Acid Shock Research Articles

Acid Shock Accumulation of Sigma S in Salmonella enterica Involves Increased Translation, Not Regulated Degradation

Enteric pathogens such as Salmonella enterica and Escherichia coli face the daunting task of surviving passage through the extremely acid pH of the stomach in order to establish an infection in the host intestinal tract. These organisms have evolved elaborate stress response systems that aid in survival. The alternative sigma factor σ^S is a key regulator of many stress responses in S. enterica and is regulated at the levels of transcription, translation, and protein stability. Of these control mechanisms, proteolysis has been considered paramount in determining σ^S levels in the cell. Until the current report, acid shock was thought to increase σ^S levels by directly regulating degradation. However, mutant strains unable to degrade σ^S still exhibited acid shock induction of σ^S. We demonstrate here that rpoS translation is a major focus of acid stress control and is responsible for the observed increase in σ^S levels. A series of deletions of the 566-nucleotide untranslated region of the rpoS mRNA were constructed to examine the importance of this regulatory region in acid shock induction of rpoS. Progressive deletions starting from the 5′ end of the rpoS message produced alternating loss and recovery of acid shock control. The results suggest that competing stem-loop structures work in concert to control the acid shock induction of rpoS. Further, the half-life of σ^S was unchanged in response to acid shock and over-expression of the MviA recognition protein resulted in constitutive σ^S degradation under acid stress conditions. The data indicate that in log phase, nonstressed cells increasing σ^S production is sufficient to increase protein half-life. In toto, these results suggest that acid shock stabilization of σ^S is the result of increased synthesis via translational control and does not involve changes in the activity of the MviA (RssB/SprE) ClpXP degradation complex. Therefore, constitutive degradation may enable the cell to reset the level of σ^S once acid stress is alleviated.

Stress response of Listeria monocytogenes isolated from cheese and other foods

The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied. Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2–3.5% (w/v) sodium chloride, also were studied. The effect of temperature on pH and sodium chloride sensitivity was also determined. The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data. Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant. A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride. All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported. Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride. Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5). The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride). In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.

Optimizing Sporulation of Clostridium perfringens

Many sporulation media have been developed for Clostridium perfringens, but none stimulates sporulation for all strains. The aim of our experiments was to develop a sporulation method using Duncan and Strong (DS) medium, which supports sporulation of a wide variety of strains. Different inoculation levels were tested, and the effects of sporulation-promoting substances and acid shock were evaluated. Furthermore, DS medium was compared with other sporulation media. Highest spore numbers in DS medium were obtained with a 10% 24-h fluid thioglycollate broth inoculum (5.0 × 105/ml). Addition of theophylline and replacement of starch by raffinose increased spore yields for some strains, but most strains were not affected (average increases in log N/ml of 0.2 and 0.3, respectively). One strain was enhanced by the addition of bile, but other strains were strongly inhibited (average decrease in log N/ml of 2.5); agar did not influence sporulation. Neither short-time acid exposure nor addition of culture supernatant fluids of well-sporulating strains resulted in higher spore numbers in DS medium. None of the tested methods enhanced sporulation in general; only strain-dependent effects were obtained. Peptone bile theophylline medium was the most promising sporulation medium tested; peptone bile theophylline starch medium yielded highest spore numbers (2.5 × 105/ml), but some strains failed to sporulate. In conclusion, adding theophylline to DS medium may optimize sporulation of C. perfringens, but peptone bile theophylline medium with or without starch is most suitable.

Stress response of Aeromonas hydrophila following environmental challenges

Two strains of Aeromonas hydrophila were studied to determine the effects of four stresses: acid adaptation, acid shock, starvation, and cold adaptation on subsequent heat tolerance (50°C) or freeze/thaw (−20°C/21°C) resistance. D -values (min or days) were calculated and interactions between stress and strain were evaluated. Survivor curves for heat tolerance and freeze/thaw resistance were nonlinear. Acid shock or acid adaptation effects on heat tolerance and freeze/thaw resistance were variable between the two strains (P<0·05). Starvation decreased heat tolerance (P<0·05) for both strains and had no effect on freeze/thaw resistance (P>0·05). Cold adaptation decreased heat tolerance for both strains and had no effect on freeze/thaw resistance. Both strains were more acid tolerant following prior exposure to acid stress (pH 5·0) in log phase (P<0·05). Log phase cells of strain ATCC 7965 were more acid tolerant than MSV1. No differences in acid tolerance were observed between strains of stationary phase cells. A. hydrophila does not appear to have a measurable phenotypic cross protective response to starvation or cold stress that enhances heat or freeze/thaw tolerance and has limited response to other stresses.

