Margaret Goodson

Habituation to normally lethal acidity by prior growth of Escherichia coli at a sub-lethal acid pH value

Both Col‐ and ColV, I‐K94+ strains of Escherichia coli, grown at pH 7–0, failed to grow after relatively short periods of exposure to pH 3·0 or 3·5. After growth in exposure medium initially at pH 5·0, both strains were almost unaffected by exposure to such acid pH values. Addition of catalase to nutrient agar only slightly increased plating efficiency after acid treatment and very slightly reduced the difference in survival, after acid treatment, between organisms grown from pH 5·0 and those grown from pH 7·0. Accordingly, acid resistance of organisms grown from pH 5·0 is not chiefly due to greater resistance to hydrogen peroxide already present in nutrient media.

Resistance of acid‐habituated Escherichia coli to organic acids and its medical and applied significance

Escherichia coli grown in broth initially at pH 5.0 (pH 5.0‐grown organisms) survived exposure to inorganic acid or to acid pH plus organic acid which prevented subsequent growth by pH 7.0‐grown organisms. This resistance of pH 5.0‐grown organisms to organic acids was observed at acid pH with lactic, propionic, benzoic, sorbic, trans‐cinnamic and acetic acids. Such resistance might allow acid‐habituated organisms to survive in acid foods or at body sites such as the urinary tract where organic acids are present at acid pH.

Habituation to alkali and increased u.v.‐resistance in DNA repair‐proficient and ‐deficient strains of Escherichia coli grown at pH 9.0

Repair‐deficient strains of Escherichia coli carrying polAI or recA mutations were more alkali‐sensitive than was their repair‐proficient parent but, like strain 1829 ColV, I‐K94, they showed habituation to alkali (induction of increased resistance) when grown at pH 9.0. Occurrence of such increased alkali resistance in the recA mutant implies that habituation to alkali does not depend on induction of SOS‐related repair mechanisms. Organisms of repair‐proficient and repair‐deficient strains also became more resistant to u.v.‐irradiation after growth at pH 9.0; this increased u.v.‐resistance also appeared to be RecA‐independent.

Regulatory aspects of alkali tolerance induction in Escherichia coli

R.J. ROWBURY, Z. LAZIM AND M. GOODSON. 1996. Escherichia coli shifted from external pH (pH) 7.0 to pH 8.5–9.5 rapidly becomes tolerant to pH 10.0–11.5, induction of tolerance (alkali habituation) being dependent on periplasmic or external alkalinization with either NaOH or KOH. Induction needs protein synthesis and makes organisms resistant to DNA damage by alkali and better able to repair any damage that occurs. Induction of tolerance was reduced by glucose (not reversed by cAMP) and by amiloride, was dependent on DNA gyrase and was abolished by fur and himA lesions (the latter suggests IHF involvement). Tolerance induction was not prevented by L‐leucine, FeCl3 or FeSO4 nor by hns or relA mutations. Habituation probably involves attachment of IHF upstream of the promoter leading to DNA bending which switches on transcription. Habituation is aberrant in nhaA mutants, so ability to resist alkali damage may only arise if NhaA is induced, with extrusion of Na+by this antiporter during alkali challenge. In accord with one tolerance component involving NhaA induction, β‐galactosidase formation from nhaA‐lacZ fusions at pH 9.0 was inhibited by glucose and amiloride.

Extracellular Sensing and Signalling Pheromones Switch-On Thermotolerance and Other Stress Responses in Escherichia Coli

