Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium.

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Modulation of lectinophagocytosis of Escherichia coli by variation of pH and temperature

The effect of variation of pH and temperature on the lectinophagocytosis of enteropathogenic Escherichia coli by polymorphonuclear leukocytes and macrophages elicited by thioglycolate medium was evaluated. The phagocytosis of enteropathogenic E. coli is dependent on pH, being maximal at pH 7.0 and reduced at pH 5.5 or 6.0. Mannan and mannose (as representative sugars that bind to phagocyte lectin receptors), are recognized by mannose receptors and reduced the phagocytic index at pH 7.0 (from 41.6 +/- 8.5 to 17.0 +/- 6.1) and at pH 6.0 (from 24.1 +/- 5.1 to 14.5 +/- 5.0), suggesting that mannose receptors, despite their reduced affinity for ligand at pH 6.0, also participate in phagocytosis of enteropathogenic E. coli. The inhibition of phagocytosis by anti-substance A antibody was also examined at pH 7.0 and at pH 6.0, decreasing (from 41.6 +/- 8.5 to 21.1 +/- 3.4) and (from 24.1 +/- 5.1 to 12.0 +/- 3.5), respectively. This antibody reduced the phagocytosis of enteropathogenic E. coli in phagocytic assays at 37 or 41 degrees C. These results suggest that the acidic pH decreased the affinity of mannose receptors to ligands on the surface of E. coli and also affected the binding of lectin from E. coli to N-acetylgalactosamine on phagocytes.     

Adaptive responses of Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and Escherichia coli O157 to low pH environments

AIMS: Cattle are a known main reservoir for acid-resistant Escherichia coli O157 and Salmonella enterica serovar Typhimurium DT104. We studied the response of S. Typhimurium DT104 to extreme low pH environments and compared their response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types. METHODS AND RESULTS: Bacteria were grown in nutrient-rich medium and subsequently acid challenged at pH 2.5. We found that stationary phase cultures of various S. Typhimurium strains were able to survive a challenge for 2 h at pH 2.5. As in E. coli, the ability of S. Typhimurium to survive at pH 2.5 was shown to be dependent on the presence of amino acids, specifically arginine. The amount of proton pumping H+/ATPase, both in E. coli O157 and S. Typhimurium strains, was lower when grown at pH values <6 than after growth at pH 7.5. Cyclo fatty acid content of membranes of bacteria grown at pH values <6 was higher than that of membranes of bacteria grown at pH 7.5. CONCLUSIONS: Various S. Typhimurium strains, both DT104 and non-DT104, are able to survive for a prolonged period of time at pH 2.5. Their response to such low pH environment is seemingly similar to that of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens like S. Typhimurium DT104 and E. coli O157 form a serious threat to public health since such strains are able to survive under extreme low pH conditions as present in the human stomach. The emergence these acid-resistant strains suggests the presence of a selection barrier. The intestinal tract of ruminants fed a carbohydrate-rich diet might be such a barrier.     

Inhibition of acetohydroxy acid synthase by leucine

The enzymatic reaction of acetohydroxy acid synthase in crude extracts of Escherichia coli K-12 is inhibited by leucine. Inhibition is most pronounced at low pH values and is low at pH values higher than 8.0. Both isoenzymes of acetohydroxy acid synthase present in E. coli K-12 (isoenzyme I and isoenzyme III) are inhibited by leucine. Isoenzyme I, which is responsible for the majority of acetohydroxy acid synthase activity in E. coli K-12 at physiological pH, is inhibited almost completely by 30 mM leucine at pH 6.25-7.0 and is not affected at all at pH values higher than 8.4. Inhibition of isoenzyme I by leucine is a mixed noncompetitive process. Leucine inhibition of isoenzyme III is pH-independent and reaches only 40% at 30 mM leucine. The inhibition of acetohydroxy acid synthase by leucine at physiological pH, observed in vitro in this study, correlates with the idea that acetohydroxy acid synthase is a target for the toxicity of the abnormally high concentrations of leucine in E. coli K-12.     