Mechanisms of acid resistance due to the urease system of Helicobacter pylori

Background & Aims:Helicobacter pylori, a neutralophile, uses acid neutralization by urease to combat gastric acidity, allowing gastric colonization. Both acute and chronic acid resistance mechanisms are present. Acute mechanisms of acid adaptation could be due to surface urease, increased inner-membrane urea permeability via UreI, or both. Slower mechanisms may involve increased nickel insertion into apoenzyme, posttranscriptional regulation, or increased enzyme synthesis. The aim of this study was to further define regulation of urease under acidic conditions. Methods: Surface-bound urease was analyzed by measurement of free and bound urease after centrifugation through a step gradient and by quantitative urease immunostaining of intact and fixed bacteria. Changes in urease synthesis or assembly were determined by incubation of the organisms at pH 5.5 or 7.0 in the absence and presence of chloramphenicol, urea, or nickel chelator and in ureI-positive and -negative organisms. Results: The amount of surface urease was below detection limits with either centrifugation washing or immunostaining. Total bacterial urease activity was increased 3–5-fold by incubation at pH 5.5 in the presence of chloramphenicol but not in nickel-free medium or in ureI knockout organisms. There was also a 3-fold increase in survival of acid shock in acid-adapted organisms. Conclusions: Surface-bound urease is too low to contribute to acid resistance. Acidic medium pH induces UreI-dependent nickel incorporation into apoenzyme. This augmentation of urease activity increases survival in acid and is part of the gastric colonization strategy of the organism.GASTROENTEROLOGY 2002;123:187-195

Auxin stimulates S6 ribosomal protein phosphorylation in maize thereby affecting protein synthesis regulation.

Auxin is known to stimulate protein synthesis in many plant tissues, but the mechanisms involved in this process are unknown. The present research inquires whether auxin might regulate selective translation of mRNAs by inducing S6 ribosomal protein phosphorylation on the 40S ribosomal subunit in maize (Zea mays L.). Maize embryonic axes auxin-stimulated by natural (IAA) or synthetic (Dicamba or 1-NAA) auxins, selectively increased ribosomal protein synthesis. This effect was not reproduced by auxin inactive analogue 2-NAA. Enhanced S6 ribosomal protein phosphorylation on the 40S ribosomal subunit was also observed after auxin stimulation, as measured by [32P] incorporation into this protein. This increment did not occur when stimulation was performed with the inactive auxin analogue. Further, increased recruitment into polysomes of two 5'TOP-like mRNAs, encoding for the initiation translation factor eIF-iso4E and the S6 ribosomal protein, was also found after auxin stimulation of maize axes. A positive correlation was established between the levels of S6 ribosomal protein phosphorylation and the S6 ribosomal protein transcript recruitment into polysomes by means of okadaic acid or heat shock application to maize axes. These data indicate that auxin stimulates S6 ribosomal protein phosphorylation on maize ribosomes, concomitant to the recruitment of specific mRNAs (5'TOP-like mRNAs) into polysomes for translation. It is proposed that by this mechanism auxin regulate the synthesis of specific proteins in maize tissues.

Autoinduction of the ompR response regulator by acid shock and control of the Salmonella enterica acid tolerance response.

Salmonella enterica serovar Typhimurium periodically experiences acid stress in a variety of host and non-host environments. An encounter with non-lethal acid stress (pH > 4) induces an assortment of physiological changes, called the acid tolerance response (ATR), that helps the cell to tolerate extreme low pH (pH 3). These physiological changes differ in log phase and stationary phase cells and are controlled by different regulatory proteins. OmpR is an acid-induced response regulator critical to the stationary phase ATR but not to the log phase ATR. As OmpR also controls the expression of the acid-induced virulence operon ssrAB, acid shock induction of ompR was examined to gain insight into how Salmonella links virulence with survival at extreme acid pH. The results indicate that acid pH induces ompR from a promoter different from that used for basal expression. Transcription from this promoter is repressed by the histone-like protein H-NS and requires OmpR-P for induction. The classic sensor kinase EnvZ and acetyl phosphate collaborate to produce the optimum level of OmpR-P needed for autoinduction. Although OmpR-P is required for acid-induced expression of ompR in wild-type cells, OmpR is not needed for ompR transcription in the absence of H-NS. Thus, the role of OmpR-P in autoinduction is to help to counteract repression by H-NS. This evidence, combined with the finding that relaxing DNA supercoiling with novobiocin also increased ompR transcription, suggests that acid stress induces ompR by altering local DNA topology, not by changing the phosphorylation status of OmpR.

