An extracellular acid stress-sensing protein needed for acid tolerance induction in Escherichia coli

An extracellular induction component (EIC), needed for acid tolerance induction at pH 5.0 in Escherichia coli, arises from an extracellular precursor which senses acid stress and is activated (forming the EIC) by such stress. The precursor, which is a heat-stable protein, was formed by cells which had not been subjected to acid stress, being present in culture media after growth at pH values from 7.0 to 9.0. This stress-sensing molecule was activated to the EIC at pH values from 4.5 to 6.0 but not at pH 6.5 and did not form EIC on incubation at an extremely acidic pH e.g. 2.0. The precursor was not inactivated at pH 2.0. Precursor activation might be reversible, as the EIC lost its ability to induce acid tolerance after incubation at pH 9.0, but regained it if subsequently incubated at pH 5.0. Whereas the sensor formed at pH 7.0 can only be activated at pH 5.0 to 6.0, that synthesized at pH 9.0 can be activated at pH 5.0 to 7.5. Accordingly, this work shows that the acid stress sensor is extracellular, and it is proposed that its presence in the medium rather than in the cells, allows more sensitive and rapid responses to acid stress.     

Induction of acid tolerance at neutral pH in log-phase Escherichia coli by medium filtrates from organisms grown at acidic pH

Escherichia coli grown at pH 5·0 became acid-tolerant (acid-habituated) but, in addition, neutralized medium filtrates from cultures of E. coli grown to log-phase or stationary-phase at pH 5·0 (pH 5·0 filtrates) induced acid tolerance when added to log-phase E. coli growing at pH 7·0. In contrast, filtrates from pH 7·0-grown cultures were ineffective. The pH 5·0 filtrates were inactivated by heating in a boiling water-bath but there was less activity loss at 75 °C. Protease also inactivated such filtrates, which suggested that a heat-resistant protein (or proteins) in the filtrates was essential for the induction of acid tolerance. Filtrates from cells grown at pH 5·0 plus phosphate or adenosine 3':5'-cyclic monophosphate (cAMP) were much less effective in inducing acid tolerance, while the conversion of pH 7·0-grown log-phase cells to acid tolerance by pH 5·0 filtrates was inhibited by cAMP and bicarbonate. It seems likely that the acid tolerance response (acid habituation) involved the functioning of the extracellular protein(s) as protease reduces tolerance induction if added during acid habituation. Most inducible responses are believed to involve the functioning of only intracellular reactions and components ; the present results suggest that this is not the case for acid habituation, as an extracellular protein (or proteins) is needed for induction.     

An extracellular sensor and an extracellular induction component are required for alkali induction of alkyl hydroperoxide tolerance in Escherichia coli

Escherichia coli K12 transferred from pH 7.0 to pH 9.0 gains alkylhydroperoxide (AHP) tolerance. The aim here was to establish whether extracellular components (ECs) are needed for such induction. Therefore, the effects of removing ECs during incubation at pH 9.0 were tested and the abilities of culture filtrates to induce tolerance were examined. First, AHP tolerance did not appear, at pH 9.0, if cultures were subjected to continuous filtration or dialysis, against the same medium, suggesting that an EC might be needed. Second, neutralized filtrates from pH 9.0-grown cultures induced tolerance at pH 7.0, and these filtrates were inactivated by dialysis, filtration or heating but not by protease. Thus, pH 9.0 filtrates have a small non-protein extracellular induction component (EIC), which acts as an alarmone, 'warning' cells of stress and preparing them to resist it. Filtrates from pH 7.0-grown cultures did not induce AHP tolerance at pH 7.0 but if incubated at pH 9.0 without organisms, gained such ability. It is proposed that pH 7.0 filtrates have an EIC precursor (termed an extracellular sensing component, ESC), which senses alkaline pH, and is converted by it to the EIC. The ESC in pH 6.0 filtrates was distinct from that in pH 7.0 filtrates; there may be several oligomeric (or conformational) forms of this ESC. As the EIC is small, it can diffuse away from the alkalinized region and induce tolerance in unstressed organisms.     

