| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Canadian Bacterial Disease Network–National Centre of Excellence and Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Correspondence
Terry J. Beveridge
tjb{at}uoguelph.ca
Natural membrane vesicles (n-MVs) produced by Pseudomonas aeruginosa 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)–1. PCR analysis using specific primers for pAK1900 sequences and a chromosomal target, oprL, 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 Escherichia coli 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–1). p-MVs also produced two bands but at two different densities (=1·250 and 1·265 g ml–1) 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-D-manno-octulosonic acid (KDO) (mg protein)–1 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.
This article has been cited by other articles:
|
|
 |
|
  S. R. Schooling, A. Hubley, and T. J. Beveridge Interactions of DNA with Biofilm-Derived Membrane Vesicles J. Bacteriol., July 1, 2009; 191(13): 4097 - 4102. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Vilain, J. M. Pretorius, J. Theron, and V. S. Brozel DNA as an Adhesin: Bacillus cereus Requires Extracellular DNA To Form Biofilms Appl. Envir. Microbiol., May 1, 2009; 75(9): 2861 - 2868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sun, X. Zhang, L. Wang, M. Prudhomme, Z. Xie, B. Martin, and J.-P. Claverys Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli J. Bacteriol., February 1, 2009; 191(3): 713 - 719. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Alaniz, B. L. Deatherage, J. C. Lara, and B. T. Cookson Membrane Vesicles Are Immunogenic Facsimiles of Salmonella typhimurium That Potently Activate Dendritic Cells, Prime B and T Cell Responses, and Stimulate Protective Immunity In Vivo J. Immunol., December 1, 2007; 179(11): 7692 - 7701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. C. Rice, E. E. Mann, J. L. Endres, E. C. Weiss, J. E. Cassat, M. S. Smeltzer, and K. W. Bayles The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus PNAS, May 8, 2007; 104(19): 8113 - 8118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. L. Simmons, J. R. Bolland, J. M. Daubenspeck, and K. Dybvig A Stochastic Mechanism for Biofilm Formation by Mycoplasma pulmonis J. Bacteriol., March 1, 2007; 189(5): 1905 - 1913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Schooling and T. J. Beveridge Membrane vesicles: an overlooked component of the matrices of biofilms. J. Bacteriol., August 1, 2006; 188(16): 5945 - 5957. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ando, H. Suzuki, S. Nishimura, T. Tanaka, A. Hiraishi, and Y. Kikuchi Characterization of Extracellular RNAs Produced by the Marine Photosynthetic Bacterium Rhodovulum sulfidophilum. J. Biochem., April 1, 2006; 139(4): 805 - 811. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Kuehn and N. C. Kesty Bacterial outer membrane vesicles and the host-pathogen interaction Genes & Dev., November 15, 2005; 19(22): 2645 - 2655. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