Practical mechanisms for interrupting the oral-fecal lifecycle of Escherichia coli

Escherichia coli is a common gut inhabitant, but it is usually out numbered by strictly anaerobic bacteria. When fecal material is exposed to oxygen, fermentation acids can be respired, and E. coli numbers increase. E. coli can survive for long periods of time in feces, but subsequent proliferation is dependent on its ability to re-enter the gastrointestinal tract via contaminated water and food. The oral-fecal lifestyle of E. coli is facilitated by its ability to survive the low pH of the human gastric stomach. Most strains of E. coli do not cause human disease, but some strains produce toxins and other virulence factors. Mature cattle carry E. coli O157:H7 without showing signs of infection, and beef can be contaminated with cattle feces at slaughter. Cattle manure is often used as a fertilizer by the vegetable industry, and E. coli from manure can migrate through the soil into water supplies. Sanitation, cooking and chlorination have been used to combat fecal E. coli, but these methods are not always effective. Recent work indicates that cattle diets can be modified overcome the extreme acid resistance of E. coli. When cattle were fed have for only a few days, colonic volatile fatty acid concentrations declined, pH increased, and the E. coli were no longer able to survive a pH shock that mimicked the human gastric stomach. E. coli in stored cattle manure eventually become highly acid resistant even if the cattle were fed hay, but these bacteria could be killed by sodium carbonate (150 mM, pH 8.5). Because the diet manipulations and carbonate treatments affected E. coli in general rather than specific serotypes, there is an increased likelihood of successful field application.     

Effect of pH oscillations on a competing mixed culture

Two microorganisms, E. coli and S. cerevisiae, competing for glucose were maintained in a stable cycle of coexistence by alternating the growth advantage between the two organisms by oscillating the pH in a Chemostat. Pure culture experiments found S. cerevisiae to be insensitive to pH between 5 and 4.3 with a maximum specific growth rate (mu(max)) of 0.4/hr; while mu(max) of E. coli decreased from 0.6 h(-1) at pH 5 to 0.1 h(-1) at pH 4.3. Steady-state and cross-inoculation chemostat runs at a dilution rate of 0.17 h(-1) confirmed the expectation that the mixed culture system is unstable at constant pH with E. coli dominating at pH 5 and S. cerevisiae dominating at pH 4.3. Three pH oscillation experiments were performed at D =0.17 h(-1) with 1 g per liter glucose feed. The 16 h/16 h cycle was stable for six periods with a stable alternating cycle of E. coli and S. cerevisiae being quickly established. A 18 h pH 5/14 h pH 4.3 cycle was found to be stable with smaller yeast concentrations. A 6 h/6 h cycle was found unstable with yeast washout. Simulation results were compared with these runs and were used to predict the onset of instability. Oscillations of pH can force stable persistence of a competing mixed culture that is otherwise unstable. Thus, varying conditions are experimentally demonstrated to be one explanation for competitive coexistence.     

Fate of Escherichia coli during ensiling of wheat and corn

A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 x 10(8) CFU/g into direct-cut wheat (348 g of dry matter kg(-1)), wilted wheat (450 g of dry matter kg(-1)), and corn (375 g of dry matter kg(-1)). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.     

Accumulation of glutamate by osmotically stressed Escherichia coli is dependent on pH.

In the present study, we measured the accumulation of glutamate after hyperosmotic shock in Escherichia coli growing in synthetic medium. The accumulation was high in the medium containing sucrose at a pH above 8 and decreased with decreases in the medium pH. The same results were obtained when the hyperosmotic shock was carried out with sodium chloride. The internal level of potassium ions in cells growing at a high pH was higher than that in cells growing in a neutral medium. A mutant deficient in transport systems for potassium ions accumulated glutamate upon hyperosmotic stress at a high pH without a significant increase in the internal level of potassium ions. When the medium osmolarity was moderate at a pH below 8, E. coli accumulated gamma-aminobutyrate and the accumulation of glutamate was low. These data suggest that E. coli uses different osmolytes for hyperosmotic adaptation at different environmental pHs.     

