Acid tolerance inLeuconostoc oenos. Isolation and characterization of an acid-resistant mutant

The acid tolerance ofLeuconostoc oenos was examined in cells surviving at pH 2.6, which is lower than the acid limit of growth (about pH 3.0). Acid-adapted cells survived better than non-adapted cells. Tolerance to acid stress was found to be dependent upon the adaptive pH. Acid resistance was increased by an order of magnitude for cultures adapted to a pH of about 2.9. Inhibiting protein synthesis with chloramphenicol prior to acid shock revealed that acid adaptation may involve two separate systems, one of which appears to be independent of protein synthesis. The acid-resistant mutant LoV8413, isolated during a long-term survival screen at pH 2.6, was found to be able to grow in acidic media and was characterized by a high H⁺-ATPase activity at low pH. The data from electrophoretic analysis of total proteins labeled with [³⁵S] methionine indicate that large amounts of a protein of 42 kDa molecular mass were produced within this acid-resistant mutant.     

Intracellular pH is a major factor in the induction of tolerance to acid and other stresses in Lactococcus lactis.

This study demonstrates that exposure of log-phase Lactococcus lactis subsp. cremoris 712 cells to mildly acid conditions induces resistance to normally lethal intensities of environmental stresses such as acid, heat, NaCl, H2O2, and ethanol. The intracellular pH (pHi) played a major role in the induction of this multistress resistance response. The pHi was dependent on the extracellular pH (pHo) and on the specific acid used to reduce the pHo. When resuspended in fresh medium, cells were able to maintain a pH gradient even at pHo values that resulted in cell death. Induction of an acid tolerance response (ATR) coincided with an increase in the ability of cells to resist change to an unfavorable pHi; nevertheless, a more favorable pHi was not the sole reason for the increased survival at acid pHo. Cells with an induced ATR survived exposure to a lethal pHo much better than did uninduced cells with a pHi identical to that of the induced cells. Survival following lethal acid shock was dependent on the pHi during induction of the ATR, and the highest survival was observed following induction at a pHi of 5.9, which was the lowest pHi at which growth occurred. Increased acid tolerance and the ability to maintain a higher pHi during lethal acid stress were not acquired if protein synthesis was inhibited by chloramphenicol during adaptation.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer.

Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

A low-pH-inducible, stationary-phase acid tolerance response in Salmonella typhimurium.

Acid is an important environmental condition encountered by Salmonella typhimurium during its pathogenesis. Our studies have shown that the organism can actively adapt to survive potentially lethal acid exposures by way of at least three possibly overlapping systems. The first is a two-stage system induced in response to low pH by logarithmic-phase cells called the log-phase acid tolerance response (ATR). It involves a major molecular realignment of the cell including the induction of over 40 proteins. The present data reveal that two additional systems of acid resistance occur in stationary-phase cells. One is a pH-dependent system distinct from log-phase ATR called stationary-phase ATR. It was shown to provide a higher level of acid resistance than log-phase ATR but involved the synthesis of fewer proteins. Maximum induction of stationary-phase ATR occurred at pH 4.3. A third system of acid resistance is not induced by low pH but appears to be part of a general stress resistance induced by stationary phase. This last system requires the alternative sigma factor, RpoS. Regulation of log-phase ATR and stationary-phase ATR remains RpoS independent. Although the three systems are for the most part distinct from each other, together they afford maximum acid resistance for S. typhimurium.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer

Induction of acid resistance (habituation) in Escherichia coli at pH 5·0 took ca 5 min in broth at 37°C and 30–60 min in minimal medium. Induction occurred at a range of pH values from 4·0 to 6·0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5·0 retained most of their resistance after 2 h growth at pH 7·0. Organisms grown at pH 5·0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7·0. DNA repair-deficient strains carrying recA, uvrA or polAl mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5·0. Organisms grown at pH 5·0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7·0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5·0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium.

