Involvement of ion channels in the transport of phage DNA through the cytoplasmic membrane of E. coli

Upon infection, phage DNA is transported through the bacterial cytoplasmic membrane. This crossing is accompanied by a transient increase in the permeability of the cytoplasmic membrane toward ions and small solutes. This has led several authors to propose that DNA might cross the cytoplasmic membrane through channels. In the first part of the review we present data that we obtained with phage T4 and that strongly support this proposal. We then present the structural and ionic characteristics of these channels. In the second part, we summarize data obtained by several authors concerning the permeability changes induced by different phages and show that these results are compatible with a model of phage DNA transfer through channels. Finally, we discuss the possible origin of these channels.     

Changes in the proton-motive force in Escherichia coli in response to external oxidoreduction potential.

The pH homeostasis and proton-motive force (Deltap) of Escherichia coli are dependent on the surrounding oxidoreduction potential (ORP). Only the internal pH value and, thus, the membrane pH gradient (DeltapH) component of the Deltap is modified, while the membrane potential (DeltaPsi) does not change in a significant way. Under reducing conditions (Eh < 50 mV at pH 7.0), E. coli decreases its Deltap especially in acidic media (21% decrease at pH 7.0 and 48% at pH 5.0 for a 850-mV ORP decrease). Measurements of ATPase activity and membrane proton conductance (CH+m) depending on ORP and pH have shown that the internal pH decrease is due to an increase in membrane proton permeability without any modification of ATPase activity. We propose that low ORP values de-energize E. coli by modifying the thiol : disulfide balance of proteins, which leads to an increase in the membrane permeability to protons.     

Interconversion of components of the bacterial proton motive force by electrogenic potassium transport.

The influence of K+ ions on the components of the transmembrane proton motive force (delta mu H+) in intact bacteria was investigated. In K+-depleted cells of the glycolytic bacterium STreptococcus faecalis the addition of K+ ions caused a depolarization of the membrane by about 60 mV. However, since the depolarization was compensated for by an increase in the transmembrane pH gradient (delta pH), the total proton motive force remained almost constant at about 120 mV. Half-maximal changes in the potential were observed at K+ concentrations at which the cells accumulated K+ ions extensively. In EDTA-treated, K+-depleted cells of Escherichia coli K-12, the addition of K+ ions to the medium caused similar, although smaller changes in the components of delta mu H+. Experiments with various E. coli K-12 K+ transport mutants showed that for the observed potential changes the cells required either a functional TrkA or Kdp K+ transport system. These data are interpreted to mean that the inward movement of K+ ions via each of these bacterial transport systems is electrogenic. Consequently, it leads to a depolarization of the membrane, which in its turn allows the cell to pump more protons into the medium.     

Mechanism of Na(+)-dependent citrate transport in Klebsiella pneumoniae.

Citrate transport via CitS of Klebsiella pneumoniae has been shown to depend on the presence of Na+. This transport system has been expressed in Escherichia coli, and uptake of citrate in E. coli membrane vesicles via this uptake system was found to be an electrogenic process, although the pH gradient is the main driving force for citrate uptake (M. E. van der Rest, R. M. Siewe, T. Abee, E. Schwartz, D. Oesterhelt, and W. N. Konings, J. Biol. Chem. 267:8971-8976, 1992). Analysis of the affinity constants for the different citrate species at different pH values of the medium indicates that H-citrate2- is the transported species. Since the electrical potential across the membrane is a driving force for citrate transport, this indicates that transport occurs in symport with at least three monovalent cations. Citrate efflux is stimulated by Na+ concentrations of up to 5 mM but inhibited by higher Na+ concentrations. Citrate exchange, however, is stimulated by all Na+ concentrations, indicating sequential events in which Na+ binds before citrate for translocation followed by a release of Na+ after release of citrate. CitS has, at pH 6.0 and in the presence of 5 mM citrate on both sides of the membrane, an apparent affinity (K(app)) for Na+ of 200 microM. The Na+/citrate stoichiometry was found to be 1. It is postulated that H-citrate2- is transported via CitS in symport with one Na+ and at least two H+ ions.     

