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1.
When grown at acidic pH, Escherichia coli cells secrete cadaverine, a polyamine known to inhibit porin-mediated outer membrane permeability. In order to understand the physiological significance of cadaverine excretion and the inhibition of porins, we isolated an OmpC mutant that showed resistance to spermine during growth and polyamine-resistant porin-mediated fluxes. Here, we show that the addition of exogenous cadaverine allows wild-type cells to survive a 30-min exposure to pH 3.6 better than cells expressing the cadaverine-insensitive OmpC porin. Competition experiments between strains expressing either wild-type or mutant OmpC showed that the lack of sensitivity of the porin to cadaverine confers a survival disadvantage to the mutant cells at reduced pH. On the basis of these results, we propose that the inhibition of porins by excreted cadaverine represents a novel mechanism that provides bacterial cells with the ability to survive acid stress. 相似文献
2.
Excretion of Endogenous Cadaverine Leads to a Decrease in Porin-Mediated Outer Membrane Permeability 总被引:6,自引:2,他引:4
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The permeability of the outer membrane of Escherichia coli to hydrophilic compounds is controlled by porin channels. Electrophysiological experiments showed that polyamines inhibit ionic flux through cationic porins when applied to either side of the membrane. Externally added polyamines, such as cadaverine, decrease porin-mediated fluxes of β-lactam antibiotics in live cells. Here we tested the effects of endogenously expressed cadaverine on the rate of permeation of cephaloridine through porins, by manipulating in a pH-independent way the expression of the cadBA operon, which encodes proteins involved in the decarboxylation of lysine to cadaverine and in cadaverine excretion. We report that increased levels of excreted cadaverine correlate with a decreased outer membrane permeability to cephaloridine, without any change in porin expression. Cadaverine appears to promote a sustained inhibition of porins, since the effect remains even after removal of the exogenously added or excreted polyamine. The cadaverine-induced inhibition is sufficient to provide cells with some resistance to ampicillin but not to hydrophobic antibiotics. Finally, the mere expression of cadC, in the absence of cadaverine production, leads to a reduction in the amounts of OmpF and OmpC proteins, which suggests a novel mechanism for the environmental control of porin expression. The results presented here support the notion that polyamines can act as endogenous modulators of outer membrane permeability, possibly as part of an adaptive response to acidic conditions. 相似文献
3.
The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane. 相似文献
4.
We have used the electrophysiological technique of patch-clamp to study the modulation of Escherichia coli porins by cadaverine. Porin channels typically have a very high probability to be open, and were not known to be inhibited by specific compounds until the present study. Experiments performed on patches of outer membrane reconstituted in liposomes reveal that cadaverine applied to the periplasmic side increases the frequency of channel closures in a concentration-dependent fashion, and thereby decreases the total amount of ion flux through a porin-containing membrane. The positive charge on cadaverine is important for inhibition, because the effect is relieved at higher pH where fewer polyamine molecules are charged. Modulation is observed only at negative pipet voltages, and therefore confers voltage dependence to porin activity. Cadaverine increases the number and duration of cooperative closures of more than one channel, suggesting that it does not merely block the pore but exerts its kinetic effect allosterically. As a biological assay of porin inhibition, E. coli behavior in chemotaxis swarm plates was tested and found to be impaired in the presence of cadaverine. Polyamines are naturally found associated with the outer membrane of E.coli, but are lost upon fractionation. We postulate that cadaverine might be a natural regulator of porin activity. 相似文献
5.
Emmanuelle Dé Arnaud Baslé Michel Jaquinod Nathalie Saint Monique Malléa Gérard Molle Jean-Marie Pagès 《Molecular microbiology》2001,41(1):189-198
In Enterobacter aerogenes, multidrug resistance involves a decrease in outer membrane permeability associated with changes in an as yet uncharacterized porin. We purified the major porin from the wild-type strain and a resistant strain. We characterized this porin, which was found to be an OmpC/OmpF-like protein and analysed its pore-forming properties in lipid bilayers. The porin from the resistant strain was compared with the wild-type protein and we observed (i) that its single-channel conductance was 70% lower than that of the wild type; (ii) that it was three times more selective for cations; (iii) a lack of voltage sensitivity. These results indicate that the clinical strain is able to synthesize a modified porin that decreases the permeability of the outer membrane. Mass spectrometry experiments identified a G to D mutation in the putative loop 3 of the porin. Given the known importance of this loop in determining the pore properties of porins, we suggest that this mutation is responsible for the novel resistance mechanism developed by this clinical strain, with changes in porin channel function acting as a new bacterial strategy for controlling beta-lactam diffusion via porins. 相似文献
6.
