Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Bacterial biofilms in nature and disease.

Acinetobacter baumannii has emerged as a highly troublesome pathogen for many institutions globally. As a consequence of its immense ability to acquire or upregulate antibiotic drug resistance determinants, it has justifiably been propelled to the forefront of scientific attention. Apart from its predilection for the seriously ill within intensive care units, A. baumannii has more recently caused a range of infectious syndromes in military personnel injured in the Iraq and Afghanistan conflicts. This review details the significant advances that have been made in our understanding of this remarkable organism over the last 10 years, including current taxonomy and species identification, issues with susceptibility testing, mechanisms of antibiotic resistance, global epidemiology, clinical impact of infection, host-pathogen interactions, and infection control and therapeutic considerations.

Acinetobacter baumannii: Emergence of a Successful Pathogen

Molecular basis of bacterial outer membrane permeability.

The complete general secretory pathway in gram-negative bacteria

The subcellular distribution of carnitine acyltransferases in mammalian liver and kidney. A new peroxisomal enzyme.

Inductively coupled plasma emission spectroscopy was used to quantitate the metal cations bound to outer and cytoplasmic membranes and to extracted lipopolysaccharide from several Escherichia coli K12 strains. The outer membrane was found to be enriched in both calcium and magnesium relative to the cytoplasmic membrane. Both membranes contained significant levels of iron, aluminum, and zinc. The multivalent cation content of the lipopolysaccharide resembled that of the intact outer membrane. Lipopolysaccharide extracted from wild-type k12 strains contained higher levels of Mg than Ca regardless of the growth medium, but the medium used for growth did affect the relative amounts of bound Mg as well as the levels of the minor cations iron, aluminum, and zinc. In contrast, lipopolysaccharide isolated from a deep rough mutant strain, D21f2, contained more Ca than Mg. Electrodialysis of lipopolysaccharide from wild-type k12 strains removed 1 mol of Mg per mol of lipopolysaccharide but did not significantly affect the level of other bound metal ions. Dialysis of lipopolysaccharide against sodium (ethylenedinitrilo)tetraacetate removed most of the Mg and Ca, resulting in a sodium salt. The equimolar replacement of divalent cations with sodium in the sodium salt resulted in a net loss of counterion change. The sodium salt was dialyzed against either tris(hydroxymethyl)aminomethane hydrochloride, CaCl2, MgCl2, or TbCl3, and the resulting lipopolysaccharide salts were analyzed for their ionic composition. It was shown that tris(hydroxymethyl)aminomethane and Ca can replace some but not all of the Na bound to the sodium salt, but all of the other multivalent cations tested replaced Na, resulting in uniform lipopolysaccharide salts. Lipopolysaccharide isolated from the deep rough mutant strain D21f2 was also converted into a sodium salt. Relative to the wild-type lipopolysaccharide, Na was able to neutralize the anionic charge to a greater extent in the mutant lipopolysaccharide. Our results suggest that the loss of specific groups in the core region of the lipopolysaccharide from the mutant strain results in a more open structure that allows the binding of larger cations and of more monovalent cations.

Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli.

Polycations, such as aminoglycoside and peptide antibiotics, and naturally occurring polyamines were found to bind to the lipopolysaccharide of Pseudomonas aeruginosa and alter its packing arrangement. Binding of cations was measured by the displacement of a cationic spin probe from lipopolysaccharide into the aqueous environment upon addition of competitive cations. The level of probe displacement was dependent on the concentration and charge of the competing cation, with the more highly charged cations being more effective at displacing probe. The relative affinity of several antibiotics for lipopolysaccharide correlated with their ability to increase outer membrane permeability, while the relative affinity of several polyamines correlated with their ability to stabilize the outer membrane. Probe mobility within the lipopolysaccharide head group was shown to be decreased by cationic antibiotics and unaltered or increased by polyamines. We propose that antibiotic permeability and disruption of outer membrane integrity by polycationic antibiotics results from binding of the antibiotic to anionic groups on lipopolysaccharide with a consequent change in the conformation of lipopolysaccharide aggregate structure.

Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa.

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different. Images

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Porin is a trimeric channel-forming protein in the outer membrane of Gram-negative bacteria. Functions of the porins OmpF, OmpC, and PhoE from Escherichia coli K12 were analyzed at various pHs. Preliminary results from bilayer lipid membrane and liposome swelling assays indicated that in vitro porin has at least two open-channel configurations with a small and a large size. The small channels were stabilized at low pH while the larger channels were detected under basic conditions. The size switch occurred over a very narrow range near neutral pH, and the two major open-channel configurations responded differently to variations in voltage. The presence of two or more pH-dependent substates of porin could explain the variability in pore diameter measured by others and suggests a more dynamic role for porin in the cell.

Estelle J. McGroarty

Papers

The subcellular distribution of carnitine acyltransferases in mammalian liver and kidney. A new peroxisomal enzyme.

Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli.

Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa.

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Effects of pH on bacterial porin function.