Isolation and characterization of OmpC porin mutants with altered pore properties

Misra, Rajeev; Benson, Spencer A.

doi:10.1128/jb.170.2.528-533.1988

Cited by 78 publications

(85 citation statements)

References 33 publications

(13 reference statements)

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“…These results are consistent with observations that altered porins, or their regulation are often responsible for cefoxitin resistance in E coli (Clarke et al, 2003). Conversely, when porins are forced to permeate larger di-or trisaccharides as growth substrates (Misra and Benson, 1988;Zhang and Ferenci, 1999), mutations in porin regulation and structure can overcome the permeability limitation, but always resulted in increased sensitivity to antibiotic and detergent (Misra and Benson, 1988;Zhang and Ferenci, 1999).…”

Section: Discussionsupporting

confidence: 80%

A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli

Phan

Ferenci

2013

The ISME Journal

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Bacterial species are internally diverse in genomic and multi-locus gene comparisons. The ecological causes of phenotypic and genotypic diversity within species are far less well understood. Here, we focus on the competitive fitness for growth on nutrients within Escherichia coli, an internally rich species. Competition experiments in nutrient-limited chemostats revealed that members of the ECOR collection exhibited a wide continuum of competitive abilities, with some fitter and some less fit than the lab strain MG1655. We observed an inverse relationship between competitiveness and the resistance of strains to detergent and antibiotic, consistent with the notion that membrane permeability and competitive fitness are linked by a trade-off between selfpreservation and nutritional competence (SPANC); high permeability has a postulated cost in antibacterial sensitivity whereas a low permeability has a cost in nutrient affinity. Isolates moved along the markedly nonlinear trade-off curve by mutational adaptation; an ECOR strain sensitive to antibacterials and a good competitor was easily converted by mutation into a mutant with higher resistance but poorer competition in the presence of low antibiotic concentrations. Conversely, a resistant ECOR strain changed into a better competitor after a short period of selection under nutrient limitation. In both directions, mutations can affect porin proteins and outer membrane permeability, as indicated by protein analysis, gene sequencing and an independent assay of outer membrane permeability. The extensive, species-wide diversity of E. coli in ecologically important traits can thus be explained as an evolutionary consequence of a SPANC trade-off driven by antagonistic pleiotropy.

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Section: Discussionsupporting

confidence: 80%

A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli

Phan

Ferenci

2013

The ISME Journal

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“…Indeed, a liposome-swelling assay with disaccharides unequivocally showed that the mutant channels were larger than the wild-type channels (571). Similar, larger-channel mutants were also isolated in OmpC (419), and the enlargement of the channels was confirmed by biochemical studies (555).…”

Section: Classical Porinsmentioning

confidence: 76%

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Nikaido

2003

Microbiol Mol Biol Rev

3,768

3,800

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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

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“…Because reliable data cannot be obtained under such conditions, we used a strain of E. coli producing a wider mutant porin channel (19,20). This strain, RAM121, allows an influx of larger maltodextrins and large antibiotics that are excluded by the normal E. coli porins (19) and shows a larger single channel conductivity (21). Indeed, it allowed a nearly 10-fold faster influx of nitrocefin, with the permeability coefficient of about 0.2 ϫ 10 Ϫ5 cm/s.…”

Section: Resultsmentioning

confidence: 99%

“…For nitrocefin efflux, we used a strain producing a mutant OmpC porin producing a large channel, RAM121 (19), a kind gift from Rajeev Misra, Arizona State University. To increase efflux we transduced ⌬acrR::kan mutation from AG100B (16) (K-12 argE3 thi-1 rpsL xyl mtl ⌬(gal-uvrB) supE44 acrR::kan), to obtain HN1157.…”

Section: Methodsmentioning

confidence: 99%

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli

Nagano

Nikaido

2009

Proc. Natl. Acad. Sci. U.S.A.

115

186

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Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic ␤-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a Km of about 5 M with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.cephalosporin ͉ drug efflux pump ͉ multidrug resistance M ultidrug eff lux pumps of the resistance-nodulationdivision (RND) family in Gram-negative bacteria can pump out a surprisingly wide range of antimicrobial compounds (1, 2). For example, AcrB of Escherichia coli, which has been studied most extensively as a prototype of similar pumps, exports a number of dyes, detergents, chloramphenicol, tetracyclines, macrolides, novobiocin, fluoroquinolones, and organic solvents. Importantly, it also pumps out ␤-lactams, some of which cannot easily cross the cytoplasmic membrane and thus stay in the periplasm (3). However, in the 13 years since their discovery (4, 5), no success was achieved in the determination of kinetic behavior of these pumps, although the affinity of some substrates could be assessed on a relative scale by their activity as competitive inhibitors in a proteoliposome reconstitution assay (6). Recently an attempt to measure the binding of dyes to purified AcrB was made by using fluorescence polarization (7); however, it is unclear if the experiments measured binding to active sites or to generally hydrophobic pockets in the protein.The knowledge of kinetic constants is essential in our attempts to understand the contribution of the pumps to drug resistance in a quantitative manner. Furthermore, recent crystallographic studies of AcrB (8-10) suggest that each protomer in this trimeric transporter undergoes a succession of conformational changes that are dependent on the conformations of the neighbors, that is, a functionally rotating mechanism. This m...

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Isolation and characterization of OmpC porin mutants with altered pore properties

Cited by 78 publications

References 33 publications

A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli

A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli

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