DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential

Abstract

Natural membrane vesicles (n-MVs) produced byPseudomonas aeruginosaPAO1 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 andEscherichia coliDH5α. 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 DH5αunder 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 (ρ=1·240 and 1·260 g ml−1). p-MVs also produced two bands but at two different densities (ρ=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)−1as 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.