RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression in Streptococcus mutans

Glucosyltransferases (Gtfs) and fructosyltransferase (Ftf), and the exopolysaccharides they produce, facilitate bacterial adherence and biofilm formation, and enhance the virulence of Streptococcus mutans. In this study, we used continuous chemostat cultures and reporter gene fusions to study the expression of ftf and gtfBC in response to carbohydrate availability and pH, and to asses the role of a protein similar to catabolite control protein A (CcpA), RegM, in regulation of these genes. Expression of ftf was efficient at pH 7.0 and 6.0, but was repressed at pH 5.0 under glucose-excess conditions. At pH 7.0, ftf expression was 5-fold lower under glucose-limiting conditions than in cells growing with an excess of glucose. Expression of gtfBC was also sensitive, albeit to a lesser extent, to pH and glucose availability. Inactivation of regM resulted in decreases of as much as 10-fold in both ftf and gtfBC expression, depending on growth conditions. These findings reinforce the importance of pH and carbohydrate availability for expression of two primary virulence attributes of S. mutans and reveal a critical role for RegM in regulation of expression of both gtfBC and ftf.     

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.     

Microbiological Survey of Adirondack Lakes with Various pH Values

Nine high-altitude oligotrophic Adirondack lakes in upstate New York having water of pH 4.3 to 7.0 were surveyed for total bacterial numbers and possible adaptation of the microbial communities to environmental pH. The number of heterotrophic bacteria from water samples recoverable on standard plate count agar were low (10¹ to 10³ per ml) for most of the lakes. Acridine orange direct counts were approximately two orders of magnitude higher than plate counts for each lake. Sediment aerobic heterotrophs recovered on standard plate count agar ranged from 1.4 × 10⁴ to 1.3 × 10⁶ per g of sediment. Direct epifluorescence counts of bacteria in sediment samples ranged from 3.0 × 10⁶ to 1.4 × 10⁷ per g. Low density values were consistent with the oligotrophic nature of all the lakes surveyed. There were no apparent differences in numbers of bacteria originally isolated at pH 5.0 and pH 7.0 between circumneutral lakes (pH > 6.0) and acidic lakes (pH < 5.0). Approximately 1,200 isolates were recultured over a range of pH from 3.0 to 7.0. Regardless of the original isolation pH (pH 5.0 or pH 7.0), less than 10% of the isolates grew at pH < 5.0. Those originally isolated at pH 5.0 also grew at pH 6.0 and 7.0. Those originally isolated at pH 7.0 preferred pH 7.0, with 98% able to grow at pH 6.0 and 44% able to grow at pH 5.0. A chi-square contingency test clearly showed (P < 0.005) that two distinct heterotrophic populations had been originally isolated at pH 5.0 and pH 7.0, although there is undoubtedly some overlap between the two populations.     

Effect of NaCl, pH, temperature, and atmosphere on growth of Salmonella typhimurium in glucose-mineral salts medium

The interactions of pH (5.0, 6.0, and 7.0), temperature (19, 28, and 37 degrees C), and atmosphere (aerobic versus anaerobic) with NaCl (0, 1, 2, 3, 4, and 5%) on the growth of Salmonella typhimurium ATCC 14028 in defined glucose-mineral salts culture medium were evaluated. Response surface methodology was used to develop equations describing the response of S. typhimurium to environmental changes. The response to an increasing concentration of NaCl at any temperature tested was nonlinear. The maximum growth was predicted to occur at an NaCl concentration of 0.5%, a temperature of 19 degrees C, and an initial pH of 7.0 under aerobic growth conditions. The relative amounts of aerobic growth at 19 degrees C, pH 7.0, and NaCl concentrations of 0, 0.5, 1, 2, 3, 4, and 5% were predicted to be 99.2, 100.0, 98.8, 90.2, 73.5, 48.6, and 15.6%, respectively. Anaerobic growth conditions repressed the amount of growth relative to that under aerobic conditions, and the effects of NaCl and pH were additive at low salt concentrations; however, at higher salt levels anaerobiosis provided protection against the effects of NaCl.     

Effect of intracellular pH on rotational speed of bacterial flagellar motors

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E. coli cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of E. coli cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered E. coli cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.     

Effect of NaCl, pH, temperature, and atmosphere on growth of Salmonella typhimurium in glucose-mineral salts medium.

The interactions of pH (5.0, 6.0, and 7.0), temperature (19, 28, and 37 degrees C), and atmosphere (aerobic versus anaerobic) with NaCl (0, 1, 2, 3, 4, and 5%) on the growth of Salmonella typhimurium ATCC 14028 in defined glucose-mineral salts culture medium were evaluated. Response surface methodology was used to develop equations describing the response of S. typhimurium to environmental changes. The response to an increasing concentration of NaCl at any temperature tested was nonlinear. The maximum growth was predicted to occur at an NaCl concentration of 0.5%, a temperature of 19 degrees C, and an initial pH of 7.0 under aerobic growth conditions. The relative amounts of aerobic growth at 19 degrees C, pH 7.0, and NaCl concentrations of 0, 0.5, 1, 2, 3, 4, and 5% were predicted to be 99.2, 100.0, 98.8, 90.2, 73.5, 48.6, and 15.6%, respectively. Anaerobic growth conditions repressed the amount of growth relative to that under aerobic conditions, and the effects of NaCl and pH were additive at low salt concentrations; however, at higher salt levels anaerobiosis provided protection against the effects of NaCl.     

