1887

Abstract

Natural membrane vesicles (n-MVs) produced by PAO1 and PAO1 carrying plasmid pAK1900 (p-MVs) were purified and analysed for DNA content. The MVs were isolated by a procedure designed to ensure no cellular contamination from the parent MV-producing cells. Fluorometry analysis revealed that p-MVs were associated with 7·80 ng DNA (20 μg MV protein). PCR analysis using specific primers for pAK1900 sequences and a chromosomal target, , indicated that only plasmid DNA was contained within the lumen of p-MVs after exogenous DNA was digested by DNase. MVs have previously been shown to be capable of fusing into the outer membrane (OM) of PAO1 and DH5. Accordingly, p-MVs should deliver the plasmid into the periplasm, where it would only have to by-pass the plasma membrane (PM) for effective transformation. It was speculated that p-MVs should increase transformation efficiency but the data suggested otherwise. p-MVs did not transform PAO1 nor DH5 under a variety of transforming conditions. To characterize p-MVs and to ensure that membrane-encapsulated pAK1900 was not derived from a small proportion of lysed cells within the culture and bound by PM instead of OM, which typically forms n-MVs, the physical and ultrastructural differences between n- and p-MVs were determined. Cryo-transmission electron microscopy (cryo-TEM) revealed that n-MVs and p-MVs closely resembled isolated OM. Buoyant density measurements using isopycnic sucrose gradients on isolated PM, OM, n- and p-MVs demonstrated that isolated OM and n-MVs both fractionated into two bands (=1·240 and 1·260 g ml). p-MVs also produced two bands but at two different densities (=1·250 and 1·265 g ml) which may be attributed to the presence of DNA. SDS-PAGE showed that p-MVs possessed most major OM proteins and also contained 43·70 nmol 3-deoxy---octulosonic acid (KDO) (mg protein) as an LPS marker. The amount of NADH oxidase activity, a PM enzyme, in the p-MVs was barely detectable. These data strongly suggest that p-MVs are OM-based, with little if any PM material associated with them. The possibility of whether exogenous plasmid DNA could enter n-MVs once the vesicles had departed from cells was also tested; surprisingly, a small amount of DNA could. Accordingly, the data suggest that DNA can be taken up by MVs using two separate routes: (1) via a periplasmic route and (2) via an extracellular, exogenous route.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26841-0
2004-07-01
2020-10-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/7/mic1502161.html?itemId=/content/journal/micro/10.1099/mic.0.26841-0&mimeType=html&fmt=ahah