Harsha Bajaj Mariano A. Scorciapino Lucile Moynié Malcolm G. P. Page James H. Naismith Matteo Ceccarelli Mathias Winterhalter 《The Journal of biological chemistry》2016,291(6):2837-2847
Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by “trapping” the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria. 相似文献
7.
Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins 总被引:2,自引:0,他引:2
Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined. 相似文献
8.
F R Sallmann S Baveye-Descamps F Pattus V Salmon N Branza G Spik D Legrand 《The Journal of biological chemistry》1999,274(23):16107-16114
The binding of lactoferrin, an iron-binding glycoprotein found in secretions and leukocytes, to the outer membrane of Gram-negative bacteria is a prerequisite to exert its bactericidal activity. It was proposed that porins, in addition to lipopolysaccharides, are responsible for this binding. We studied the interactions of human lactoferrin with the three major porins of Escherichia coli OmpC, OmpF, and PhoE. Binding experiments were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds to the purified native OmpC or PhoE trimer with molar ratios of 1.9 +/- 0.4 and 1.8 +/- 0.3 and Kd values of 39 +/- 18 and 103 +/- 15 nM, respectively, but not to OmpF. Furthermore, preferential binding of lactoferrin was observed on strains that express either OmpC or PhoE. It was also demonstrated that residues 1-5, 28-34, and 39-42 of lactoferrin interact with porins. Based on sequence comparisons, the involvement of lactoferrin amino acid residues and porin loops in the interactions is discussed. The relationships between binding and antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological studies revealed that lactoferrin can act as a blocking agent for OmpC but not for PhoE or OmpF. However, a total inhibition of the growth was only observed for the PhoE-expressing strain (minimal inhibitory concentration of lactoferrin was 2.4 mg/ml). These data support the proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the stability and/or the permeability of the bacterial outer membrane. 相似文献
9.
A possible role of cadaverine in the biosynthesis of polyamines in the Japanese newt testis 总被引:1,自引:0,他引:1
Polyamine content in testes of various vertebrates was studied extensively. Putrescine, spermidine and spermine were detected in all the animals examined, although the distribution pattern varied greatly from animal to animal. Cadaverine was detected only in amphibian testes; sym-homospermidine was found not only in testes but also in various other tissues of amphibians and of some reptiles. In the newt testis the concentration of cadaverine was lower than that of any other polyamines in summer, but there was a great increase in cadaverine content from autumn to winter. The testicular content of cadaverine was greater than that of other polyamines in winter. There was a gradual decrease in the cadaverine content in spring. The spermidine and spermine levels, which were rather low in winter, increased in spring and reached a peak in summer when spermatogenesis was active. The testicular concentration of putrescine that was much higher than that of spermidine or spermine throughout the year, increased only a little in summer. There was a significant negative correlation between the cadaverine levels and four other polyamine levels. Exogenous cadaverine decreased the testicular levels of putrescine. Mammalian gonadotropins decreased the cadaverine levels and increased the levels of other polyamines. A partially purified LH fraction from pituitaries of bullfrog, Rana catesbeiana, was also potent in depleting cadaverine of the testes of newts kept at 8 degrees C. These results suggest that testicular cadaverine suppresses the biosynthesis of polyamines, especially spermidine and spermine which are closely associated with spermatogenesis. 相似文献
10.
The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins. 相似文献
11.