ISOLATION AND PARTIAL CHARACTERIZATION OF CATALYTIC ANTIBODIES WITH OLIGONUCLEASE ACTIVITY FROM BOVINE COLOSTRUM

ABSTRACT Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA fromEscherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75°C for 52 min.

The Helicobacter pylori homologue of the ferric uptake regulator is involved in acid resistance.

The only known niche of the human pathogen Helicobacter pylori is the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid are important for successful colonization. One of the few regulatory proteins in the H. pylori genome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation of iron homeostasis. However, in Salmonella enterica serovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the H. pylori Fur homologue in acid resistance. Isogenic fur mutants were generated in three H. pylori strains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent strains. Under acidic conditions, growth of the fur mutants was severely impaired. No differences were observed between the survival of the fur mutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not improve the growth of the fur mutant at acidic pH. This indicates that the phenotype of the fur mutant at low pH was not due to increased iron sensitivity. Transcription of fur was repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of H. pylori; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.

Human macrophage activation programs induced by bacterial pathogens.

Understanding the response of innate immune cells to pathogens may provide insights to host defenses and the tactics used by pathogens to circumvent these defenses. We used DNA microarrays to explore the responses of human macrophages to a variety of bacteria. Macrophages responded to a broad range of bacteria with a robust, shared pattern of gene expression. The shared response includes genes encoding receptors, signal transduction molecules, and transcription factors. This shared activation program transforms the macrophage into a cell primed to interact with its environment and to mount an immune response. Further study revealed that the activation program is induced by bacterial components that are Toll-like receptor agonists, including lipopolysaccharide, lipoteichoic acid, muramyl dipeptide, and heat shock proteins. Pathogen-specific responses were also apparent in the macrophage expression profiles. Analysis of Mycobacterium tuberculosis-specific responses revealed inhibition of interleukin-12 production, suggesting one means by which this organism survives host defenses. These results improve our understanding of macrophage defenses, provide insights into mechanisms of pathogenesis, and suggest targets for therapeutic intervention.

Complementation of cold shock proteins by translation initiation factor IF1 in vivo.

The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For B. subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the B. subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from E. coli in a B. subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.

Cell density modulates acid adaptation in Streptococcus mutans: implications for survival in biofilms.

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.

Analysis of expression of GroEL (Hsp60) of Clostridium difficile in response to stress

Our laboratory has previously shown that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses and that GroEL, a heat shock protein, serves an adhesive function in this bacterium. In this communication, RT–PCR, SDS–PAGE and immunoblotting were used to study the stress response in C. difficile following heat, acid or osmotic shock, iron deprivation or presence of a subinhibitory concentration of ampicillin in the culture medium. All these stresses increased transcription of groEL and production of GroEL to various degrees. Furthermore, the protein was found in membrane fractions and in the extracellular space after heat stress.

Effect of carbon starvation and proteolytic activity on stationary-phase acid tolerance of Streptococcus mutans.

Previous research with Streptococcus mutans and other oral streptococci has demonstrated that the acid shock of exponential-phase cells (pH 7.5 to 5.5) resulted in the induction of an acid tolerance response (ATR) increasing survival at low pH (3.5-3.0). The current study was designed to determine whether two fresh isolates, H7 and BM71, and two laboratory strains, Ingbritt and LT11, were capable of a stationary-phase ATR as estimated by a survival test at pH 3.5 for 3 h. All four strains were unable to generate a stationary-phase ATR under control conditions at pH 7.5, with the exception of a burst of survivors in the transition between the exponential and stationary phases when the carbon source (glucose) was depleted. Adaptation at pH 5.5 resulted in the expected pH-dependent exponential-phase ATR, but only the fresh isolates exhibited a stationary-phase ATR at this pH. Glucose starvation of cells in complex medium was shown to enhance acid tolerance for the fresh isolates, but not the laboratory strains. This tolerance was, however, greatly diminished for all strains in a defined medium with a low concentration of amino acids. Growth of strain H7 in complex medium resulted in the formation of at least 56 extracellular proteins, nine of which were degraded in the early stationary phase following the induction of proteolytic activity during the transition period. No proteolytic activity was observed with strain LT11 and only 19 extracellular proteins/peptides were apparent in the medium with only one being degraded in the early stationary phase. Strain H7 was also shown to have two- to fourfold higher levels of intracellular glycogen in the stationary phase than strain LT11. These results suggest that S. mutans H7 possessed the required endogenous metabolism to support amino acid/peptide uptake in the early-stationary phase, which resulted in the formation of basic end products that, in turn, contributed to enhanced intracellular pH homeostasis.