Changes in membrane potential and transport of ions through the S. typhimurium LT2 membrane induced by bacteriophages

Bacteriophages P22 and dp8 cause the membrane potential depolarization for 10-30 mV, reversal rapid H+ influx into bacteria and K+ exit from S. typhimurium LT2, these effects depend on infection plural and are observed only in the presence of Ca+2 in the medium. delta psi depolarization and K+ efflux induced by phage dp8 are increased with the growth of Mg+2 concentration from 0 to 2 mM. Changes of delta pH and also Na+,Ca+2 concentrations are not observed. In the presence of glucose phage infection leads to changes in H(+)-K(+)-exchange. The phages P22 and dp8 adsorption on bacteria causes changes in the form or turn of the channels in S. typhimurium membrane.     

Conversion of Escherichia coli cell-produced metabolic energy into electric form.

The formation of membrane potential in energized E. coli cells has been investigated by means of ionic penetrants. The fluxes of anions and cations in opposite directions have been observed: anions moved out and cations moved into the cells. The energy-linked uptake of cations was stoichiometrically coupled with the outflow of H+ ions from the cells. The value of a membrane potential in the energized cells calculated from a distribution of permanent cations was in the range of -140 mV (inside minus). The uptake of penetrating cations by deenergized cells has been observed following the non-enzymatic generation of a membrane potential. The influx of synthetic and natural (lactose) penetrants collapsed the non-enzymatic membrane potential. The effect of lactose was sensitive to N-ethyl maleimide. These results are in favour of the conception that in the energized E. coli cells an energy-linked H+-pump generates a membrane potential which is a driving force for the transport of synthetic and some natural penetrants.     

Activation by calcium of membrane channels for potassium in exocrine gland cells

In the rat parotid salivary gland, fluid secretion is regulated by alterations in fluxes of monovalent ions. , stimulation of muscarinic, α-adrenergic or substance P receptors provokes a biphasic increase in membrane permeability to K⁺ which can be conveniently assayed as efflux of ⁸⁶Rb. The increased ⁸⁶Rb flux is thought to arise in response to a receptor mediated elevation in [Ca²⁺]_i which activates Ca²⁺-activated K⁺-channels. The biphasic nature of the response is presumably due to a biphasic mode of Ca²⁺ mobilization by secretagogues; a transient response reflects release of a finite pool of Ca from an intracellular store while a more sustained phase results from Ca entry through receptor operated Ca channels or gates. Calcium also mediates an increased Na⁺ entry which in turn activates the Na⁺, K⁺-pump. The mechanism involved in the regulation of monovalent ion channels by Ca²⁺ is not understood.     

Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4

In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally. The phage caused a reversible depolarization of cell membrane, while phage 'ghosts' induced an irreversible depolarization. The phage infection was strictly dependent on E. coli membrane potential value when phage/cell ratio was 5 and higher. When the ratio was close to 1, the decrease in the membrane potential up to -100 mV caused practically no effect on the phage infection. The infection inhibition was observed when the membrane potential was lowered below this 'threshold' value. On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient. The phage DNA transfer through the membrane of ATPase-deficient cells was reversibly inhibited by switching off the respiratory chain - the sole generator of a protonmotive force in these mutant cells. The membrane should be kept in the energized state during the phage DNA entrance into the cell. Adsorption of the phage on E. coli was followed by the reversible release of the respiratory control. Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E. coli. They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sps. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bungori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperate phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.     

Ionic currents and secretion in the exocrine glands

Exocrine glands extrude both proteins and salt. Fluid secretion is related to a modification of the membrane permeability of secreting cells. This permeability change may be measured as an increase of labelled ion fluxes or as a rise of membrane conductance. It involves Na+, K+, Cl- and Ca2+ ions. Intracellular Ca2+ acts as "second messenger" in the development of the electrical response. Recent recordings using the "patch-clamp" technique have revealed three types of ion channel activated by secretory agents. These channels are sensitive to internal Ca2+ ions. They are respectively selective to K+, Cl- and positively charged monovalent ions. Two models suggesting possible roles for these channels in the secretion process are presented. However, evaluation of such models is presently restricted by numerous uncertainties on the function of secreting cells in vivo. Information is notably lacking concerning the exact composition of the secreted fluid, and the exchanges between exocrine glands and blood circulation.     