The role of regulatory gene products in alkali sensitization by extracellular medium components in Escherichia coli

Organisms of Escherichia coli 1829 become alkali sensitized on transfer from pH 7·0 to pH 5·5 but they also secrete extracellular agents which induce alkali sensitivity when added (in neutralized filtrates) to organisms growing at pH 7·0. In contrast, filtrates from cultures grown at pH 7·0 have no effect. Filtrates were inactivated by protease but not by heat treatment in a boiling water-bath, suggesting that a very heat-stable protein is involved in alkali sensitivity induction. A heatstable low molecular weight component (or components) may also be needed for induction, or the induction protein itself may be of low molecular weight. Strains with lesions in hns, fur or himA produced almost inactive filtrates and it therefore appears that H-NS, Fur and IHF are involved in synthesis of the induction components. As the presence of protease during incubation at pH 5·5 totally abolished alkali sensitization of strain 1829 while inhibition of sensitization induction occurred if the induction components were removed by filtration or dialysis during pH 5·5 incubation, it is proposed that the extracellular induction components (EICs) are essential for the original sensitization response. These results suggest that sensitization induction occurs by a different mechanism to that which is believed to occur for most bacterial inducible response systems; these are claimed to involve exclusively intracellular reactions and components whereas the present response involves functioning of extracellular components.     

Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP

Escherichia coli shifted from broth at external pH (pH₀) 7·0 to pH₀ 7·0 broth plus glucose rapidly induced marked acid tolerance which also appeared, albeit to a lesser extent, plus maltose, sucrose or lactose. Tolerance appeared without the medium pH becoming acidic. Tolerance was most substantial when glucose was added at pH₀ 7·0 but was also appreciable at pH₀ 7·5, 8·0 and 8·5. Induction of tolerance by glucose was markedly reduced by cyclic AMP and essentially abolished plus NaCl or sucrose ; the induction process was also reduced but not fully inhibited by chloramphenicol, tetracycline and nalidixic acid. Glucose-induced organisms showed less acid damage to DNA and β-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH₀. In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E. coli at pH₀ 7·0 ; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.     

Susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0·1, 0·2 and 0·5 mg l⁻¹), pH (6, 7 and 8) and temperature (4, 21 and 32 °C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH 6 than at pH 8 for both strains. Under the standard conditions of temperature 21 °C, pH 7 and chlorine concentration 0·2 mg l⁻¹, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Sensitization of thermally injured spores of Bacillus stearothermophilus to sodium benzoate and potassium sorbate

The effects of sodium benzoate and potassium sorbate added to the recovery medium, at different pH values (6·5, 6·0 and 5·0), on the recovery rates and heat resistance of Bacillus stearothermophilus spores (ATCC 12980, 7953, 15951 and 15952) were investigated. Heated spores of strains 12980 and 7953 were inhibited by sorbate concentrations over 0·05%. Potassium sorbate at concentrations as low as 0·025%, and sodium benzoate at 0·1%, were very effective inhibitory agents for heat-damaged spores. Their effectiveness always increased at pH 5·0, at which no growth occurred, with sodium benzoate for strains 7953, 15951 and 15952, and with potassium sorbate for strains 15951 and 15952. Decimal reduction times, whenever recovery was possible, were not significantly ( P  > 0·05) affected. None of these compounds modified the z -values obtained for the spores of the four strains, which had a mean value of 7·53 ± 0·28.     