The findings reviewed here overturn a major tenet of bacterial physiology, namely that stimuli which switch-on inducible responses are always detected by intracellular sensors, with all other components and stages in induction also being intracellular. Such an induction mechanism even applies to quorum-sensed responses, and some others which involve functioning of extracellular components, and had previously been believed to occur in all cases. In contrast, for the stress responses reviewed here, triggering is by a quite distinct process, pairs of extracellular components being involved, with the stress sensing component (the extracellular sensing component, ESC) and the signalling component, which derives from it and induces the stress (the extracellular induction component, EIC), being extracellular and the stimulus detection occurring in the growth medium. The ESCs and EICs can also be referred to as extracellular sensing and signalling pheromones, since they are not only needed for induction in the stressed culture, but can act as pheromones in the same region activating other organisms which fail to produce the extracellular component (EC) pair. They can also diffuse to other regions and there act as pheromones influencing unstressed organisms or those which fail to produce such ECs. The cross-talk occurring due to such interactions, can then switch-on stress responses in such unstressed organisms and in those which cannot form the ESC/EIC pair. Accordingly, the ESC/EIC pairs can bring about a form of intercellular communication between organisms. If the unstressed organisms, which are induced to stress tolerance by such extracellular components, are facing impending stress challenge, then the pheromonal activities of the ECs provide an early warning system against stress. The specific ESC/EIC pairs switch-on numerous responses; often these pairs are proteins, but non-protein ECs also occur and for a few systems, full induction needs two ESC/EIC pairs. Most of the above ECs needed for response induction are highly resistant to irreversible inactivation by lethal agents and conditions and, accordingly, many killed cultures still contain ESCs or EICs. If these killed cultures come into contact with unstressed living organisms, the ECs again act pheromonally, altering the tolerance to stress of the living organisms. It has been claimed that bacteria sense increased temperature using ribosomes or the DnaK gene product. The work reviewed here shows that, for thermal triggering of thermotolerance and acid tolerance in E. coli, it is ESCs which act as thermometers.

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.

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.

An extracellular stress-sensing protein is activated by heat and u.v. irradiation as well as by mild acidity, the activation producing an acid tolerance-inducing protein

During growth of Escherichia coli in broth at pH 5·0, an extracellular protein termed an extracellular induction component (EIC) appears in the medium, this component being essential for acid tolerance induction. The present study establishes that the EIC arises from an extracellular precursor which is formed during growth at pH 7·0, and that conversion of the precursor to the EIC occurs at pH 5·0 (and other mildly acidic pH values) in the absence of organisms. On the basis that this precursor is produced by non‐stressed cells as well as by stressed ones, and that it is converted to the EIC (which in turn induces the tolerance response) by the stress, the precursor can be considered to be a stress sensor, the first extracellular stimulus sensor to be reported. The EIC formed at pH 5·0 was inactivated at pH 9·0. This inactivation probably involved conversion back to the precursor as EIC was reformed if the alkali‐inactivated component was incubated at pH 5·0. Both mild heat treatments (exposure to 40–55 °C) and u.v. irradiation also activated the precursor; the active induction component formed by the mild heat treatments was reversibly inactivated at pH 9·0 and so it seems likely that the component formed by heat treatment is similar or identical to the EIC produced at acidic pH. In contrast, the EIC produced by u.v. irradiation was not inactivated at pH 9·0, suggesting that it is different in some way to the EICs produced from the precursor by acidity or by heat treatment. It is likely that many responses affecting stress tolerance involve the functioning of such extracellular sensors, as similar components were shown to be involved in the acid tolerance responses induced at pH 7·0 by glucose, l‐aspartate and l‐glutamate. Extracellular stimulus sensors may also be needed for other inducible responses.

Novel acid sensitivity induced in Escherichia coli at alkaline pH

Transfer of pH 7.0‐grown Escherichia coli to pH 9.0 led to rapid acid sensitivity induction (ASI), the response being fully accomplished within 15 min at 37°C in broth. Only a slight increase in acid sensitivity occurred at pH 8.2 but the response was substantial at pH 8.4 and complete at pH 9.0 with no further sensitization at pH 9.5–10.5. ASI was not prevented by lesions in rpoH, katF, ompR, relA, spoT, fur, phoU, phoM (CreC), phoB/R, unc(atp), phoP or cadA and was unaffected by nalidixic acid, L‐leucine or iron starvation or excess. Full acid sensitivity was maintained for at least 2 h after a shift from pH 9.0 back to pH 7.0. ASI did not depend to a major extent on PhoE derepression and increased acid sensitivity of alkali‐induced strain C75a (phoE+) probably did not involve use of a new outer membrane proton pore.

Habituation to alkali in Escherichia coli

Escherichia coli grown at pH 9·0 was much more resistant to extremes of alkaline pH (10·5–11·5) than when grown at pH 7·0. This is termed habituation to alkali. It was not due to ability to reduce the pH of the medium during exposure but was due to a phenotypic change during growth at pH 9·0. Habituation occurred within 60 min at pH 9·0.