Genetic and physiologic analysis of the groE operon and role of the HrcA repressor in stress gene regulation and acid tolerance in Streptococcus mutans.

Our working hypothesis is that the major molecular chaperones DnaK and GroE play central roles in the ability of oral bacteria to cope with the rapid and frequent stresses encountered in oral biofilms, such as acidification and nutrient limitation. Previously, our laboratory partially characterized the dnaK operon of Streptococcus mutans (hrcA-grpE-dnaK) and demonstrated that dnaK is up-regulated in response to acid shock and sustained acidification (G. C. Jayaraman, J. E. Penders, and R. A. Burne, Mol. Microbiol. 25:329-341, 1997). Here, we show that the groESL genes of S. mutans constitute an operon that is expressed from a stress-inducible sigma(A)-type promoter located immediately upstream of a CIRCE element. GroEL protein and mRNA levels were elevated in cells exposed to a variety of stresses, including acid shock. A nonpolar insertion into hrcA was created and used to demonstrate that HrcA negatively regulates the expression of the groEL and dnaK operons. The SM11 mutant, which had constitutively high levels of GroESL and roughly 50% of the DnaK protein found in the wild-type strain, was more sensitive to acid killing and could not lower the pH as effectively as the parent. The acid-sensitive phenotype of SM11 was, at least in part, attributable to lower F(1)F(0)-ATPase activity. A minimum of 10 proteins, in addition to GroES-EL, were found to be up-regulated in SM11. The data clearly indicate that HrcA plays a key role in the regulation of chaperone expression in S. mutans and that changes in the levels of the chaperones profoundly influence acid tolerance.

Acid Stress, Starvation, and Cold Stress Affect Poststress Behavior of Escherichia coli O157:H7 and Nonpathogenic Escherichia coli

The effects of acid shock, acid adaptation, starvation, and cold stress of Escherichia coli O157:H7 (ATCC 43895), an rpo S mutant (FRIK 816-3), and nonpathogenic E. coli (ATCC 25922) on poststress heat resistance and freeze–thaw resistance were investigated. Following stress, heat tolerance at 56°C and freeze–thaw resistance at −20 to 21°C were determined. Heat and freeze–thaw resistance of E. coli O157:H7 and nonpathogenic E. coli was enhanced after acid adaptation and starvation. Following cold stress, heat resistance of E. coli O157:H7 and nonpathogenic E. coli was decreased, while freeze–thaw resistance was increased. Heat and freeze–thaw resistance of the rpoS mutant was enhanced only after acid adaptation. Increased or decreased tolerance of acid-adapted, starved, or cold-stressed E. coli O157:H7 cells to heat or freeze–thaw processes should be considered when processing minimally processed or extended shelf-life foods.

Differences in Escherichia coli Culture Conditions Can Have a Large Impact on the Induction of Extreme Acid Resistance

A recently isolated Escherichia coli strain (3TF4) survived an acid shock that mimicked the low pH of the human gastric stomach (pH 2, 1 h), but this survival was highly influenced by prior growth conditions. Only 0.01% of the stationary phase cells that had been grown anaerobically in a carbonate medium (2 mg glucose and 0.25 mg yeast extract per ml, 40 mm sodium carbonate, final pH 6.5) survived the acid shock, and the survival of exponential phase cultures was even lower (0.0001%). Small amounts of Trypticase (1.5 mg/ml) increased the survival as much as 5000-fold, but cultures that were provided with higher concentrations of Trypticase (7.5 mg/ml) did not reach the stationary phase in 24 h and were more acid sensitive. Sodium acetate (50 mm) also increased acid resistance, and the increased acid shock survival was greater for the cells that had reached the stationary phase (100 versus 1000-fold, respectively). E. coli 3TF4 cultures that had been grown aerobically in Luria broth were already so acid resistant (survivals greater than 40%) that they did not respond to sodium acetate. E. coli 3TF4 cultures that were refrigerated (5 degrees C, 7 days) were nearly as acid resistant as those that were immediately subjected to acid shock (pH 2.0, 1 h).

