Cadaverine induces closing of E. coli porins.

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.     

Excretion of Endogenous Cadaverine Leads to a Decrease in Porin-Mediated Outer Membrane Permeability

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.     

Adaptive functions of <Emphasis Type="Italic">Escherichia coli</Emphasis> polyamines in response to sublethal concentrations of antibiotics

Escherichia coli exposure to sublethal antibiotic concentrations induced an increase in cell polyamine contents. Maximum accumulation of putrescine and spermidine in response to antibiotics-induced oxidative stress preceded the increment of cadaverine, the content of which was dependent on the rpoS expression level and reached the maximum in response to fluoroquinolones. The polyamine positive modulating effects on rpoS expression increased in the following order: cadaverine-putrescine-spermidine. The reason for cadaverine accumulation was the increase in activities of lysine decarboxylases CadA and Ldc. High cadaverine accumulation in the cells exposed to fluoroquinolones and cephalosporins resulted in the reduction of porin permeability; so it was considered as a response aimed at cell protection against antibiotic penetration into the cell. Netilmycin, unlike other antibiotics, did not substantially affect the lysine decarboxylase activity and cellular polyamine pools.     

Cadaverine inhibition of porin plays a role in cell survival at acidic pH

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.     

Lysine decarboxylase activity as a factor of fluoroquinolone resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Exposure of E. coli cells to sublethal concentrations of fluoroquinolones induced synthesis of lysine decarboxylase LdcC, which was previously considered to be a constitutive enzyme. Under these conditions, a key role in this process is played by RNA polymerase σ ^S subunit (RpoS); its quantity increased substantially in the presence of antibiotics. Fluoroquinolones of the second and third generations had a more pronounced effect on rpoS expression and LdcC activity than the first-generation antibiotics. A direct correlation was shown between the level of cadaverine, the product of lysine decarboxylase reaction in E. coli cells, and their resistance to fluoroquinolones. An increase in endogenous cadaverine reduced effectiveness of the second and third-generation fluoroquinolones, but had no effect on antimicrobial activity of the first-generation antibiotics. This is in good agreement with the hydrophilic properties of antibiotics of different generations and, consequently, with different mechanisms of their penetration into bacterial cells.     

Distinct sensitivities of OmpF and PhoE porins to charged modulators

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.     

Polyamines decrease Escherichia coli outer membrane permeability.

The permeability of the outer membranes of gram-negative bacteria to hydrophilic compounds is mostly due to the presence of porin channels. We tested the effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on two processes known to depend on intact porin function: fluxes of beta-lactam antibiotics in live cells and chemotaxis. In both cases, inhibition was observed. Measurements of the rate of permeation of cephaloridine and of chemotaxis in swarm plates and capillary assays were used to determine the concentration dependence of this modulation. The effective concentration ranges depended on the nature of the polyamine and varied from submillimolar for spermine to tens of millimolar for cadaverine. Both OmpC and OmpF porins were inhibited, although the effects on OmpC appeared to be milder. These results are in agreement with our observations that polyamines inhibit porin-mediated ion fluxes in electrophysiological experiments, and they suggest that a low-affinity polyamine binding site might exist in these porins. These results reveal the potential use of porins as targets for blocking agents and suggest that polyamines may act as endogenous modulators of outer membrane permeability.     

Microscopic Mechanism of Antibiotics Translocation through a Porin

OmpF from the outer membrane of Escherichia coli is a general porin considered to be the main pathway for beta-lactam antibiotics. The availability of a high-resolution crystal structure of OmpF and new experimental techniques at the single-molecule level have opened the way to the investigation of the microscopic mechanisms that allow the passage of antibiotics through bacterial pores. We applied molecular dynamics simulations to investigate the translocation process of ampicillin (Amp) through OmpF. Using a recent algorithm capable of accelerating molecular dynamics simulations we have been able to obtain a reaction path for the translocation of Amp through OmpF. The mechanism of passage depends both on the internal degrees of freedom of Amp and on interactions of Amp with OmpF. Understanding this mechanism would help us design more efficient antibiotics and shed light on nature's way of devising channels able to enhance the transport of molecules through membranes.     

Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli

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.     

The selectivity filter of voltage-dependent channels formed by phosphoporin (PhoE protein) from E. coli.

Phosphoporin, an Escherichia coli outer membrane-spanning protein re-incorporated in phospholipid planar bilayers generates aqueous channels similar to those of matrix porin. One phosphoporin trimer contains three pores which are induced simultaneously but fluctuate separately between open and closed states. Membrane potential shifts this two-state equilibrium in favour of closed channels. This negative resistance occurs at lower potentials than with matrix porin channels. The phosphoporin channel is poorly anion selective for small solutes. Polyphosphates and other phosphorylated molecules specifically inhibit phosphoporin pore conductance to small ions, a property which is specific to phosphoporin. There is an excellent correlation between the effect of such solutes measured in planar bilayers and their inhibitory effect on beta-lactam antibiotic uptake in vivo by phosphoporin. It is concluded that the phosphoporin channel contains a selectivity filter which is only efficient for larger molecules, most probably through basic residues.     

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic-resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channels.     

Porin channels in intact cells of Escherichia coli are not affected by Donnan potentials across the outer membrane

Donnan potential (interior negative) across the outer membrane of Escherichia coli was measured by the distribution of [14C]choline in a mutant with a deletion through the genes for the active transport of choline. Calculation showed that the presence of membrane-derived oligosaccharides in the periplasm could quantitatively explain the magnitude of the Donnan potential and the periplasmic volume. By measuring the permeability of porin channels in intact cells suspended in solutions of widely different ionic strengths, it was shown that changing Donnan potential from 5 mV to approximately 100 mV had no effect on the permeability of either OmpF or OmpC porin channel toward a zwitterionic compound, cephaloridine. Thus, the "voltage-dependent gating" of porin channel, previously reported from another laboratory, is likely to be an artifact of in vitro reconstitution. The influx of negatively charged compounds, however, was affected by the Donnan potential as expected from the electrolyte diffusion theory.     

Porin channels in Escherichia coli: studies with beta-lactams in intact cells

Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.     

生物法合成戊二胺研究进展

随着经济快速发展,大气污染和全球变暖的趋势日益恶化。世界上每年消耗大量石化资源来源的聚酰胺,戊二胺作为聚酰胺的重要组成单体,生物法合成戊二胺具有经济学和生态学双重意义。目前,生物法合成戊二胺的工程菌主要有谷氨酸棒状杆菌和大肠杆菌,文中从微生物中戊二胺的代谢、戊二胺合成途径的关键酶和转运蛋白、戊二胺生产最佳代谢途径和戊二胺产量的预测、代谢工程研究进展等方面综述了生物法合成戊二胺的最新研究现状和进展,并对其前景进行了展望。     

Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli

Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.     

The acrAB homolog of Haemophilus influenzae codes for a functional multidrug efflux pump.

Disruption of gene HI0894 or HI0895 in Haemophilus influenzae Rd, homologs of Escherichia coli acrAB multidrug efflux genes, caused hypersusceptibility to erythromycin, rifampin, novobiocin, and dyes such as ethidium bromide and crystal violet and increased accumulation of radioactive erythromycin, showing that these genes are expressed and contribute to the baseline level resistance of this organism through active drug efflux. The gene disruption did not produce detectable changes in susceptibility to several other antibiotics, possibly because rapid influx of small antibiotic molecules through the large H. influenzae porin channels counterbalances their efflux.