The inhibition of the anion-selective PhoE porin by ATP and of the cation-selective OmpF porin by polyamines has been previously documented. In the present study, we have extended the comparison of the inhibitor-porin pairs by investigating the effect of anions (ATP and aspartate) and positively charged polyamines (spermine and cadaverine) on both OmpF and PhoE with the patch-clamp technique, and by comparing directly the gating kinetics of the channels modulated by their respective substrates. The novel findings reported here are (1) that the activity of PhoE is completely unaffected by polyamines, and (2) that the kinetic changes induced by ATP on PhoE or polyamines on OmpF suggest different mechanisms of inhibition. ATP induces a high degree of flickering in the PhoE-mediated current and appears to behave as a blocker of ion flow during its presumed transport through PhoE. Polyamines modulate the kinetics of openings and closings of OmpF, in addition to promoting a blocker-like flickering activity. The strong correlation between sensitivity to inhibitors and ion selectivity suggests that some common molecular determinants are involved in these two properties and is in agreement with the hypothesis that polyamines bind inside the pore of cationic porins. 相似文献
12.
Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF-ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection. 相似文献
13.
Lou H Chen M Black SS Bushell SR Ceccarelli M Mach T Beis K Low AS Bamford VA Booth IR Bayley H Naismith JH 《PloS one》2011,6(10):e25825
Antibiotic-resistant bacteria, particularly gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane. 相似文献
14.
Poor permeability of the lipopolysaccharide‐based outer membrane of Gram‐negative bacteria is compensated by the existence of protein channels (porins) that selectively admit low molecular weight substrates, including many antibiotics. Improved understanding of the translocation mechanisms of porin substrates could help guide the design of antibiotics capable of achieving high intracellular exposure. Energy barriers to channel entry and exit govern antibiotic fluxes through porins. We have previously reported a hypothesis that the costs of transferring protein solvation to and from bulk medium underlie the barriers to protein‐ligand association and dissociation, respectively, concomitant with the gain and loss of protein‐ligand interactions during those processes. We have now applied this hypothesis to explain the published rates of entry (association) and exit (dissociation) of six antibiotics to/from reconstituted E. coli porin OmpC. WaterMap was used to estimate the total water transfer energies resulting from transient occupation by each antibiotic. Our results suggest that solvation within the porin cavity is highly energetically favorable, and the observed moderately fast entry rates of the antibiotics are consistent with replacement of protein‐water H‐bonds. The observed ultrafast exit kinetics is consistent with the lack of intrachannel solvation sites that convey unfavorable resolvation during antibiotic dissociation. These results are aligned with known general relationships between antibiotic efficacy and physicochemical properties, namely unusually low logP, reflecting an abundance of H‐bond partners. We conclude that antibiotics figuratively “melt” their way through porin solvation at a rate determined by the cost of exchanging protein‐solvent for protein‐antibiotic H‐bonds. Proteins 2013. © 2012 Wiley Periodicals, Inc. 相似文献
15.
Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC.
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The immunochemistry and structure of enteric bacterial porins are critical to the understanding of the immune response to bacterial infection. We raised 41 monoclonal antibodies (MAbs) to Salmonella typhimurium OmpD and OmpC porin trimers and monomers. Enzyme-linked immunosorbent assays, immunoprecipitations, and/or Western immunoblot techniques indicated that 39 MAbs (11 anti-trimer and 28 anti-monomer) in the panel are porin specific and one binds to the lipopolysaccharide; the specificity of the remaining MAb probably lies in the porin-lipopolysaccharide complex. Among the porin-specific MAbs, 10 bound cell-surface-exposed epitopes, one reacted with a periplasmic epitope, and the remaining 28 recognized determinants that are buried within the outer membrane bilayer. Many of the MAbs reacting with surface-exposed epitopes were highly specific, recognizing only the homologous porin trimers; this suggests that the cell-surface-exposed regions of porins tends to be quite different among S. typhimurium OmpF, OmpC, and OmpD porins. Immunological cross-reaction showed that S. typhimurium OmpD was very closely related to Escherichia coli NmpC and to the Lc porin of bacteriophage PA-2. Immunologically, E. coli OmpG and protein K also appear to belong to the family of closely related porins including E. coli OmpF, OmpC, PhoE, and NmpC and S. typhimurium OmpF, OmpC, and OmpD. It appears, however, that S. typhimurium "PhoE" is not closely related to this group. Finally, about one-third of the MAbs that presumably recognize buried epitopes reacted with porin domains that are widely conserved in 13 species of the family Enterobacteriaceae, but apparently not in the seven nonenterobacterial species tested. These data are evaluated in relation to host immune response to infection by gram-negative bacteria. 相似文献
16.