The acid tolerance response (ATR) is an adaptive system triggered at external pH (pHo) values of 5.5 to 6.0 that will protect cells from more severe acid stress (J. Foster and H. Hall, J. Bacteriol. 172:771-778, 1990). Correlations between the internal pH (pHi) of adapted versus unadapted cells at pHo of 3.3 indicate that the ATR system produces an inducible pH-homeostatic function. This function serves to maintain the pHi above 5 to 5.5. Below this range, cells rapidly lose viability. Development of this pH homeostasis mechanism was sensitive to protein synthesis inhibitors and operated only to augment the pHi at pHo values below 4. In contrast, classical constitutive pH homeostasis was insensitive to protein synthesis inhibitors and was efficient only at pHo values above 4. Physiological studies indicated an important role for the Mg(2+)-dependent proton-translocating ATPase in affording ATR-associated survival during exposure to severe acid challenges. Along with being acid intolerant, cells deficient in this ATPase did not exhibit inducible pH homeostasis. We speculate that adaptive acid tolerance is important to Salmonella species in surviving acid encounters in both the environment and the infected host.     

Effect of pH on the Protective Action of Interferon in L Cells

The pH of the solution in which interferon was applied to L cells determined the level of resistance developed against challenge with vesicular stomatitis virus (VSV). No inhibition of challenge virus was observed when interferon was applied to cells at pH 6.0. At pH 6.5, partial inhibition of VSV replication was observed and inhibition was maximum at pH 7.0. Evidence was obtained that interferon interacted with L cells at pH 6.0, but that resistance did not develop until the cells were placed in a medium at pH 7.0. These effects were explained by data showing that exposure of cells to a medium at pH 6.0 reversibly inhibited both ribonucleic acid and protein synthesis.     

Acid adaptation induces cross-protection against some environmental stresses in Vibrio parahaemolyticus

The relationship of acid adaptation to the resistance of other environmental stresses was examined in Vibrio parahaemolyticus. Acid-adapted cells were found to have increased resistance to various stresses, including heat, crystal violet, bile, and deoxy cholic acid. However, heat-adapted cells showed no increased resistance against acid stress. Adaptation required protein synthesis, since treatment with chloramphenicol during adaptation to pH 5.3 prevented the development of acid resistance. Acid-adapted cells showed an increased amount of outer membrane protein with an apparent molecular weight of 27,000. These results show that acid-induced cross-protection involved changes in outer membrane protein composition and the known enhancement of intracellular pH homeostasis.     

Effect of Pre-Stressing on the Acid-Stress Response in Bifidobacterium Revealed Using Proteomic and Physiological Approaches

Weak acid resistance limits the application of Bifidobacteria as a probiotic in food. The acid tolerance response (ATR), caused by pre-stressing cells at a sublethal pH, could improve the acid resistance of Bifidobacteria to subsequent acid stress. In this study, we used Bifidobacterium longum sub. longum BBMN68 to investigate the effect of the ATR on the acid stress response (ASR), and compared the difference between the ATR and the ASR by analyzing the two-dimensional-PAGE protein profiles and performing physiological tests. The results revealed that a greater abundance of proteins involved in carbohydrate metabolism and protein protection was present after the ASR than after the ATR in Bifidobacterium. Pre-stressing cells increased the abundance of proteins involved in energy production, amino acid metabolism, and peptidoglycan synthesis during the ASR of Bifidobacterium. Moreover, after the ASR, the content of ATP, NH₃, thiols, and peptidoglycan, the activity of H⁺-ATPase, and the maintenance of the intracellular pH in the pre-stressed Bifidobacterium cells was significantly higher than in the uninduced cells. These results provide the first explanation as to why the resistance of Bifidobacterium to acid stress improved after pre-stressing.     

Stabilization of neocarzinostatin nonprotein chromophore activity by interaction with apoprotein and with HeLa cells

The methanol-extracted, nonprotein chromophore of the protein antibiotic neocarzinostatin (NCS), which possesses the full in vitro and in vivo deoxyribonucleic acid (DNA) strand-breaking activities and the ability to inhibit DNA synthesis and growth in HeLa cells of the holoantibiotic, is much more labile to inactivation by heat, 2-mercaptoethanol, long-wavelength UV light, and pH values above 4.8. Inactivation is inversely related to the methanol concentration. The pH activity profile of the isolated chromophore extends to pH values below 7.0. Chromophore inactivation is specifically blocked by the apoprotein of NCS; 100-fold higher concentrations of the apoprotein of another protein antibiotic, auromomycin, gave similar protection, whereas bovine serum albumin is even less effective. The chromophore, and not the apoprotein, is inactivated by heat or light (360 nm) as determined by both activity and isoelectric focusing experiments. In contrast to other chromophoric antibiotic substances (daunorubicin and the extracted chromophore of aurodomomycin), the NCS chromophore interacts irreversibly with HeLa cells at 0 degrees C in serum-free medium so as to inhibit subsequent DNA synthesis at 37 degrees C. Such interaction at 0 degrees C is very rapid, reaching 50% completion in about 15 s, and is not found with native NCS or when apo-NCS is added before the chromophore or when serum is included in the preincubation at 0 degrees C. Washing with apo-NCS or serum-containing (or-free) medium after preincubation of the cells with the chromophore at 0 degrees C fails to reverse the subsequenct inhibition of DNA synthesis.     

