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.

Journal Article 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 Get access R.J. Rowbury R.J. Rowbury Biology Department, University College London, London, UK Search for other works by this author on: Oxford Academic Google Scholar Journal of Applied Microbiology, Volume 90, Issue 5, 18 May 2001, Pages 677–695, https://doi.org/10.1046/j.1365-2672.2001.01312.x Published: 18 May 2001 Article history Received: 08 November 2000 Revision received: 22 January 2001 Accepted: 30 January 2001 Published: 18 May 2001

Escheichia 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 DOG, 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 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.

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.

Several acid tolerance responses of Escherichia coli were associated with secretion into the growth media of components (frequently proteins) which altered acid tolerance of other cultures. First, medium filtrates from cultures induced to acid tolerance by several conditions converted pH 7.0-grown organism to tolerance and, for most such responses, filtrate proteins were needed for full induction. Secondly, filtrates from cultures induced to acid sensitivity at alkaline pH produced sensitisation of resistant cultures. Thirdly, filtrates from inherently tolerant or sensitive strains altered tolerance or sensitivity of normal strains. In many cases, filtrate components were essential for the original response, e.g. acid habituation at pH 5.0. Extracellular components may function as intermediates only in stress tolerance responses, but other adaptive responses must be tested as such components may function in other inducible processes.

It is confirmed that for a large number of inducible stress tolerance and sensitization responses in Escherichia coli, induction is associated with the secretion of components, often proteins, which can induce related responses in other organisms under normally non-inducing conditions. For most responses, the original induction is also dependent on the functioning of the extracellular agent. It is proposed that the extracellular induction components (EICs) not only induce appropriate responses in organisms in the vicinity of those challenged by the stress, but also act as extracellular alarmones. It is predicted that the switching-on of all other inducible stress tolerances and stress cross-protection and cross-sensitization responses will be found to require similar EICs produced on exposure to appropriate stresses. It is also suggested that cultures will respond to many inhibitory chemicals such as antibiotics, chlorine, other biocides and NO (and possibly to inhibitory biological agents) by producing appropriate EICs i.e. that organisms have evolved so that on exposure to any lethal challenge or a challenge that might be expected to become lethal, they produce diffusible secreted components (EIC alarmones) which both act as a warning to other potentially vulnerable organisms and prepare those organisms to resist the expected challenge. One major stress involves transfer to higher oxygen levels and it is suggested that EIC alarmones will be involved in the rapid habituation to such conditions; such EICs may function not only to induce protection against oxidative components formed as the O2 level rises, but may also be involved in induction/repression of metabolic pathways needed to accomodate to the conditions by giving the most favourable growth at high O2. It is also predicted that envelope alterations induced by environmental changes will prove to be dependent on secreted EICs. The possibility that other inducible processes, including those involving induction or repression of enzymes or pathways by changes in nutrient level, might need EICs has also been discussed. It is concluded that the evolution of EICs now allows organisms to make a meaningful response to almost all inhibitory chemical, physical and, possibly, biological agents and agencies. Also, however, it can be argued that it would be beneficial if nutrient-regulated inducible and repressible processes involving changes in enzyme levels also depended on EICs and, accordingly, in view of such advantages, evolution may have ensured that enzyme inductions and repressions commonly involve such extracellular induction components. It is essential that this be tested. Even those responses unlikely to be associated with EIC secretion (e.g., rapid and reversible processes such as chemotactic responses) may be indirectly influenced by EICs.

ED1829ColV, I-K 94, a derivative of Escherichia coli K-12, were injured with hydrogen peroxide, acetic and formic acid for various length of time at acidic pH(s) in Nutrient Broth # 2. The acid injured organicism recovered significantly better in the media containing phosphate than the media without phosphate.Phosphate helped in plating efficiency of the inured bacterial population and somehow expedite the recovery process of the acid stressed organisms.

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 degrees 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.

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 heat-stable 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.

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.

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.

Escherichia coli shifted from external pH (pH(O)) 7.0 to pH(O) 8.5-9.5 rapidly becomes tolerant to pH(O) 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, beta-galactosidase formation from nhaA-lacZ fusions at pH(O) 9.0 was inhibited by glucose and amiloride.

