Pore functioning of outer membrane protein PhoE of Escherichia coli: mutagenesis of the constriction loop L3

Gelder, Patrick Van; Saint, Nathalie; Boxtel, Ria van; Rosenbusch, J P; Tommassen, Jan

doi:10.1093/protein/10.6.699

Cited by 49 publications

(43 citation statements)

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“…We show here that in E. aerogenes, the activities of imipenem and cefepime, two zwitterionic molecules, are only weakly affected compared to large negatively charged drugs, and similar observations were reported with OmpK35 and OmpK36 (12,20,25). Interestingly, an S residue is located in the PhoE eyelet and has been reported to be involved in the rate of uptake of large negatively charged cephalosporins (29). Taking into account the determination of Omp35 channel properties, the characteristics of Omp36 previously determined (9), and the analyses of ␤-lactam drug diffusions through the E. coli porins (31), it appears that the WNY sequence could be involved in cephalosporin uptake.…”

Section: Discussionsupporting

confidence: 86%

“…Omp35 forms cation selective channels in planar lipid bilayers, similarly to Omp36 (9). The Omp35 channel tended to close at a lower critical voltage than Omp36 channels and behaved more like OmpF and PhoE (28,29). It had a high channel conductance, 1,430 pS in 1 M KCl for the monomer, quite similar to the OmpF channel (26).…”

Section: Discussionmentioning

confidence: 96%

“…In OmpF, the corresponding residues play a critical role in determining the characteristics of the pore (4,16,17). Various mutations located in these residues which perturb the electrostatic field acting in the eyelet strongly change the physicochemical and biological channel properties (4,26,28,29). The representation of the OmpK36, based on crystallographic structure (13), shows that the three residues Y116, G117, and S118, polarapolar-polar residues, participate in the hydrogen-bond network in the eyelet due to their special location and could be involved in the porin properties.…”

Section: Discussionmentioning

confidence: 99%

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Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins

Bornet

Saint

Fetnaci

et al. 2004

Antimicrob Agents Chemother

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In Enterobacter aerogenes, ␤-lactam resistance often involves a decrease in outer membrane permeability induced by modifications of porin synthesis. In ATCC 15038 strain, we observed a different pattern of porin production associated with a variable antibiotic susceptibility. We purified Omp35, which is expressed under conditions of low osmolality and analyzed its pore-forming properties in artificial membranes. This porin was found to be an OmpF-like protein with high conductance values. It showed a noticeably higher conductance compared to Omp36 and a specific location of WNYT residues in the L3 loop. The importance of the constriction region in the porin function suggests that this organization is involved in the level of susceptibility to negative large cephalosporins such as ceftriaxone by bacteria producing the Omp35 porin subfamily.The bacterial porins are major outer membrane proteins that form water-filled channels, allowing the diffusion across the outer membrane of small polar molecules, amino acids, and nutrients (14,18,23). General nonspecific porins, such as OmpF and OmpC from Escherichia coli, form homotrimers in the outer membrane. These porin trimers are constituted by large ␤-barrel monomer subunits which are constricted about halfway through the membrane by an internal loop, loop 3 (L3) (18). Resolution of the three-dimensional structure of the E. coli OmpF and Klebsiella pneumoniae OmpK36 has led to identification of the functional domains of the enterobacterial channels (5,8,13,17).Enterobacter aerogenes is one of the most frequently described gram-negative pathogens responsible for nosocomial respiratory tract infections in France and Belgium (2, 6, 10). This bacterial pathogen harbors a variety of antibiotic resistance mechanisms (7,9,19,24,30). Among them, the modification of outer membrane permeability, a porin deficiency associated with the expression of cephalosporinase activity, is frequently detected in clinical resistant isolates (7,19). We recently isolated a strain with an unusual porin phenotype: this EA3 strain synthesized a mutated Omp36 porin containing a G112D substitution in the L3 domain (19). Despite this prominent role of porins in drug susceptibility, only one porin, Omp36, is today functionally characterized in E. aerogenes (19).The aim of this work was to investigate the structural and functional properties of the second major pore-forming protein produced by the E. aerogenes strain. The sequence and the channel properties of the new porin Omp35 were determined and agree with the biological differences concerning the ␤-lactam susceptibility, observed between the bacteria producing either Omp35 or Omp36. MATERIALS AND METHODSBacterial strains, growth conditions, and antibiotic susceptibility tests. The E. aerogenes ATCC 15038 strain was used as the standard strain. E. aerogenes EAEP289 is a Kan s derivative of the previously described clinical strain EA27 (24). Bacteria were routinely grown in Luria-Bertani (LB) medium or nutrient broth (NB). For the determination of MI...