Bacteriocin production by Pseudomonas syringae pv. ciccaronei NCPPB2355. Isolation and partial characterization of the antimicrobial compound

Pseudomonas syringae pv. ciccaronei strain NCPPB2355 was found to produce a bacteriocin inhibitory against strains of Ps. syringae subsp. savastanoi , the causal agent of olive knot disease. Treatments with mitomycin C did not substantially increase the bacteriocin titre in culture. The purification of the bacteriocin obtained by ammonium sulphate precipitation of culture supernatant fluid, membrane ultrafiltration, gel filtration and preparative PAGE, led to the isolation of a high molecular weight proteinaceous substance. The bacteriocin analysed by SDS-PAGE revealed three protein bands with molecular weights of 76, 63 and 45 kDa, respectively. The bacteriocin was sensitive to heat and proteolytic enzymes, was resistant to non-polar organic solvents and was active between pH 5·0–7·0. Plasmid-DNA analysis of Ps. syringae ciccaronei revealed the presence of 18 plasmids; bacteriocin-negative variants could not be obtained by cure experiments.     

Electrophoretic profile of hybrids between cryotolerant and non-cryotolerant Saccharomyces strains

Escherichia coli O157 : H7 (O157) has unusual acid tolerance. The influence of heat shock on acid tolerance of O157 was studied. Seven strains of O157 and E. coli K-12 were tested for their ability to survive in minimum glucose medium (pH 2·5) at 37 °C. The survival of heat-shocked (10 min at 48 °C) cells was about 10–100 times greater compared with untreated cells depending on the strain. No significant difference ( P > 0·05) for O157 strain 932 was observed between heat shock-induced and acid adaptation-induced (pH 5·0) acid tolerance. Chloramphenicol prevented heat shock-induced acid tolerance, indicating the requirement of newly synthesized protein(s). Two outer membrane proteins (OMP) (22 and 15 kDa) were synthesized within 10 min of heat shock and were expressed for at least 6 h by cells held at 37 °C. N-terminal amino acid sequence analysis suggested that the 22 kDa OMP is a component of an alkyl hydroperoxide reductase. This protein contains a redox active disulphide, which is probably involved in H⁺ transport. Results indicate that sublethal heat treatment of O157 cells substantially increases their tolerance to acidic conditions. This could have practical implications for foods that receive a mild heat treatment and rely on acid as a preservative.     

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

Mathematical modelling of the heat resistance of Listeria monocytogenes

The heat resistance of Listeria monocytogenes phagovar 2389/2425/3274/2671/47/108/340 (1992 French outbreak strain) in broth was studied at 55, 60 and 65 °C. Experiments were carried out on bacterial cultures in three different physiological states: cultures at the end of the log phase, cultures heat-shocked at 42 °C for 1 h, and subcultures of cells resistant to prolonged heating. Survivor curves were better fitted using a sigmoidal equation than the classical log-linear model. This approach was justified by the existence of heat resistance distributions within the bacterial populations. Peaks (log₁₀ of heating time) of heat resistance distributions of untreated, heat-shocked, and selected cultures at 55, 60 and 65 °C were 0·34, −0·90 and −1·84 min, 0·74, −0·51 and −1·24 min, and 0·17, −0·94 and−1·45 min, respectively. The widths of the distributions are proportional to 0·29, 0·36and 0·41 min^0·5, 0·26, 0·36 and 0·41 min^0·5, and 0·34, 0·44 and 0·41 min^0·5. An increase in thethermal tolerance could then be induced by sublethal heat shock or by selection of heatresistant cells.     

Properties of an L-glutamate-induced acid tolerance response which involves the functioning of extracellular induction components

Escherichia coli became more acid tolerant following incubation for 60 min in a medium containing L-glutamate at pH 7.0, 7.5 or 8.5. Several agents, including cAMP, NaCl, sucrose, SDS and DOC, prevented tolerance appearing if present with L-glutamate. Lesions in cysB, hns, fur, himA and relA, which frequently affect pH responses, failed to prevent L-glutamate-induced acid tolerance but a lesion in L-glutamate decarboxylase abolished the response. Induction of acid tolerance by L-glutamate was associated with the accumulation in the growth medium of a protein (or proteins) which was able to convert pH 7.0-grown cultures to acid tolerance, and the original L-glutamate-induced tolerance response was dependent on this component(s). Acid tolerance was also induced by L-aspartate at pH 7.0 and induction of such tolerance was dependent on an extracellular protein (or proteins). The L-glutamate and L-aspartate acid tolerance induction processes are further examples of a number of stress tolerance responses which differ from most inductions in that extracellular components, including extracellular sensors, are required.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE 905 from ready-to-eat salad

