 1987 Aug;169(8):3836–3839. doi: 10.1128/jb.169.8.3836-3839.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rottem S., Greenberg A. S. Changes in composition, biosynthesis, and physical state of membrane lipids occurring upon aging of Mycoplasma hominis cultures. J Bacteriol. 1975 Feb;121(2):631–639. doi: 10.1128/jb.121.2.631-639.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rottem S., Linker C., Wilson T. H. Proton motive force across the membrane of Mycoplasma gallisepticum and its possible role in cell volume regulation. J Bacteriol. 1981 Mar;145(3):1299–1304. doi: 10.1128/jb.145.3.1299-1304.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rottem S. Membrane lipids of mycoplasmas. Biochim Biophys Acta. 1980 May 27;604(1):65–90. doi: 10.1016/0005-2736(80)90585-4. [DOI] [PubMed] [Google Scholar]
  20. Salman M., Tarshis M., Rottem S. Small unilamellar vesicles are able to fuse with Mycoplasma capricolum cells. Biochim Biophys Acta. 1991 Apr 2;1063(2):209–216. doi: 10.1016/0005-2736(91)90373-g. [DOI] [PubMed] [Google Scholar]
  21. Salvado J. C., Barroso G., Labarère J. Involvement of a Spiroplasma citri plasmid in the erythromycin-resistance transfer. Plasmid. 1989 Sep;22(2):151–159. doi: 10.1016/0147-619x(89)90024-3. [DOI] [PubMed] [Google Scholar]
  22. Sladek T. L., Maniloff J. Transfection of REP- mycoplasmas with viral single-stranded DNA. J Virol. 1985 Jan;53(1):25–31. doi: 10.1128/jvi.53.1.25-31.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stegmann T., Doms R. W., Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. doi: 10.1146/annurev.bb.18.060189.001155. [DOI] [PubMed] [Google Scholar]

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