Naturally produced membrane vesicles (MVs), isolated from 15 strains of gram-negative bacteria (Citrobacter,Enterobacter, Escherichia,Klebsiella, Morganella, Proteus,Salmonella, and Shigella strains), lysed many gram-positive (including Mycobacterium) and gram-negative cultures. Peptidoglycan zymograms suggested that MVs contained peptidoglycan hydrolases, and electron microscopy revealed that the murein sacculi were digested, confirming a previous modus operandi (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 174:2767–2774, 1996). MV-sensitive bacteria possessed A1α, A4α, A1γ, A2α, and A4γ peptidoglycan chemotypes, whereas A3α, A3β, A3γ, A4β, B1α, and B1β chemotypes were not affected. Pseudomonas aeruginosa PAO1 vesicles possessed the most lytic activity.

The mechanism of uptake of aminoglycosides across the outer membrane of Escherichia coli was reevaluated. Porin-deficient mutants showed no alteration in gentamicin or kanamycin susceptibility. Furthermore, the influence of kanamycin on intrinsic tryptophan fluorescence of porin OmpF (Y. Kobayashi, and T. Nakae, Eur. J. Biochem. 151:231-236, 1985) was shown to be strongly influenced by protein concentration and EDTA. This led to the hypothesis that aminoglycoside-mediated increases and decreases in intrinsic tryptophan fluorescence were due to aggregation-disaggregation of OmpF mediated by interaction at a divalent cation binding site on OmpF. Gentamicin, kanamycin, and polymyxin B increased E. coli outer membrane permeability to the hydrophobic fluorescent compound 1-N-phenyl-naphthylamine (NPN) and the peptidoglycan-degrading enzyme lysozyme. Addition of Mg2+ blocked these permeabilizing activities. Furthermore, gentamicin and polymyxin B bound to Mg2+-binding sites on E. coli lipopolysaccharide, as determined in dansyl polymyxin displacement experiments. A polymyxin-resistant, lipopolysaccharide-altered pmr mutant of E. coli had a fourfold-lower MIC of gentamicin and kanamycin and was more poorly permeabilized to 1-N-phenylnaphthylamine than was its parent strain. These data were consistent with uptake of aminoglycosides across the E. coli outer membrane by the self-promoted uptake mechanism.The outer membrane of gram-negative bacteria is a semipermeable barrier which restricts the access of all antibiotics to their targets (8,9,23). Hydrophilic molecules of sizes below a given exclusion limit can pass through the waterfilled channels of proteins called porins. Included among these molecules are the ,B-lactam antibiotics. The most convincing data suggesting that r-lactams are taken up through porin channels are the increases in MICs of P-lactams in mutants with deficiencies in porins (8,13,15,16,23), although this conclusion is supported by liposome swelling data showing the uptake of P-lactams into liposomes reconstituted with porins (32).In contrast, polycations, some of which are larger than the exclusion limit of the outer membrane, can be taken up by another mechanism, which we have termed the self-promoted uptake pathway (7,11,22). In this pathway, the polycations act to competitively displace divalent cations which cross-bridge adjacent lipopolysaccharide (LPS) molecules, thus disrupting these important outer membrane stabilizing sites. This, then, permeabilizes the outer membrane and is proposed to promote uptake of other molecules of the permeabilizing polycation. Mutants which interact more poorly with polymyxin B have been shown to resist killing by that polycation (11,22,25,29,30), while a mutant (totA) with LPS which better interacted with polycations was supersusceptible (26). These data show that self-promoted uptake is the first stage of uptake leading to cell killing by certain polycationic antibiotics.In the case of aminoglycosides, the mechanism of uptake across the outer membrane has been...

A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures. In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases. Immunogold labeling of thin sections of P. aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm. It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled. Since division is due to cell constriction in this P. aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled. Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm. Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide.

The surfaces of bacteria are highly interactive with their environment. Whether the bacterium is Gram-negative or Gram-positive, most surfaces are charged at neutral pH because of the ionization of the reactive chemical groups which stud them. Since prokaryotes have a high surface area-to-volume ratio, this can have surprising ramifications. For example, many bacteria can concentrate dilute environmental metals on their surfaces and initiate the development of fine-grained minerals. In natural environments, it is not unusual to find such bacteria closely associated with the minerals which they have helped develop. Bacteria can be free-living (planktonic), but in most natural ecosystems they prefer to grow on interfaces as biofilms; supposedly to take advantage of the nutrient concentrative effect of the interface, although there must also be gained some protective value against predators and toxic agents. Using a Pseudomonas aeruginosa model system, we have determined that lipopolysaccharide is important in the initial attachment of this Gram-negative bacterium to interfaces and that this surface moiety subtly changes during biofilm formation. Using this same model system, we have also discovered that there is a natural tendency for Gram-negative bacteria to concentrate and package periplasmic components into membrane vesicles which bleb-off the surface. Since some of these components (e.g., peptidoglycan hydrolases) can degrade other surrounding cells, the vesicles could be predatory; i.e., a natural system by which neighboring bacteria are targeted and lysed, thereby liberating additional nutrients to the microbial community. This obviously would be of benefit to vesicle-producing bacteria living in biofilms containing mixed microbial populations.

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