Modelling inactivation of Escherichia coli by low pH: application to passage through the stomach of young and elderly people

AIM: To estimate the survival of enteropathogenic Escherichia coli after passage through the stomach of young and elderly people. METHODS AND RESULTS: Using enterohaemorrhagic E. coli O157 and a non-pathogenic laboratory strain, inactivation in a pH range between 1.5 and 4.0 was experimentally quantified. Gastric pH and transport have previously been studied in human volunteers following consumption of a solid meal. Combining all these findings, time series of surviving bacteria were mathematically predicted and subsequently, the predictions were validated with in vitro experiments using a pH-controlled fermentor. On average, 20-80% of ingested E. coli are estimated to arrive in the small intestine without inactivation by low pH. The mean overall gastric passage was similar for young and elderly subjects. CONCLUSIONS: The tested E. coli strains can survive the human stomach with a high probability. SIGNIFICANCE AND IMPACT OF THE STUDY: Survival of E. coli under conditions of changing pH in the stomach may be predicted by batch experiments at constant pH. The effectiveness of the gastric acid barrier strongly depends on buffering effects of food.     

Solubility and antimicrobial efficacy of protamine on Listeria monocytogenes and Escherichia coli as influenced by pH

The antimicrobial efficacy of protamine on Listeria monocytogenes and Escherichia coli was evaluated at concentrations from 50 to 10 000 microgram ml-1 and pH levels from 5.5 to 8.0. The minimum inhibitory concentrations decreased with increasing pH. Protamine inhibited E. coli at all pH values while L. monocytogenes was inhibited at pH 6.5 and above. The antimicrobial efficacy of protamine decreased in the presence of negatively charged gelatine B but remained almost unchanged with addition of the positively charged gelatine A. Binding studies showed that the amount of protamine adsorbed to culture media components in tryptic soy broth and bacterial cells increased with increasing pH values. The increased efficacy of protamine at alkaline pH may be explained on the basis of an increase in electrostatic affinity for the cell surface of target cells. E. coli produced a protamine-degrading enzyme, however, was still susceptible to protamine.     

Effect of Glutaraldehyde on the Outer Layers of Escherichia coli

S ummary : Sodium lauryl sulphate (SLS) at pH 3 and 8 lysed cell walls of Escherichia coli. Pretreatment with glutaraldehyde at pH 3 and at pH 8 prevented this lysis. SLS induced maximum lysis of E. coli cells at 40°; pretreatment of cells with glutaraldehyde prevented this lysis also. Electrophoretic studies indicated that glutaraldehyde accumu lated on the surface of E. coli cells more rapidly in acid than in alkaline conditions, and that it blocked amino groups on the surface layer of Bacillus subtilis spores. The relationship of these findings to the bactericidal efficiency of glutaraldehyde in acid and alkaline solution is discussed.     

Protective effects of organic acids on survival of Escherichia coli O157:H7 in acidic environments

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.     

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.     

Mechanisms of acid resistance in enterohemorrhagic Escherichia coli.