Osmotic regulation of porin expression: a role for DNA supercoiling 总被引:24,自引:8,他引:16
The OmpC and OmpF porins are major outer membrane proteins of Escherichia coli and Salmonella typhimurium. Their expression is affected by many environmental factors and by mutations in a variety of independent genes. The pair of regulatory proteins, OmpR and EnvZ, are required for normal porin expression. Despite intensive investigation, the mechanisms by which porin expression is regulated remain unclear. Mutations which alter supercoiling, as well as inhibitors of DNA gyrase, show that porin expression is extremely and specifically sensitive to the level of DNA supercoiling. Our data lead us to suggest that environmentally induced changes in DNA supercoiling may play a role in determining the level of porin expression. These findings have implications for current models of porin regulation. 相似文献
17.
Michèle Kervella Jean-Louis Fauchère Didier Fourel Jean-Mari Pagès 《FEMS microbiology letters》1992,99(2-3):281-286
Immunocrossreactivity between the major outer membrane protein (MOMP) of Campylobacter jejuni 85H and the OmpC porin of Escherichia coli K-12 was observed. These results indicate that a common antigenic domain is conserved in both MOMP and OmpC. This antigenic region is detected only after a 96 degrees C treatment suggesting that it is buried in the native conformation of the respective porins. In addition, differences were observed between the major outer membrane proteins from various C. jejuni strains. About 60% of the C. jejuni pathogenic strains tested contained a protein exhibiting a similar electrophoretic profile to the 85H porin. 相似文献
18.
O. P. Vostrikova M. P. Isaeva G. N. Likhatskaya O. D. Novikova N. Yu. Kim V. A. Khomenko T. F. Solov’eva 《Biochemistry. Biokhimii?a》2013,78(5):496-504
OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the “warm” variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the “cold” variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 β-strands connected by short “periplasmic” and longer “extracellular” loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the “extracellular” loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential. 相似文献
19.
Outer membrane porins are important in maintenance of the surface structure of Escherichia coli cells. 总被引:10,自引:8,他引:2
Escherichia coli cells lacking the OmpF and OmpC proteins, porin proteins of the outer membrane, are often unstable and easily revert to strains which either have regained one or both of these proteins or contain a new outer membrane protein. The structural importance of porin proteins in the cell surface was studied in the present work. Tris-hydrochloride buffer at a concentration of 120 mM caused deformation of the cell surface of a strain lacking these porins; the undulated appearance of the negatively stained cell surface changed to a smooth and expanded form. The Tris-induced deformation was seldom observed with either the wild-type strain or a pseudorevertant that possessed the OmpF protein. The role of the OmpF protein in stabilizing the cell surface against Tris treatment could be slightly taken over by the LamB protein, which shares a number of unique properties with the former proteins. The deformation of the cell surface by Tris-hydrochloride buffer was accompanied by a loss of viability, the lethal damage being especially significant when the cells lacked porins. Upon induction with maltose, cells with the undulated appearance could absorb lambda phages, whereas the deformed cells could not. These results suggest that the instability of cells lacking porins is primarily due to a structural defect of the outer membrane. 相似文献
20.
A putative contribution of polyamines to the control of peptidase activity expression during re-growth was studied in source organs (roots and stolons) of defoliated white clover (Trifolium repens L.). Endopeptidase activity increased in roots during the first 6 days following complete defoliation, while exopeptidase expression seemed to be restricted to the early hours of re-growth. These changes correlated with an immediate 80% decline in the content of total free polyamines, mainly represented by the diamine cadaverine. The inhibitory capacities of cadaverine and spermine were tested on enzyme activity in vitro in order to elucidate whether the endogenous polyamine level was associated with the cut-induced endopeptidase expression. Cadaverine seemed to inhibit endopeptidase activity of stolons but not root endopeptidase activity. These data support the view that polyamines may play a role in the regulation of peptidase expression in source organs of white clover during post-clipping re-growth. The existence of different endopeptidase isoforms in roots and stolons is discussed in relation to the molecular mechanisms by which polyamines may regulate their activities.Abbreviations AP
aminopeptidase
- Cad
cadaverine
- CP
carboxypeptidase
- EP
endopeptidase
- PA(s)
polyamine(s)
- Spm
spermine 相似文献