R.J. Rowbury

An assessment of environmental factors influencing acid tolerance and sensitivity in Escherichia coli, Salmonella spp. and other enterobacteria

Environmental factors such as temperature, pH and nutrient level affect enterobacterial acid sensitivity, as do the presence of phosphate and Na+ and the extent of aeration. The mechanisms governing these effects are partially understood and the involvement of phoE, fur and atp in acid tolerance, of phoE, envZ, tonB, (p)ppGpp and cAMP in salt‐induced acid sensitivity and of rpoS in stationary‐phase acid tolerance are of particular interest. It should be noted that surface attachment enhances acid resistance.

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.

Regulatory components, including integration host factor, CysB and H‐NS, that influence pH responses in Escherichia coli

This review describes a range of pH responses. Some are only induced if relevant DNA is brought to an appropriately supercoiled configuration by DNA gyrase and bent by the action of, for example, integration host factor (IHF). Bending may allow transcription by bringing activators into juxtaposition with RNA polymerase, which is CysB‐associated in several of the responses. Control of arginine decarboxylase (AdiA) synthesis at acid pH is of the above type, with dependence on the presence of gyrase, H‐NS, IHF and CysB ; acid induction of LysU has similar requirements but also needs Lrp ; lysine decarboxylase (CadA) formation at acid pH is controlled quite differently, needing the CadC activator and interaction of lysine/lysine permease ; H‐NS probably reverses induction by CadC. The Hyd components of formic hydrogenlyase are induced by acid under anaerobiosis ; a transcriptional activator is involved and Fur may also function in regulation. Acid tolerance induced at low pH in log‐phase cells needs CysB and PhoE but not DNA gyrase ; tolerance is reduced by NaCl but not affected by Fe3+, Fe2+ glucose/cAMP or by lrp, him, fur, hns or nhaA/B lesions. Alkali tolerance (habituation), induced at pH0 8·5–9·0, probably involves DNA supercoiling and bending ; the induction process needs IHF, CysB, PhoE, NhaA, TonB and Fur and is glucose‐repressed ; tolerance may result from Na+ efflux catalysed by the NhaA antiporter, which is induced at pH0 9·0. Alkali sensitivity induced at pH0 5·5 also requires gyrase, IHF and CysB, but H‐NS, Lrp, NhaA and OmpC are also needed and induction is abolished by NaCl. Salt‐induced acid sensitivity results from PhoE formation and is blocked by glucose (reversed by cAMP), FeCl3 and hns and relA lesions, the effect of relA being envZ‐suppressed. Acid sensitivity induction (ASI) at pH0 9·0 needs H‐NS, is inhibited by FeCl3 and amiloride, and is associated with alkyl hydroperoxide reductase synthesis. Leucine‐induced acid sensitivity needs gyrase, CysB, H‐NS, Fur, OmpA and RelA, is inhibited by Fe3+, Fe2+, tetracycline, glucose and nalidixic acid, but not by chloramphenicol ; increased outer membrane proton passage may result from OmpA modification.

Cross‐talk involving extracellular sensors and extracellular alarmones gives early warning to unstressed Escherichia coli of impending lethal chemical stress and leads to induction of tolerance responses

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Heat tolerance of Salmonella typhimurium DT104 isolates attached to muscle tissue

Eight separate experiments were performed with three isolates of Salmonella typhimurium DT104 to examine the impact that attachment to pork muscle tissue has on heat tolerance. Infive experiments, attachment to muscle increased heat tolerance. For example, in one experimentthe D (58°C) value increased from approximately 2 min for free cells, to >10 min forattached cells. In three other experiments, differences between free and attached cells were not sopronounced, although attached cells were still more tolerant. This suggests that muscleattachment, which may occur naturally during the preparation of comminuted meat products,could permit greater survival during subsequent cooking and thus may be a possible explanationfor the involvement of cooked foods in outbreaks/cases of infection with Salm. typhimurium DT104.

Morphological changes to Escherichia coli O157:H7, commensal E. coli and Salmonella spp in response to marginal growth conditions, with special reference to mildly stressing temperatures.

Certain rod-shaped bacteria have been reported to form elongated filamentous cells when exposed to marginal growth conditions, including refrigeration temperatures. To expand upon these observations, the filamentation of commensal Escherichia coli, E. coli O157:H7 and Salmonella spp was investigated, following exposure to certain, mildly stressing, levels of temperature, pH or water activity (aw), with levels of cellular protein being monitored during cell elongation, in some experiments. Our studies indicated that cellular filamentation could be demonstrated in all 15 strains of the above organisms tested, following exposure to marginal conditions achieved by incubation at high or low temperatures, high or low pH values and low aw. The level of environmental stress causing filamentation tended to be specific to the particular organisms. For example, Salmonella spp formed filamentous cells at 44°C, whereas E. coli strains, including 0157, grew by binary fission at that temperature, but formed filamentous cells at 46°C. In addition, plate count techniques to enumerate bacteria during filamentation, failed to reflect the increase in cell biomass that was occurring, whereas measurements of protein concentration demonstrated the increase quite strikingly. These findings have important implications for our understanding of the ability of food-borne pathogens to cause disease, since the infectious dose of a microorganism implicated in an outbreak of such disease is typically determined by a viable count method, which could underestimate the number of potential infectious units present in a food that had been stored in such a way as to provide marginal growth conditions.

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.

Acid habituation in Salmonella enteritidis PT4: impact of inhibition of protein synthesis

Incubation of Salmonella enteritidis PT4 in media at pH values between 3.0–6.0 resulted in a marked and rapid increase in acid resistance, manifested when cells were subsequently challenged at pH 2.5‐2.9. Habituation involves two separate systems, one of which is independent of protein synthesis. The relative importance of the two systems is governed by the pH of the challenge medium. Thus in broth at pH 2.7 or above, inhibition of protein synthesis during habituation did not lessen subsequent acid resistance. At pH values of 2.6 and below, the addition of chloramphenicol during habituation was found to reduce subsequent acid resistance although cells were not as sensitive as control cultures grown at pH 7.0.