Characterization of the groESL operon in Listeria monocytogenes: utilization of two reporter systems (gfp and hly) for evaluating in vivo expression.

The ability of intracellular pathogens to sense and adapt to the hostile environment of the host is an important factor governing virulence. We have sequenced the operon encoding the major heat shock proteins GroES and GroEL in the gram-positive food-borne pathogen Listeria monocytogenes. The operon has a conserved orientation in the order groES groEL. Upstream of groES and in the opposite orientation is a gene encoding a homologue of the Bacillus subtilis protein YdiL, while downstream of groEL is a gene encoding a putative bile hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and transcriptional fusions to the UV-optimized Aequorea victoria green fluorescent protein (GFP(UV)) to analyze expression of groESL under various environmental stress conditions, including heat shock, ethanol stress, and acid shock, and during infection of J774 mouse macrophage cells. Strains harboring GFP(UV) transcriptional fusions to the promoter region of groESL demonstrated a significant increase in fluorescence following heat shock that was detected by both fluorimetry and fluorescence microscopy. Using both RT-PCR and GFP technology we detected expression of groESL following internalization by J774 cells. Increased intracellular expression of dnaK was also determined using RT-PCR. We have recently described a system which utilizes L. monocytogenes hemolysin as an in vivo reporter of gene expression within the host cell phagosome (C. G. M. Gahan and C. Hill, Mol. Microbiol. 36:498-507, 2000). In this study a strain was constructed in which hemolysin expression was placed under the control of the groESL promoter. In this strain hemolysin expression during infection also confirms transcription from the groESL promoter during J774 and murine infection, albeit at lower levels than the known virulence factor plcA.

Induction of vap genes encoded by the virulence plasmid of Rhodococcus equi during acid tolerance response

The response of the intracellular pathogen Rhodococcus equi to acid shock, a stress potentially encountered after phagocytosis by macrophages, was analyzed. The wild-type and its avirulent plasmid-cured strain acquired increased acid tolerance during the exponential growth phase upon exposure to sublethal acid stress, a response referred to as the acid tolerance response. Maximal adaptation was observed when cells were pretreated for 90 min at pH 5.0 before exposure to the pH challenge. Search for plasmid-encoded proteins regulated by an acidic pH was performed using two-dimensional gel electrophoresis, and enabled us to detect several membrane and cytoplasmic proteins with altered expression during the adaptation phase, but none of them were plasmid-encoded. However, using a strategy based on plasmid-encoded gene expression, we showed that two operons located on the virulence plasmid of strain 85F were upregulated by acid pHs with a maximal induction at pH 5.0. One operon, containing vapA, was monocistronic whereas the other was polycistronic composed of vapD and an unknown open reading frame. Our combined results suggest that these genes may play an important role in the pathogenicity of R. equi.

Identification of stress-responsive genes in Streptococcus mutans by differential display reverse transcription-PCR.

Streptococcus mutans, which causes dental caries in the human oral cavity and occasionally causes infective endocarditis in the heart, withstands adverse environmental stress through diverse alterations in protein synthesis. Differential gene expression in response to environmental stress was analyzed by RNA fingerprinting using arbitrarily primed PCR with a panel of 11mer primers designed for differential display in Enterobacteriaceae. Dot and Northern blot hybridization confirmed that the transcription of several genes was up- or down-regulated following exposure to acid shock from pH 7.5 to 5.5. RNA of a gene designated AP-185 (acid-stress protein) was induced specifically by acid treatment, while RNA of GSP-781 (general-stress protein) was up-regulated significantly when bacteria were exposed to high osmolarity and temperature, as well as low pH. The deduced amino acid sequence of AP-185 shares homology (78% identity) with branched-chain amino acid aminotransferase. Cloning and sequence analysis of GSP-781 revealed a potential secreted protein of a molecular mass of about 43 kDa and with a pI predicted to be 5.5. Transcriptional levels of another gene, designated AR-186 (acid-repressed protein), which encodes putative aconitase, were repressed by acid treatment but were enhanced by plasma or serum components. Analogous results were identified in icd and citZ genes, and repression of these genes, along with AR-186, was also observed when they were exposed to high osmolarity and temperature. These results indicate that differential regulation of specific genes at the transcriptional level is triggered by different stress and that genes responsible for glutamate biosynthesis in the citrate pathway are coordinately regulated during the stress response of S. mutans.