References

  1. Adrian M., Dubochet J., Fuller S. D., Harris J. R.. 1998; Cryo-negative staining. Micron29:145–160[CrossRef]
    [Google Scholar]
  2. Beveridge T. J.. 1999; Structures of Gram-negative cell walls and their derived membrane vesicles. J Bacteriol181:4725–4733
    [Google Scholar]
  3. Beveridge T. J., Kadurugamuwa J. L.. 1996; Periplasm, periplasmic spaces, and their relation to bacterial wall structure: novel secretion of selected periplasmic proteins from Pseudomonas aeruginosa. Microb Drug Resist2:1–8[CrossRef]
    [Google Scholar]
  4. Beveridge T. J., Makin S. A., Kadurugamuwa J. L., Li Z.. 1997; Interactions between biofilms and the environment. FEMS Microbiol Rev20:291–303[CrossRef]
    [Google Scholar]
  5. Ciofu O., Beveridge T. J., Kadurugamuwa J. L., Walther-Rasmussen J., Høiby N.. 2000; Chromosomal β-lactamase is packaged into membrane vesicles secreted from Pseudomonas aeruginosa. J Antimicrob Chemother45:9–13
    [Google Scholar]
  6. Dorward D. W., Garon C. F.. 1989; DNA binding proteins in cells and membrane blebs of Neisseria gonorrhoeae. J Bacteriol171:4196–4201
    [Google Scholar]
  7. Dorward D. W., Garon C. F., Judd R. C.. 1989; Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J Bacteriol171:2499–2505
    [Google Scholar]
  8. Dubnau D.. 1999; DNA uptake in bacteria. Annu Rev Microbiol53:217–244[CrossRef]
    [Google Scholar]
  9. Forsberg C. W., Beveridge T. J., Hellstrom A.. 1981; Cellulase and xylanase release from Bacteroides succinogenes and its importance in the rumen environment. Appl Environ Microbiol42:886–896
    [Google Scholar]
  10. Hancock R. E. W., Nikaido H.. 1978; Outer membrane of Gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of permeability barrier. J Bacteriol136:381–390
    [Google Scholar]
  11. Hancock R. E. W., Siehnel R., Martin N.. 1990; Outer membrane proteins of Pseudomonas. Mol Microbiol4:1069–1075[CrossRef]
    [Google Scholar]
  12. Horstman A. L., Kuehn M. J.. 2000; Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. J Biol Chem275:12489–12496[CrossRef]
    [Google Scholar]
  13. Horstman A. L., Kuehn M. J.. 2002; Bacterial surface association of heat-labile enterotoxin through LPS after secretion via the general secretory pathway. J Biol Chem277:32538–32545[CrossRef]
    [Google Scholar]
  14. Jansons I., Touchie G., Sharp R., Almquist K., Farina M., Lam J. S., Kropinski A. M.. 1994; Deletion and transposon mutagenesis and sequence analysis of the pRO1600 OriR region found in the broad-host range plasmids of the pQF series. Plasmid31:265–274[CrossRef]
    [Google Scholar]
  15. Kadurugamuwa J. L., Beveridge T. J.. 1995; Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion. J Bacteriol177:3998–4008
    [Google Scholar]
  16. Kadurugamuwa J. L., Beveridge T. J.. 1996; Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics. J Bacteriol178:2767–2774
    [Google Scholar]
  17. Kadurugamuwa J. L., Beveridge T. J.. 1997; Natural release of virulence factors in membrane vesicles by Pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release. J Antimicrob Chemother40:615–621[CrossRef]
    [Google Scholar]
  18. Kadurugamuwa J. L., Beveridge T. J.. 1998; Delivery of nonmembrane-permeative antibiotic gentamicin into mammalian cells by using Shigella flexneri membrane vesicles. Antimicrob Agents Chemother42:1476–1483
    [Google Scholar]
  19. Kadurugamuwa J. L., Beveridge T. J.. 1999; Membrane vesicles derived from Pseudomonas aeruginosa and Shigella flexneri can be integrated into the surfaces of other Gram-negative bacteria. Microbiology145:2051–2060[CrossRef]
    [Google Scholar]
  20. Kadurugamuwa J. L., Mayer A., Messner P., Sleytr U. B., Beveridge T. J, Sára M.. 1998; S-layered Aneurinibacillus and Bacillus spp. are susceptible to the lytic action of Pseudomonas aeruginosa membrane vesicles. . J Bacteriol180:2306–2311
    [Google Scholar]
  21. Kahn M. E., Maul G., Goodgal S. H.. 1982; Possible mechanism for donor DNA binding and transport in Haemophilus. Proc Natl Acad Sci U S A79:6370–6374[CrossRef]
    [Google Scholar]
  22. Kahn M. E., Barny F., Hamilton O. S.. 1983; Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation. Proc Natl Acad Sci U S A80:6927–6931[CrossRef]
    [Google Scholar]
  23. Karkhanis Y. D., Zeltner J. Y., Jackson J. J., Carlo D. L.. 1978; A new and improved microassay to determine 2-keto-3-deoxyoctonate in LPS of Gram-negative bacteria. Anal Biochem85:595–601[CrossRef]
    [Google Scholar]
  24. Kato S., Yusuke K., Demuth D. R.. 2002; Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin. Microb Pathog32:1–13[CrossRef]
    [Google Scholar]
  25. Kolling G. L., Matthews K. R.. 1999; Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157 : H7. . Appl Environ Microbiol65:1843–1848
    [Google Scholar]
  26. Li Z., Clarke A. J., Beveridge T. J.. 1996; A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division, and secretion in surface membrane vesicles. J Bacteriol178:2479–2488
    [Google Scholar]
  27. Li Z., Clarke A. J., Beveridge T. J.. 1998; Gram-negative bacteria produce membrane vesicles which are capable of killing other bacteria. J Bacteriol180:5478–5483
    [Google Scholar]
  28. MacDonald K. L., Beveridge T. J.. 2002; Bactericidal effect of gentamicin-induced membrane vesicles derived from Pseudomonas aeruginosa PAO1 on Gram-positive bacteria. Can J Microbiol48:810–820[CrossRef]
    [Google Scholar]
  29. Osborn M. J., Gander J., Parisi E., Carson J.. 1972; Mechanism and assembly of the outer membrane of Salmonella typhimurium. J Biol Chem247:3962–3972
    [Google Scholar]
  30. Stoica O., Tuanyok A., Yao X., Jericho M. H., Pink D., Beveridge T. J.. 2003; Elasticity of membrane vesicles isolated from Pseudomonas aeruginosa. Langmuir19:10916–10924[CrossRef]
    [Google Scholar]
  31. Tavares F., Sellstedt A.. 2001; DNase-resistant DNA in the extracellular and cell wall-associated fractions of Frankia strains R43 and Cc13. Curr Microbiol42:168–172[CrossRef]
    [Google Scholar]
  32. Wai S. N., Takade A., Amako K.. 1995; The release of outer membrane vesicles from the strains of enterotoxigenic Escherichia coli. Microbiol Immunol39:451–456[CrossRef]
    [Google Scholar]
  33. Whitchurch C. B., Tolker-Nielsen T., Ragas P. C., Mattick J. S.. 2002; Extracellular DNA required for bacterial biofilm formation. Science295:1487[CrossRef]
    [Google Scholar]
  34. Yaron S., Kolling G. L., Simon L., Matthews K. R.. 2000; Vesicle-mediated transfer of virulence genes from Escherichia coli O157 : H7 to other enteric bacteria. Appl Environ Microbiol66:4414–4420[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26841-0
Loading
/content/journal/micro/10.1099/mic.0.26841-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error