Enterohemorrhagic strains of Escherichia coli must pass through the acidic gastric barrier to cause gastrointestinal disease. Taking into account the apparent low infectious dose of enterohemorrhagic E. coli, 11 O157:H7 strains and 4 commensal strains of E. coli were tested for their abilities to survive extreme acid exposures (pH 3). Three previously characterized acid resistance systems were tested. These included an acid-induced oxidative system, an acid-induced arginine-dependent system, and a glutamate-dependent system. When challenged at pH 2.0, the arginine-dependent system provided more protection in the EHEC strains than in commensal strains. However, the glutamate-dependent system provided better protection than the arginine system and appeared equally effective in all strains. Because E. coli must also endure acid stress imposed by the presence of weak acids in intestinal contents at a pH less acidic than that of the stomach, the ability of specific acid resistance systems to protect against weak acids was examined. The arginine- and glutamate-dependent systems were both effective in protecting E. coli against the bactericidal effects of a variety of weak acids. The acids tested include benzoic acid (20 mM; pH 4.0) and a volatile fatty acid cocktail composed of acetic, propionic, and butyric acids at levels approximating those present in the intestine. The oxidative system was much less effective. Several genetic aspects of E. coli acid resistance were also characterized. The alternate sigma factor RpoS was shown to be required for oxidative acid resistance but was only partially involved with the arginine- and glutamate-dependent acid resistance systems. The arginine decarboxylase system (including adi and its regulators cysB and adiY) was responsible for arginine-dependent acid resistance. The results suggest that several acid resistance systems potentially contribute to the survival of pathogenic E. coli in the different acid stress environments of the stomach (pH 1 to 3) and the intestine (pH 4.5 to 7 with high concentrations of volatile fatty acids). Of particular importance to the food industry was the finding that once induced, the acid resistance systems will remain active for prolonged periods of cold storage at 4 degrees C.     

Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.

Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.     

Influence of pH treatments on survival of Escherichia coli O157:H7 in continuous cultures of rumen contents

The pH (i.e., 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, and 7.25) effect on Escherichia coli O157:H7 in an artificial rumen model was investigated. Eight fermenters were inoculated with bovine rumen fluid and were supplied with a diet (75 g of dry matter daily in 12 equal portions [every 2 hr]) containing similar forage-to-concentrate ratio. After an adaptation period (i.e., 3 days for adjusting the rumen fluid [pH 6.2] microbial population to the test pH and 4 days for adjustment to the diet at the test pH), each fermenter was inoculated with 10(9) cells of E. coli O157:H7. Samples were collected hourly for 12 hr and every 2 hr for an additional 12 hr and were analyzed by flow cytometer. E. coli O157:H7 could not be quantified after 24 hr, and detection was only possible after enrichment. Because the pathogen could not be detected 5 days postinoculation (i.e., Day 13), the fermenters were reinoculated with E. coli O157:H7 on Days 17 and 22. E. coli O157:H7 numbers decreased from 10(6) to 10(4)/ml of fermenter contents in a quadratic (P < 0.05) fashion over the 24-hr sampling period, and the rate of reduction was slower (P < 0.05) for pH 7.0 than for other pH treatments. Results suggested that E. coli O157:H7 population were decreased by competitive exclusion and were not affected by culture pH.     

Effect of intracellular pH on rotational speed of bacterial flagellar motors

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E. coli cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of E. coli cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered E. coli cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.     

Microbial volatile organic compound emissions from Stachybotrys chartarumgrowing on gypsum wallboard and ceiling tile

In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis. Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress. Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli.     

Pulsed electric fields cause sublethal injury in Escherichia coli

AIMS: The objective was to investigate the occurrence of sublethal injury in Escherichia coli by pulsed electric fields (PEF) at different pH values. METHODS AND RESULTS: The occurrence of sublethal injury in PEF-treated E. coli cells depended on the pH of the treatment medium. Whereas a slight sublethal injury was detected at pH 7, 99.95% of survivors were injured when cells were treated at pH 4 for 400 micros at 19 kV. The PEF-injured cells were progressively inactivated by a subsequent holding at pH 4. CONCLUSIONS: PEF cause sublethal injury in E. coli. The measurement of sublethal injury using a selective medium plating technique allowed prediction of the number of cells that would be inactivated by subsequent storage in acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could be useful for improving food preservation by PEF technology and contributes to the knowledge of the mechanism of microbial inactivation by PEF.