Lactobacillus plantarum BFE 905 isolated from 'Waldorf' salad produced a bacteriocin termed plantaricin D which was active against Lact. sake and Listeria monocytogenes strains. Plantaricin D was heat stable, retaining activity after heating at 121 °C. The bacteriocin was inactivated by α-chymotrypsin, trypsin, pepsin and proteinase K, but not by papain and other non-proteolytic enzymes tested. Plantaricin D was stable at pH values ranging from 2·0 to 10·0. The bacteriocin inhibited growth of L. monocytogenes in automated turbidity assays. Although Lact. plantarum BFE 905 harboured plasmids ranging in size from 3 to 55 kilobase pairs, loss of bacteriocin production could not be correlated with plasmid loss. A role for bacteriocin-producing Lact. plantarum of vegetable origin in assuring the safety of vegetable foods is suggested.     

Cultural conditions for the production of bacteriocin by a native isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 in milk medium

The effect of growth parameters and the molecular basis for antibacterial activity by a natural isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 was studied. The inhibition was tested against a toxigenic strain of Bacillus cereus F 4810. When grown in milk medium, the activity was highest at an incubation temperature of 37 °C in 48 h. The antibacterial activity appeared to be produced between late logarithmic and early stationary phases. The active principle was proteinaceous in nature (bacteriocin) and stable to low pH (3·8–5·0) and heat (75 °C for 30 min). There was also the possible role of hydrogen peroxide in bringing about inhibition. The strain of Lact. delbruecki ssp. bulgaricus CFR 2028 revealed the presence of plasmid DNA bands of 9·4 and 6·5 kbp, respectively, in agarose gel electrophoresis. The above strain has the potential to be used as a biopreservative in popular Indian fermented foods.     

Drought tolerance by lentil rhizobia (Rhizobium leguminosarum) from arid and semiarid areas of Pakistan

Ten strains of lentil rhizobia (Rhizobium leguminosarum ) were evaluated for drought tolerance by exposing them to soil moisture potentials of −0·03, −1·0 and −1·5 MPa. Water availability, rhizobial strain and time of exposure to drought had a significant ( P ≤ 0·001) effect on the number of surviving rhizobia g⁻¹ of soil. Highest cell counts were observed at −0·03 MPa, followed by soil maintained at −1·0 and −1·5 MPa. Five strains originating from saline areas showed significantly ( P ≤ 0·05) better survival under low water potential after 35 days. Two strains exhibited greatest survival under low water potential and produced viable cell counts of more than 10⁷ rhizobia g⁻¹ of soil. These strains could probably be used successfully as inoculants for lentil production in arid and semi-arid environments.     

Heat resistance of Salmonella enteritidis PT4: the influence of prior exposure to alkaline conditions

Salmonella enteritidis phage type 4 incubated in broth at pH 9·2 ± 0·2 for 5 min or longer became significantly more heat-resistant ( P < 0·001) when subsequently heated at pH 7·0 but not when heated at pH 9·0. The induction of enhanced heat resistance was not associated with an increase in cell numbers, occurred rapidly and was probably phenotypic.     