Citrate transport in Klebsiella pneumoniae

Sodium ions were specifically required for citrate degradation by suspensions of K. pneumoniae cells which had been grown anaerobically on citrate. The rate of citrate degradation was considerably lower than the activities of the citrate fermentation enzymes citrate lyase and oxaloacetate decarboxylase, indicating that citrate transport is rate limiting. Uptake of citrate into cells was also Na+ -dependent and was accompanied by its rapid metabolism so that the tricarboxylic acid was not accumulated in the cells to significant levels. The transport could be stimulated less efficiently by LiCl. Li+ ions were cotransported with citrate into the cells. Transport and degradation of citrate were abolished with the uncoupler [4-(trifluoromethoxy)phenylhydrazono]propanedinitrile (CCFP). After releasing outer membrane components and periplasmic binding proteins by cold osmotic shock treatment, citrate degradation became also sensitive towards monensin and valinomycin. The shock procedure had no effect on the rate of citrate degradation indicating that the transport is not dependent on a binding protein. Citrate degradation and transport were independent of Na+ ions in K. pneumoniae grown aerobically on citrate and in E. coli grown anaerobically on citrate plus glucose. An E. coli cit+ clone obtained by transformation of K. pneumoniae genes coding for citrate transport required Na specifically for aerobic growth on citrate indicating that the Na-dependent citrate transport system is operating. Na+ and Li+ were equally effective in stimulating citrate degradation by cell suspensions of E. coli cit+. Citrate transport in membrane vesicles of E. coli cit+ was also Na+ dependent and was energized by the proton motive force (delta micro H+). Dissipation of delta micro H+ or its components delta pH or delta psi by ionophores either totally abolished or greatly inhibited citrate uptake. It is suggested that the systems energizing citrate transport under anaerobic conditions are provided by the outwardly directed cotransport of metabolic endproducts with protons yielding delta pH and by the decarboxylation of oxaloacetate yielding delta pNa+ and delta psi. In citrate-fermenting K. pneumoniae an ATPase which is activated by Na+ was not found. The cells contain however a proton translocating ATPase and a Na+/H+ antiporter in their membrane.     

Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4 degrees C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

Luria effect in bacteriophages and the role of the products of recA+ and lexA+ genes in its induction

An analysis of UV-damages accumulation in the phages as revealed by delay of intracellular growth is represented using temperate lambda phage. The maximum of growth delay of phage lambda at given UV-dose was found with lambda red+, infecting Escherichia coli AB1886 uvrA strain. The growth delay was absent, when a strain RH-1 uvrA-recA- was infected with UV-irradiated phage lambda red3. A moderate growth delay was obtained with the phages lambda red+, infecting E. coli RH-1 uvrA-recA- or phage lambda red3, infecting E. coli AB1886 uvrA-. THe growth delay was also absent when wild type, recA- and uvrA mutants of E. coli were infected with phage lambda after 8-metnoxypsoralen + light (lambda > 310 nm) treatment. It is known that the crosslinks appear to be the DNA defects which give rise to the observed biological inactivation following psoralen + light treatment. However, a considerable growth delay of phage lambda, treated by 8-metnoxypsoralen + light, was only found under condition of crosslinks repair (W-reactivation and prophage-reactivation). The results obtained are best explained by the assumption that the growth delay reflects the time required for the postreplication repair (RecA, LexA, Red) of any lethal UV-lesion.     

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces

AIM: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot. METHODS AND RESULTS: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (approximately 97%) contained phage that infected E. coli B or O157:H7. CONCLUSIONS: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.     

