Introduction. Responses to weak acids, alkalinisation, biocides, UV irradiation, and toxic metal ions.
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Acids↗
Biomedical subjects
R J Rowbury
Publications and source records attributed to R J Rowbury.
Extracellular sensing components and extracellular induction component alarmones give early warning against stress in Escherichia coli.
The work reported here follows from the proposal that, for efficient induction of numerous extracellular stress responses, cultures contain extracellular stress-sensing molecules, termed extracellular sensing components (ESCs). These are directly converted to extracellular induction components (EICs) by stresses, thus providing an early warning system against stress, with very rapid responses occurring on exposure to increasing levels of stress. Although some stress responses appear to involve activation of intracellular sensors, the proposed ESCs and EICs function for many stress tolerance and sensitization responses and for several cross-tolerance and cross-sensitization responses. Because EICs can induce responses in unstressed cells, and because they are small molecules that can diffuse away from the site of formation, they can be considered to be 'alarmones', both warning unstressed organisms of future stress and preparing both stressed and unstressed ones to resist it. Therefore, EICs produced by one group of organisms could affect another group i.e. there could be 'cross-talk' (cell-to-cell communication) with other organisms in an area, to which the EICs diffuse, that has not yet faced the stress. In particular, stimuli that switch on acid tolerance, alkali tolerance, pH sensitization responses and alkylhydroperoxide tolerance are detected by ESCs; these molecules can give rise to EICs in the presence of the stress without organisms needing to be present. Not only does the ESC-EIC interconversion allow rapid switching on of responses, but for some responses it also allows rapid switching off. For some ESCs, the sensor can be modified by the culture conditions, modification leading to altered responsiveness to stress; such sensor changes appear to have evolved to allow the most efficient responses to stress to occur, under defined sets of conditions. In addition, the receptors on the organisms that interact with EICs are modified by culture conditions, so that extracellular components that function as ESCs for some cultures can act as EICs for others. In view of their role in early warning of stress, EICs and ESCs are likely to have important functions in the natural environment, especially in natural waters, in foods and food preparation and production, in hospital, domestic and commercial locations, and in the animal and human body. Findings of major importance relate to the extreme stress tolerance of some EICs. For example, because the acid-tolerance EIC formed at pH 5.0 is a heat-resistant molecule, heat-killed suspensions of acid-tolerant cultures can confer acid tolerance on living E. coli; cultures killed by extreme acidity and alkalinity and by exposure to high levels of UV irradiation or novobiocin are also able to confer acid tolerance on living E. coli. Extracellular components that inhibit induction of stress responses also occur in enterobacteria, since it has been found that AMP and HCO3-, which inhibit acid-tolerance induction, do so by forming extracellular agents that block the functioning of EICs. Similar agents to the above EICs and ESCs may occur in other non-stress-related processes. Systems using these extracellular components are quite distinct in their properties from quorum-sensing systems in Gram-negative bacteria and from those systems that use small peptides in intercellular communication and which induce virulence-related enzyme synthesis in Staphylococcus aureus and competence in streptococci and bacilli. Additionally, probably because the ESCs have evolved to become modified by cultural conditions, the components in the stress-related systems, although relatively small proteins, are much larger than the extracellular components used in the quorum-sensing processes and related systems. It is possible that the extracellular 'protectants' of Nikolaev, which protect E. coli from stress, act similarly to the EICs described here, e.g. by inducing stress tolerance. The antimutagenic factor of Vorobjeva may act similarly, although there is no evidence, so far, to suggest that it acts by inducing tolerance to mutagens.
Anti-Bacterial Agents↗
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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Acids↗
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.
Culture Media↗
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.
Alkalies↗
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.
Acids↗
Acid tolerance induced by metabolites and secreted proteins, and how tolerance can be counteracted.
Several metabolites and salts including glucose, L-glutamate, L-aspartate, FeCl3, KCl and L-proline induce acid tolerance at neutral external pH (pHo) in log phase Escherichia coli. For induction by glucose and L-glutamate, the processes are independent of integration host factor (IHF), H-NS, CysB, ferric uptake regulator (Fur) and RelA. For most of the above, tolerance does not appear if induction occurs and NaCl, sucrose, SDS or DOC are present. For several responses, cAMP inhibits induction. For many established acid tolerance and sensitization processes, including those tolerance responses switched on at pH 5.0 and by glucose, glutamate or aspartate, induction is associated with secretion of extracellular induction proteins. These proteins bring about the response if added to organisms under normally non-inducing conditions. Secreted components also influence inherent acid tolerances and sensitivities. Analysis of some established tolerance responses indicates that induction is a two-stage process, secreted extracellular proteins playing an obligate role in induction. For example, the functioning of the acid-induced medium protein(s) is essential for acid habituation at pHo 5.0. It seems likely that such two-stage mechanisms are essential for many inducible processes in bacteria.
Acids↗
Extracellular sensors and inducible protective mechanisms.
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Escherichia coli↗
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.
Aspartic Acid↗
Extracellular proteins and other components as obligate intermediates in the induction of a range of acid tolerance and sensitisation responses in Escherichia coli.
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.
Culture Media↗
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 beta-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.
Adaptation, Physiological↗
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 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.
Bacterial Proteins↗
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 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.
Bacterial Proteins↗
Recent advances in biology: intercellular communication and quorum sensing in micro-organisms.
N-acyl-L-homoserine lactones are involved as intercellular signalling agents controlling a wide range of physiological responses in Gram-negative bacteria. They function especially in vibrios, pseudomonads and erwinias as well as in Rhizobium and Agrobacterium spp, particularly where the bacteria are in symbiotic or parasitic relationships with higher organisms. Several Gram-negatives, such as Escherichia coli, do not, however, appear to produce or respond to AHLs and they may have other intercellular signalling molecules. The present review reports that several stress related responses in E. coli can be induced by supernatant fluids from cultures which have already induced the response. In some cases at least, the active agents in the supernatant fluids are proteins rather than AHLs.
Cell Division↗
Life sciences up-date. Do we need to rethink our ideas on the mechanisms of inducible processes in bacteria?
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.
Adaptation, Physiological↗
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.
Anaerobiosis↗
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. In five experiments, attachment to muscle increased heat tolerance. For example, in one experiment the D (58 degrees C) value increased from approximately 2 min for free cells, to > 10 min for attached cells. In three other experiments, differences between free and attached cells were not so pronounced, although attached cells were still more tolerant. This suggests that muscle attachment, which may occur naturally during the preparation of comminuted meat products, could permit greater survival during subsequent cooking and thus may be a possible explanation for the involvement of cooked foods in outbreaks/cases of infection with Salm. typhimurium DT104.
Animals↗
More chaos on the microbiology front: the E. coli O157 outbreaks.
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Cross-Sectional Studies↗