Protective effect of salicylates against hydrogen peroxide stress in yeast

Aims:  To investigate the effects of salicylates in Saccharomyces cerevisiae exposed to oxidative stress induced by hydrogen peroxide (H₂O₂).
Methods and Results:  Saccharomyces cerevisiae was cultured through to the postlogarithmic phase of growth. Stress was induced by the addition of 1·5 mmol l⁻¹ H₂O₂ for 1 h, while N-acetyl-l-cysteine (NAC) and glutathione (GSSG) were used as control agents that affect the redox balance. Sodium salicylate, at 0·01–10 mmol l⁻¹or acetylsalicylic acid, at 0·02–2·5 mmol l⁻¹ was administered at various times before hydrogen peroxide stress. Both agents conferred resistance to a subsequent hydrogen peroxide stress, similarly to the induction of the adaptive response observed upon pretreatment with NAC and GSSG. Sodium salicylate was more potent as a short-term, but not as a long-term pretreatment agent, compared to acetylsalicylic acid.
Conclusions:  Pharmacological pretreatment with salicylates resulted in dose related increases in cell survival, indicating the induction of the protective response in yeast.
Significance and Impact of the study:  The possible role of salicylates in the modulation of the hydrogen peroxide stress response in eukaryotic cells address questions on the effects of these commonly used therapeutic agents in a number of disorders exhibiting an oxidative stress component.     

Inactivation of Salmonella enterica serovar enteritidis in shell eggs by sequential application of heat and ozone

Aims:  To assess the contribution of ozone to lethality of Salmonella enterica serovar Enteritidis in experimentally inoculated whole shell eggs that are sequentially treated with heat and gaseous ozone in pilot-scale equipment.
Methods and Results:  Whole shell eggs were inoculated with small populations of Salmonella Enteritidis (8·5 × 10⁴–2·4 × 10⁵ CFU per egg) near the egg vitelline membrane. Eggs were subjected to immersion heating (57°C for 21 min), ozone treatment (vacuum at 67·5 kPa, followed by ozonation at a maximum concentration of approx. 140 g ozone m⁻³ and 184–198 kPa for 40 min) or a combination of both treatments. Survivors were detected after an enrichment process or enumerated using modified most probable number technique. Ozone, heat and combination treatments inactivated 0·11, 3·1 and 4·2 log Salmonella Enteritidis per egg, respectively.
Conclusions:  Sequential application of heat and gaseous ozone was significantly more effective than either heat or ozone alone. The demonstrated synergy between these treatment steps should produce safer shell eggs than the heat treatment alone.
Significance and Impact of the Study:  Shell eggs are the most common vehicle for human infection by Salmonella Enteritidis. Many cases of egg-related salmonellosis are reported annually despite efforts to reduce contamination, including thermal pasteurization of shell eggs and egg products. Treatment with ozone-based combination should produce shell eggs safer than those treated with heat alone.     

Thermal tolerance of a northern population of striped bass Morone saxatilis

Thermal tolerance of age 0+ year Shubenacadie River (Nova Scotia, Canada) striped bass Morone saxatilis juveniles (mean ± s . e . fork length, L _F, 19·2 ± 0·2 cm) acclimated in fresh water to six temperatures from 5 to 30° C was measured by both the incipient lethal technique (72 h assay), and the critical thermal method ( C _m). The lower incipient lethal temperature ranged from 2·4 to 11·3° C, and the upper incipient lethal temperature ( I _U) from 24·4 to 33·9° C. The area of thermal tolerance was 618° C². In a separate experiment, the I _U of large age 2+ year fish (34·4 ± 0·5 cm L _F) was 1·2 and 0·6° C lower ( P < 0·01) than smaller age 1+ year fish (21·8 ± 0·5 cm L _F) at acclimation temperatures of 16 and 23° C. Using the C _m, loss of equilibrium occurred at 27·4–37·7° C, loss of righting response at 28·1–38·4° C and onset of spasms at 28·5–38·8° C, depending on acclimation temperature. The linear regression slopes for these three responses were statistically similar (0·41; P > 0·05), but the intercepts differed (25·3, 26·0 and 26·5° C; P < 0·01). The thermal tolerance of this northern population appears to be broader than southern populations.