Induction of Acid Resistance of Salmonella typhimurium by Exposure to Short-Chain Fatty Acids

Exposure to short-chain fatty acids (SCFA) is one of the stress conditions Salmonella typhimurium encounters during its life cycle, because SCFA have been widely used as food preservatives and SCFA are also present at high concentrations in the gastrointestinal tracts of host animals. The effects of SCFA on the acid resistance of the organism were examined in an attempt to understand the potential role of SCFA in the pathogenesis of S. typhimurium. The percent survival of S. typhimurium at pH 3.0 was determined after exposure to SCFA for 1 h at pH 7.0. The percent acid survival, which varied depending on the SCFA species and the concentration used, was 42 after exposure to 100 mM propionate at pH 7.0 under aerobic incubation conditions, while less than 1% could survive without exposure. The SCFA-induced acid resistance was markedly enhanced by anaerobiosis (64%), lowering pH conditions (138% at pH 5.0), or increasing incubation time (165% with 4 h) during exposure to propionic acid. When protein synthesis during exposure to propionate was blocked by chloramphenicol, the percent acid survival was less than 1, indicating that the protein synthesis induced by exposure to propionate is required for the induction of the acid resistance. The percent acid survival determined with the isogenic mutant strains defective in acid tolerance response revealed that AtrB protein is necessary for the full induction of acid resistance by exposure to propionate, while unexpectedly, inactivation of PhoP significantly increased acid resistance over that of the wild type (P < 0.05). The results suggest that the virulence of S. typhimurium may be enhanced by increasing acid resistance upon exposure to SCFA during its life cycle and further enhanced by anaerobiosis, low pH, and prolonged exposure time.     

Characterization of the protein-synthesis dependent adaptive acid tolerance response in Lactobacillus acidophilus

Exposure of L. acidophilus CRL 639 cells to sublethal adaptive acid conditions (pH 5.0 for 60 min) was found to confer protection against subsequent exposure to lethal pH (pH 3.0). Adaptation, which only occurred in complex media, was dependent on de novo protein synthesis and was inhibited by amino acid analogues. There was no modification in the protein synthesis rate during adaptation, but the protein degradation rate decreased. Synthesis of acid stress proteins may increase the stability of pre-existing proteins. By 2D-PAGE, induction of nine acid stress proteins and repression of several housekeeping proteins was observed. Putative heat shock proteins DnaK, DnaJ, GrpE, GroES and GroEL (70, 43, 24, 10 and 55 kDa, respectively) were among the proteins whose synthesis was induced in response to acid adaptation.     

Peptidoglycan Synthesis in Cocci and Rods of a pH-Dependent, Morphologically Conditional Mutant of Klebsiella pneumoniae

Mir M7 is a spontaneous morphologically conditional mutant of Klebsiella pneumoniae which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [(3)H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to beta-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Erythromycin inhibition of cell proliferation and in vitro mitochondrial protein synthesis in human HeLa cells is pH dependent

The growth of HeLa cells in Hepes-buffered medium was significantly more sensitive to the inhibitory effects of erythromycin than in medium buffered by the more conventional bicarbonate-CO₂ system. Since growth inhibition by erythromycin became more pronounced as the pH of the medium was increased the difference in erythromycin sensitivity between the Hepes-buffered medium vs. the bicarbonate-CO₂-buffered medium is most likely due to pH effects. The relative growth sensitivity to erythromycin of ERY2301, an erythromycin-resistant mutant of HeLa, was also affected by elevated pH of the growth medium. However, ERY2301 cells were able to proliferate to a greater extent in the presence of erythromycin than HeLa cells grown under the same conditions. The selective growth advantage of ERY2301 (in the presence of erythromycin) is best seen in medium of pH 7.4, or in the Hepes-buffered medium. In vitro protein synthesis by intact mitochondria isolated from HeLa cells was relatively insensitive to erythromycin inhibition at pH 7.4 and 7.6, but at high pH values was inhibited approx. 50%. Although the erythromycin sensitivity of ERY2301 mitochondrial protein synthesis was also affected by increasing the pH, the incorporation of [³H]leucine was more resistant to erythromycin than that observed for HeLa mitochondria over the pH range tested. Increasing the concentration of erythromycin at a given pH did not result in a further increase in the inhibition of either HeLa or ERY2301 mitochondrial protein synthesis. When the mitochondrial membranes were disrupted by Triton X-100, erythromycin inhibition of HeLa mitochondrial protein synthesis was pH dependent and, at the lower pH values tested, greater inhibition was observed as the erythromycin concentration was increased. ERY2301 mitochondrial protein synthesis under the same conditions displayed a high level of erythromycin-resistant activity independent of both pH and erythromycin concentration. It is suggested that, as has been proposed for bacterial systems, only the non-protonated molecule of erythromycin is effective in inhibiting mitochondrial protein synthesis. The ability of erythromycin to permeate the mitochondrial membranes and the plasma membres may also be facilitated by a higher pH.     