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins

Bornet

Saint

Fetnaci

et al. 2004

Antimicrob Agents Chemother

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“…To examine the permeability of porins in vivo, we measured the hydrolysis of the chromogenic ␤-lactam antibiotic nitrocefin in cells expressing periplasmic ␤-lactamase. In this assay, the diffusion of the antibiotics across the outer membrane is the ratelimiting step in their hydrolysis (41). L3481, a ␤-lactamase-producing PIB strain, was used as a recipient for the mtrR and porB-G120K resistance determinants.…”

Section: Sugar Permeation Through Recombinant Wild-type and Variant Pmentioning

confidence: 99%

Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae : Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations

et al. 2006

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Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46: 2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or ␤-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci.In the 1970s and early 1980s, Neisseria gonorrhoeae, the etiological agent of the sexually transmitted infection gonorrhea, gradually became more resistant to penicillin and tetracycline until these antibiotics were discontinued as a first-line therapy. Resistance to these antibiotics can be either plasmid mediated or chromosomally mediated. Plasmid-mediated resistance to penicillin or tetracycline involves expression of a TEM-1-like ␤-lactamase (8, 30) or the TetM protein (25), respectively. In contrast, chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains arise from cumulative mutations in endogenous genes or loci that gradually increase resistance until treatment failure occurs (3).Four genes or loci (penA, mtrR, penB, and ponA) are known to be involved in high-level penicillin resistance (MICs, Ն2 g/ml) in CMRNG strains, while the existence of a fifth resistance gene is inferred but not yet identified (3, 9, 32). penA (38, 39) and ponA (32, 33) encode alterations in the two essential penicillin-binding proteins (PBP 2 and PBP 1, respectively) that decrease their rates of acylation by ␤-lactam antibiotics. A single-base-pair deletion in the mtrR promoter abolishes transcription of the mtrR...

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“…1988; Rocque & McGroarty, 1990;Lou et al, 1996;Saint et al, 1996a), and site-directed mutations (Bauer et al, 1989;Bishop et al, 1996;Le Dain et al, 1996;Saint et al, 1996b: Gokce et al, 1997Srikumar et a]., 1997; Van Gelder et al, 1997). As a general result, point mutations altering the charge at the pore eyelet gave rise to ion conductivity and selectivity changes, while growth selection for maltodextrin usage resulted among others in deletions within the constriction loop L3, presumably giving rise to larger eyelet cross sections.…”

mentioning

confidence: 99%

Porin mutants with new channel properties

et al. 1998

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The general diffusion porin from Rhodopseudomonas blastica was produced in large amounts in Escherichia coli inclusion bodies and (re)natured to the exact native structure. Here, we report on 13 mutants at the pore eyelet giving rise to new diffusion properties as measured in planar lipid bilayer experiments. The crystal structures of seven of these mutants were established. The effects of charge-modifying mutations at the pore eyelet are consistent with the known selectivity for cations. Deletions of 16 and 27 residues of the constriction loop L3 resulted in labile trimers and pores. The reduction of the eyelet cross section by introducing tryptophans gave rise to a closely correlated decrease of the conductivities. A mutant with six newly introduced tryptophans in the eyelet closed its pore in a defined manner within seconds under a voltage of 20 mV, suggesting the existence of two states. The results indicate that the pore can be engineered in a rational manner.Keywords: lipid bilayer experiments; membrane channel; pore eyelet mutants; Rhodopseudomonas blastica; voltage gating; X-ray structure analysis General diffusion porins are water-filled passive channels spanning the outer membrane of Gram-negative bacteria. They are permeable for small polar solutes with exclusion limits around 600 Da, but exclude nonpolar molecules of comparable sizes. Porins are particularly stable toward heat, detergents, and proteases. Usually porins are found as homotrimeric proteins with molecular masses ranging from 30 to 50 kDa per subunit (Benz & Bauer, 1988;Nikaido, 1994;Schulz, 1996). An initial structure analysis of the major porin from Rhodobacfer capsulatus demonstrated that the pore of each subunit is formed by a 16-stranded P-barrel constricted to an eyelet near its center (Weiss et al., 1990). The constricting loop L3 contains around 40 residues connecting the fifth with the sixth strand of the @barrel. The same applies for other general diffusion porins (Schulz, 1993), among them the major porin from Rhodopsrudomonas blastica , which is the object of the reported investigation (Fig. I).Previously reported porin modifications can be subdivided into chemical labeling (Przybylski et al., 1996), growth-selected mutations, for example, for colicin resistance (Jeanteur et al., 1994) Rocque & McGroarty, 1990;Lou et al., 1996;Saint et al., 1996a), and site-directed mutations (Bauer et al., 1989; Bishop et al., 1996;Le Dain et al., 1996;Saint et al., 1996b: Gokce et al., 1997 Srikumar et a]., 1997; Van Gelder et al., 1997). As a general result, point mutations altering the charge at the pore eyelet gave rise to ion conductivity and selectivity changes, while growth selection for maltodextrin usage resulted among others in deletions within the constriction loop L3, presumably giving rise to larger eyelet cross sections. Here, we report on the properties and structures of site-directed point mutations as well as deletions in the constriction loop L3 that were meant to alter the charge pattern at the eyelet and to incr...

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Pore functioning of outer membrane protein PhoE of Escherichia coli: mutagenesis of the constriction loop L3

Cited by 49 publications

References 0 publications

Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins

Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins

Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae : Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations

Porin mutants with new channel properties

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