Short-tailed stx phages exploit the conserved YaeT protein to disseminate Shiga toxin genes among enterobacteria

Infection of Escherichia coli by Shiga toxin-encoding bacteriophages (Stx phages) was the pivotal event in the evolution of the deadly Shiga toxin-encoding E. coli (STEC), of which serotype O157:H7 is the most notorious. The number of different bacterial species and strains reported to produce Shiga toxin is now more than 500, since the first reported STEC infection outbreak in 1982. Clearly, Stx phages are spreading rapidly, but the underlying mechanism for this dissemination has not been explained. Here we show that an essential and highly conserved gene product, YaeT, which has an essential role in the insertion of proteins in the gram-negative bacterial outer membrane, is the surface molecule recognized by the majority (ca. 70%) of Stx phages via conserved tail spike proteins associated with a short-tailed morphology. The yaeT gene was initially identified through complementation, and its role was confirmed in phage binding assays with and without anti-YaeT antiserum. Heterologous cloning of E. coli yaeT to enable Stx phage adsorption to Erwinia carotovora and the phage adsorption patterns of bacterial species possessing natural yaeT variants further supported this conclusion. The use of an essential and highly conserved protein by the majority of Stx phages is a strategy that has enabled and promoted the rapid spread of shigatoxigenic potential throughout multiple E. coli serogroups and related bacterial species. Infection of commensal bacteria in the mammalian gut has been shown to amplify Shiga toxin production in vivo, and the data from this study provide a platform for the development of a therapeutic strategy to limit this YaeT-mediated infection of the commensal flora.     

The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophages

Seven phages highly active in vitro and in vivo against one or other of seven bovine enteropathogenic strains of Escherichia coli belonging to six different serotypes were isolated from sewage. Severe experimentally induced E. coli diarrhoea in calves could be cured by a single dose of 10(5) phage organisms. It could be prevented by doses as low as 10(2), by spraying the litter in the calf rooms with aqueous phage suspensions or simply by keeping the calves in uncleaned rooms previously occupied by calves whose E. coli infections had been treated with phage. Microbiological examinations of calves used in these experiments revealed that the phage organisms multiplied rapidly and profusely after gaining entry to the E. coli-infected small intestine, quickly reducing the E. coli to numbers that were virtually harmless. The only phage-resistant E. coli that emerged in the studies on calves infected with one or other of the seven E. coli strains were K-. These organisms were much less virulent than the K+ organisms from which they were derived and did not present a serious problem in calves given adequate amounts of colostrum. Infections produced by oral inoculation of a mixture of six strains of the E. coli could be controlled by administration of a pool of the six phages that were active against them but, in general, the control was less complete than that observed in the single-strain infections. K+ phage-resistant bacteria emerged in some of the calves used in these mixed infections and they were as virulent as their parent organisms; evidence from in vitro studies suggested that they might have arisen by genetic transfer between organisms of the different infecting strains. Infections produced by these K+ mutants and their parents could be controlled by the use of mutant phages derived from phages that were active on their parents. During the experiments with mixed E. coli infection, an extraneous phage active against one of the six E. coli strains suddenly appeared in calves kept in the same rooms. Microbiological examinations revealed that this phage was effectively controlling the multiplication of organisms of that particular strain of E. coli in the small intestines of the calves.     

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.     

The highly virulent 2006 Norwegian EHEC O103:H25 outbreak strain is related to the 2011 German O104:H4 outbreak strain

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.     

The capacity of anaerobically grown bacteria to exchange the 2H+ of the cell for the K+ of the medium and to maintain a high K+ distribution between cell and medium

The N,N'-dicyclohexylcarbodiimide sensitive exchange of 2H+ of a cell for K+ of medium stable to pH, K+ activity and temperature changes has been discovered in anaerobically grown gram-negative Escherichia coli, Salmonella typhimurium. S. enteritidis, Proteus mirabilis, P. vulgaris, anaerobic gram-positive bacteria Streptococcus faecalis, Lactobacillus salivarius, L. lactis in the presence of exogenic energy source. This exchange in gram-negative bacteria is operating only at increase of medium osmolarity. The high K+ distribution between cell and medium has been reached during the exchange of 2H+ for one K+ and the corresponding potassium equilibrium potential is much more than the measured delta psi. In aerobically grown E. coli, S. typhimurium, Brevibacterium flavum and aerobic Micrococcus luteus exchange of 2H+ for K+ does not take place, the K+ distribution is lower and in good conformity with the measured delta psi. It is assumed that exchange of 2H+ for K+ in anaerobic bacteria is carried out by the H+-ATPase complex and the Trk (or Trk-like) system of K+ absorption united into the same membrane supercomplex which functions as the H+-K+-pump and supports the high K+ distribution between cell and medium.