NaCl-induced acid sensitivity is not a response to high osmotic pressure but is triggered by a high internal sodium ion concentration, evidenced by the finding that conditions which enhance Na+ influx or reduce Na+ efflux, led to increased internal Na+ and allowed induction at lower external sodium ion concentrations and that induction could occur with no osmotic upshock. NaCl-induced acid sensitivity was not observed in Escherichia coli strain MC4100 and its relA lesion was investigated as a possible cause. Transformation of this strain and two other relA mutants to relA+ allowed sensitization by NaCl and whereas strain CF1648 (relA+) was sensitized, its relA deletion derivative was not. Additionally, transduction of two relA+ strains to relA produced derivatives which were not sensitized by salt. Although MC4100 was not sensitized by NaCl, envZ derivatives of it were sensitized. Lesions in fur and tonB did not prevent sensitization by NaCl, although extent of sensitization was slightly increased by fur and considerably increased by tonB. Acetate strongly inhibited sensitization, which was also subject to glucose repression, reversible by cAMP. The possibility was discussed that (p)ppGpp and cAMP both positively affect transcription of the genes encoding the acid sensitization components; in accord with this, high concentrations of cAMP suppressed the effect of the relA lesion of sensitization. Salt-induced organisms are more sensitive to acid damage to DNA, to acid inhibition of enzyme synthesis and DNA transfer and slightly less able to repair acid-damaged transforming DNA.

Escherichia coli transferred from pHo 7.0 to pHo 5.5 or 6.0 became alkali-sensitive by a rapidly induced phenotypic response. Alkali sensitization was reduced at pHo 5.0 and virtually abolished at pHo 6.5. The response was triggered by cytoplasmic rather than external or periplasmic acidification and de novo protein synthesis was needed. Alkali sensitivity failed to appear at pHo 5.5 plus DNA gyrase inhibitors and was markedly reduced by himA, himD, hns, ompC and nhaA lesions. A tonB deletion mutant showed alkali sensitivity at pHo 7.0. Alkali sensitivity induction was not subject to catabolite repression nor was it appreciably affected by a relA lesion. Acid-induced cells were more sensitive to alkali damage to both DNA and beta-galactosidase and to alkali inhibition of beta-galactosidase induction. Alkali sensitization induced at pHo 5.5 may involve NhaB loss.

Organisms grown in low salt broth (LSB) are acid resistant but become sensitive on growth for 30-60 min with 300 mmol l-1 added NaCl. Salt-induced acid sensitivity only occurs in relA+ strains and sensitization is abolished by glucose, this catabolite repression effect being reversed by cAMP. The finding that sensitization did not occur in a phoE strain but did occur in a phoE+ derivative of it suggested that the response might result from PhoE induction, since PhoE acts as the major outer membrane (OM) proton pore under most conditions. In agreement with this, low-salt broth (LSB)-grown cells of a chromosomally lac- strain carrying pJP102 (phoE-lacZ) produced low levels of beta-galactosidase but growth with added NaCl led to rapid and appreciable induction. Also, a phoA mutant carrying a phoE-phoA fusion produced little alkaline phosphatase after growth in LSB but much more in LSB with added NaCl. Increased beta-galactosidase synthesis (in phoE-lacZ strains) in the presence of NaCl was abolished by glucose, this effect being reversible by cAMP, and there was more NaCl-induced synthesis of this enzyme in relA+ strains. Accordingly, it appears that addition of NaCl to LSB leads to acid sensitivity because it induces synthesis of the OM proton pore PhoE.

Escherichia coli grown in low-salt broth (LSB) showed rapid induction of acid sensitivity when L-leucine was added. Induction was strongly inhibited by nalidixic acid and by tetracycline but only slightly by chloramphenicol and rifampicin. Regulation of the effect appeared to involve H-NS, CysB, the ferric uptake regulator (Fur) and (p)ppGpp; ompA mutants were also unable to mount the response. Acid sensitization was greatly reduced by glucose, ferric chloride and ferrous sulphate but was not affected appreciably by phosphate or amiloride. Lesions abolishing synthesis of integration host factor, the PhoE porin and NhaA antiporter, did not prevent sensitization by L-leucine and lrp lesions only slightly affected the response. Organisms exposed to L-leucine were less susceptible to phage K3. Acid sensitivity depends on appearance of a new outer membrane (OM) pathway for protons and L-leucine-induced organisms may use a new pore formed from a modified OmpA protein.

In a comparative study of different Salmonella enteritidis phage type 4 isolates we found that those isolates with enhanced heat tolerance also survived better than isolates that were heat sensitive either at pH 2.6, in 10 mM H2O2, or on surfaces. Culture to the stationary phase increased the heat tolerance of all isolates and the acid and H2O2 tolerance of heat-tolerant isolates. With heat-sensitive isolates, however, extended culture had no impact on survival in H2O2 and only a marginal impact on acid tolerance. The growth phase had no appreciable impact on the surface survival of any of the isolates.