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

Outer Membrane Permeability and Antibiotic Resistance

The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane alpha-helices, but instead fold into antiparallel beta-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2000.01983.x

Structure and function of bacterial outer membrane proteins: barrels in a nutshell

The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

/pdf/the-challenge-of-efflux-mediated-antibiotic-resistance-in-3le37l35cs.pdf

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies

Acinetobacter baumannii contains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system of Escherichia coli. We expressed adeABC and adeIJK of A. baumannii, as well as E. coli acrAB, in an E. coli host from which acrAB was deleted. The A. baumannii pumps were functional in E. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in the E. coli host.

/pdf/properties-of-adeabc-and-adeijk-efflux-systems-of-1n0rw615e7.pdf

Properties of AdeABC and AdeIJK Efflux Systems of Acinetobacter baumannii Compared with Those of the AcrAB-TolC System of Escherichia coli

Pore-forming activity of OmpA protein of Escherichia coli.

An investigation was made into the oligomerization, the ability to form pores and the secretion‐related properties of the 45 kDa C‐terminal domain of the IgA protease (C‐IgAP) from Neisseria gonorrhoeae . This protease is the best studied example of the autotransporters (ATs), a large family of exoproteins from Gram‐negative bacteria that includes numerous virulence factors from human pathogens. These proteins contain an N‐terminal passenger domain that em bodies the secreted polypeptide, while the C‐domain inserts into the outer membrane (OM) and trans locates the linked N‐module into the extracellular medium. Here we report that purified C‐IgAP forms an oligomeric complex of ∼500 kDa with a ring‐like structure containing a central cavity of ∼2 nm diameter that is the conduit for the export of the N‐domains. These data overcome the previous model for ATs, which postulated the passage of the N‐module through the hydrophilic channel of the β‐barrel of each monomeric C‐domain. Our results advocate a secretion mechanism not unlike other bacterial export systems, such as the secretins or fimbrial ushers, which rely on multimeric complexes assembled in the OM.

https://www.embopress.org/doi/pdf/10.1093/emboj/21.9.2122

Export of autotransported proteins proceeds through an oligomeric ring shaped by C‐terminal domains

http://www.jbc.org/content/269/27/17981.full.pdf

OmpA protein of Escherichia coli outer membrane occurs in open and closed channel forms.

When purified without the use of ionic detergents, both OmpA and OprF proteins contained nearly 20% alpha-helical structures, which disappeared completely upon the addition of sodium dodecyl sulfate. This result suggests that the proteins fold in a similar manner, with an N-terminal, membrane-spanning beta-barrel domain and a C-terminal, globular, periplasmic domain.

Etsuko Sugawara

Papers

Properties of AdeABC and AdeIJK Efflux Systems of Acinetobacter baumannii Compared with Those of the AcrAB-TolC System of Escherichia coli

Pore-forming activity of OmpA protein of Escherichia coli.

Export of autotransported proteins proceeds through an oligomeric ring shaped by C‐terminal domains

OmpA protein of Escherichia coli outer membrane occurs in open and closed channel forms.

Secondary structure of the outer membrane proteins OmpA of Escherichia coli and OprF of Pseudomonas aeruginosa.