Changes in the proton-motive force in Escherichia coli in response to external oxidoreduction potential.

The pH homeostasis and proton-motive force (Deltap) of Escherichia coli are dependent on the surrounding oxidoreduction potential (ORP). Only the internal pH value and, thus, the membrane pH gradient (DeltapH) component of the Deltap is modified, while the membrane potential (DeltaPsi) does not change in a significant way. Under reducing conditions (Eh < 50 mV at pH 7.0), E. coli decreases its Deltap especially in acidic media (21% decrease at pH 7.0 and 48% at pH 5.0 for a 850-mV ORP decrease). Measurements of ATPase activity and membrane proton conductance (CH+m) depending on ORP and pH have shown that the internal pH decrease is due to an increase in membrane proton permeability without any modification of ATPase activity. We propose that low ORP values de-energize E. coli by modifying the thiol : disulfide balance of proteins, which leads to an increase in the membrane permeability to protons.     

Adaptation to life at micromolar nutrient levels: the regulation of Escherichia coli glucose transport by endoinduction and cAMP

Abstract: Free-living bacteria are expert in adapting to variations in nutrient availability, often using an array of transport systems of different affinities to scavenge for particular substrates (multiport). This review concentrates on the regulation of expression of different transporters contributing to multiport in response to varying nutrient levels. A novel mechanism of controlling bacterial transport affinity under sugar limitation is described. In particular, switching from glucose-rich to glucose-limited conditions results in Escherichia coli orchestrating outer membrane changes as well as the induction of a periplasmic binding protein-dependent (ABC-type) transport system. The changes leading to the high affinity transport pathway are directed towards uptake of rapidly utilisable concentrations and are optimal close to 10⁻⁶ M medium glucose. High affinity transport is absent under both glucose-rich 'feast' and glucose-starved 'famine' conditions hence high affinity transporters are not simply repressed by excess nutrient. Rather, the improvement in glucose scavenging involves induction of genes in 2 distinct regulons ( mgl/gal and mal/lamB ) through synthesis of 2 different endogenous inducer molecules (galactose, maltotriose). Endoinducer levels are tightly controlled by extracellular glucose concentration at different glucose-limited growth rates. Aside from endoinducers, the elevated intracellular level of cAMP plays a role in induction of the high-affinity pathway but CAMP-mediated relief from catabolite repression is not itself sufficient for high affinity transport. In contrast to the repressive role of glucose when present at millimolar concentrations, micromolar glucose also leads to the induction of transport systems for other sugars, further broadening the scavenging potential of nutrient-limited bacteria for other substrates.     

Mechanism of proton gating of a urea channel

The size and complexity of many pH-gated channels have frustrated the development of specific structural models. The small acid-activated six-membrane segment urea channel of Helicobacter hepaticus (HhUreI), homologous to the essential UreI of the gastric pathogen Helicobacter pylori, enables identification of all the periplasmic sites of proton gating by site-directed mutagenesis. Exposure to external acidity enhances [(14)C]urea uptake by Xenopus oocytes expressing HhUreI, with half-maximal activity (pH(0.5)) at pH 6.8. A downward shift of pH(0.5) in single site mutants identified four of six protonatable periplasmic residues (His-50 at the boundary of the second transmembrane segment TM2, Glu-56 in the first periplasmic loop, Asp-59 at the boundary of TM3, and His-170 at the boundary of TM6) that affect proton gating. Asp-59 was the only site at which a protonatable residue appeared to be essential for pH gating. Mutation of Glu-110 or Glu-114 in PL2 did not affect the pH(0.5) of gating. A chimera, where the entire periplasmic domain of HhUreI was fused to the membrane domain of Streptococcus salivarius UreI (SsUreI), retained the pH-independent properties of SsUreI. Hence, proton gating of HhUreI likely depends upon the formation of hydrogen bonds by periplasmic residues that in turn produce conformational changes of the transmembrane domain. Further studies on HhUreI may facilitate understanding of other physiologically important pH-responsive channels.     

Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium: characterization of the dipeptide permease (Dpp) and the dipeptide-binding protein

The dipeptide permease (Dpp) is one of three genetically distinct peptide-transport systems in enteric bacteria. Dpp also plays a role in chemotaxis towards peptides. We have devised three selections for dpp mutations based on resistance to toxic peptides (bacilysin, valine-containing peptides, and bialaphos). All dpp mutations mapped to a single chromosomal locus between 77 and 78 min in Salmonella typhimurium and at 79.2 min in Escherichia coli. Expression of dpp was constitutive in both species but the absolute level of expression varied widely between strains. At least in part this difference in expression levels is determined by cis-acting sequences. The dpp locus of E. coli was cloned. The first gene in the operon, dppA, encodes a periplasmic dipeptide-binding protein (DBP) required for dipeptide transport and chemotaxis. Downstream of dppA are other genes required for transport but not for chemotaxis. The dipeptide-binding protein was found to share 26.5% sequence identity with the periplasmic oligopeptide-binding protein OppA.     

Exposure of Escherichia colito acid habituation conditions sensitizes it to alkaline stress

R.J. ROWBURY AND N.H. HUSSAIN. 1996. Escherichia coli transferred from pH 7.0 to pH 5.5 or 6.0 became alkali-sensitive by a rapidly induced phenotypic response. Alkali sensitization was reduced at pH 5.0 and virtually abolished at pH 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 pH 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 pH 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 β-galactosidase and to alkali inhibition of β-galactosidase induction. Alkali sensitization induced at pH 5.5 may involve NhaB loss.     

The Effect of pH and NaCl Levels on the Physicochemical and Emulsifying Properties of a Cruciferin Protein Isolate

The influence of pH (3.0, 5.0, and 7.0) and ionic strength (0, 50, 100 mM NaCl) on the physicochemical and emulsifying properties of a cruciferin-rich protein isolate (CPI) was investigated. Surface charge on the CPI was found to substantially reduced in the presence of NaCl. Surface hydrophobicity was found to be the lowest for CPI at pH 7.0 with 100 mM NaCl, and highest at pH 3.0 without NaCl. Solubility was found to be lowest at pH 5.0 and 7.0 without NaCl (<20 %), however greatly improved for all other pH and NaCl conditions (>80 %). Interfacial tension was found to be lowest at 10–11 mN/m for pH 5.0–0 mM NaCl and pH 7.0–50/100 mM NaCl, whereas under all other conditions interfacial tension was higher (15+ mN/m). Overall, NaCl has no effect on EAI at pH 3.0 where it ranged between 18.8 and 19.4 m²/g. At pH 5.0, EAI decreased from 21.1 to 12.8 m²/g as NaCl levels increased from 0 to 100 mM. At pH 7.0, EAI values were found to decrease from 14.9 to 5.2 m²/g as NaCl levels were raised from 0 to 100 mM. Overall, ESI was reduced with the addition of NaCl from ~15.7 min at 0 mM NaCl to ~11.6 min and ~12.0 min for the 50 and 100 mM NaCl levels, respectively.     

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.     

pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli

Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.     

The sodium ion translocating adenosinetriphosphatase of Propionigenium modestum pumps protons at low sodium ion concentrations

The purified ATPase (F1F0) of Propionigenium modestum has its pH optimum at pH 7.0 or at pH 6.0 in the presence or absence of 5 mM NaCl, respectively. The activation by 5 mM NaCl was 12-fold at pH 7.0, 3.5-fold at pH 6.0, and 1.5-fold at pH 5.0. In addition to its function as a primary Na+ pump, the ATPase was capable of pumping protons. This activity was demonstrated with reconstituted proteoliposomes by the ATP-dependent quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine. No delta pH was formed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone or by blocking the ATPase with dicyclohexylcarbodiimide. In the presence of valinomycin and K+, the delta pH increased, in accord with the operation of an electrogenic proton pump. The proton pump was only operative at low Na+ concentrations (less than 1 mM), and its activity increased as the Na+ concentration decreased. Parallel to the decrease of H+ pumping, the velocity of the Na+ transport increased about 6-fold from 0.1 to 4 mM NaCl, indicating a switch from H+ to Na+ pumping, as the Na+ concentration increases. Due to proton leaks in the proteoliposomal membranes, fluorescence quenching was released after blocking the ATPase with dicyclohexylcarbodiimide, by trapping residual ATP with glucose and hexokinase, or by the Na+-induced conversion of the proton pump onto a Na+ pump. Amiloride, an inhibitor of various Na+-coupled transport systems, was without effect on the kinetics of Na+ transport by the P. modestum ATPase.     

Cellular mechanisms of pH tolerance in Rhizobium loti

Seven strains of Rhizobium loti were tested for acid tolerance in yeast-extract mannitol (YEM) broth at pH values ranging from 4.0 to 8.0. The strains that grew at pH 4.0 showed the slowest generation time when grown at pH above 7.0 and also produced the most acid. The acid tolerance was related to the composition and structure of the membrane. pH influenced protein expression in acid-tolerant strains growing at pH 4.0 or 7.0. Acid tolerant strains showed one membrane protein of 49.5 kDa and three soluble proteins of 66.0, 58.0 and 44.0kDa; their expression increased when the cells grew at pH 4.0. It is suggested that acid tolerance in Rhizobium loti involves constitutive mechanisms, such as permeability of the outer membrane together with adaptive responses, including the state of bacterial growth and concomitant changes in protein expression.