Sensitivity of Synchronized Chinese Hamster Cells to Ultraviolet Light

The age response for lethality of Chinese hamster cells to ultraviolet light shows that they are resistant in G(1), sensitive as they move into and through the S phase and resistant again in G(2) and mitosis. Survival curves determined at different times in the cycle reveal that mitotic cells are the most resistant fraction, much more resistant than S cells, and more resistant than either G(1) or G(2) cells. The extent to which the age response is ilfluenced by nucleic acid and protein synthesis was investigated by using inhibitors of these processes. In the presence of inhibitors of DNA or protein synthesis added to G(1) cells before exposure, cell survival neither declines to the minimum survival of S cells nor rises subsequently to the resistance of G(2) cells. If, before exposure, DNA synthesis is arrested in the middle of S, when survival is at a minimum, the subsequent rise in survival during G(2) is not prevented. However when cycloheximide is added before exposure, during the middle of S, this rise is prevented. When actinomycin D, an inhibitor of RNA synthesis is added prior to exposure the age response is affected only slightly. Postirradiation treatment of G(1) and mid-S cells with inhibitors of DNA or protein synthesis maintains survival at a level characteristic of the age of the cells.     

Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli.

Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.     

Acid response of exponentially growing Escherichia coli K-12

Induction of acid tolerance response (ATR) of exponential-phase Escherichia coli K-12 cells grown and adapted at different conditions was examined. The highest level of protection against pH 2.5 challenges was obtained after adaptation at pH 4.5-4.9 for 60 min. To study the genetic systems, which could be involved in the development of log-phase ATR, we investigated the acid response of E. coli acid resistance (AR) mutants. The activity of the glutamate-dependent system was observed in exponential cells grown at pH 7.0 and acid adapted at pH 4.5 in minimal medium. Importantly, log-phase cells exhibited significant AR when grown in minimal medium pH 7.0 and challenged at pH 2.5 for 2 h without adaptation. This AR required the glutamate-dependent AR system. Acid protection was largely dependent on RpoS in unadapted and adapted cells grown in minimal medium. RpoS-dependent oxidative, glutamate and arginine-dependent decarboxylase AR systems were not involved in triggering log-phase ATR in cells grown in rich medium. Cells adapted at pH 4.5 in rich medium showed a higher proton accumulation rate than unadapted cells as determined by proton flux assay. It is clear from our study that highly efficient mechanisms of protection are induced, operate and play the main role during log-phase ATR.     

Synthesis of peptidoglycan and teichoic acid in Bacillus subtilis: role of the electrochemical proton gradient.

The effects of several ionophores and uncouplers on glycerol and N-acetylglucosamine incorporation by Bacillus subtilis 61360, a glycerol auxotroph, were tested at different pH values. In particular, the effect of valinomycin on the synthesis of teichoic acid and peptidoglycan was examined in more detail in both growing cells and in vitro biosynthetic systems. Valinomycin inhibited synthesis of wall teichoic acid and peptidoglycan in whole cells but not in the comparable in vitro systems. It did not inhibit formation of free lipid or lipoteichoic acid. The results were consistent with a role for the electrochemical proton gradient in maintaining full activity of cell wall synthetic enzymes in intact cells. Such an energy source would be required for a model in which rotation or reorientation of synthetic enzyme complexes is envisaged for the translocation of wall precursor molecules across the cytoplasmic membrane (Harrington and Baddiley, J. Bacteriol. 155:776-792, 1983).     

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.