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 °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.     

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 organisms 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.O. 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.     

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.     

Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes. Possible relationship of this protein to physiologic glutamate receptors

Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approximately 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger Mr protein in synaptic membranes. Protease activity during protein purification was prevented by introducing five protease inhibitors, and a three-step purification procedure was developed that yielded a high degree of purification of glutamate-binding proteins. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 microM, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L-[3H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes.     

Characterization of a binding protein-dependent glutamate transport system of Rhodobacter sphaeroides.

The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (Kt of 1.2 microM), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH. From the shock fluid, an L-glutamate-binding protein was isolated and purified. The protein binds L-glutamate (apparent Kd of 1.3 microM) and L-glutamine (Ki of 15 microM) with high affinity. The expression level of this binding protein is maximal at limiting concentrations of glutamine in the growth medium. The glutamate-binding protein restores the uptake of L-glutamate in spheroplasts. L-Aspartate is a strong competitive inhibitor of L-glutamate uptake (Ki of 3 microM) but competes only poorly with L-glutamate for binding to the binding protein (Ki of > 200 microM). The uptake of L-aspartate in R. sphaeroides also involves a binding protein which is distinct from the L-glutamate-binding protein. These data suggest that in R. sphaeroides, the L-glutamate- and L-aspartate-binding proteins interact with the same membrane transporter.     

Transient treatments with L-glutamate and threo-beta-hydroxyaspartate induce swelling of rat cultured astrocytes

We characterized swelling of rat cultured astrocytes induced by L-glutamate and its analogues. Among L-glutamate receptor agonists, L-glutamate, L-aspartate, L-cysteic acid, DL-homocysteic acid, quisqualate and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) increased astrocytic intracellular volume (3H-OMG space), while kainate, and N-methyl-D-aspartate did not. Threo-beta-hydroxyaspartate (TBHA), D-aspartate and L-trans-pyrrolidine-2,4-dicarboxylic acid, high-affinity substrates for Na+-dependent L-glutamate transporters, increased astrocytic 3H-OMG space. L-Glutamate (0.5 mM) increased astrocytic 3H-OMG space to 300% of control in 40-60 min. The increase in 3H-OMG space by 1 mM TBHA was comparable to the L-glutamate-induced one. After a 10 min-exposure to 0.5 mM L-glutamate, astrocytic 3H-OMG space was further increased to 200% even in the absence of L-glutamate. Astrocytes transiently exposed to L-glutamate did not increase their cell volume in K+-free medium and in the presence of 1 mM ouabain, a Na+-K+ ATPase inhibitor. The increase after a transient exposure was also observed by a treatment of 1 mM TBHA, but not by 0.5 mM quisqualate. These results suggest that the volume increases after a transient treatment are mediated by activation of Na+-dependent L-glutamate transporter.     

Possible linkage between glutamate transporter and mitogen-activated protein kinase cascade in cultured rat cortical astrocytes

The mitogen-activated protein kinases (MAPKs) play a pivotal role in the mediation of cellular responses to a variety of signalling molecules. In the present study, we investigated possible linkage between glutamate signalling and the MAPK cascade in cultured rat cortical astrocytes. Exposure of the cells to L-glutamate (100-1000 microM) resulted in an increase in phosphorylated p44/42 MAPK (ERK1/2) in a concentration- and time-dependent manner. The glutamate-induced ERK1/2 phosphorylation was blocked by U0126 and PD98059, specific inhibitors of the MAPK-activating enzyme MEK. Furthermore, L-glutamate-induced ERK1/2 phosphorylation was not mimicked by glutamate receptor agonists and was not blocked by glutamate receptor antagonists. In contrast, the effect of L-glutamate was mimicked by D- and L-aspartate and transportable glutamate uptake inhibitors. These results suggest that the MEK/ERK cascade is activated by a mechanism related to glutamate transporters. We propose that the glutamate transporter functions as a receptor transmitting extracellular glutamate signal to intracellular messengers.     

The role of calcium ion in the L-glutamate-induced depolarization in the frog spinal cord

1. The role of Ca2+ in L-glutamate-induced depolarization was investigated in the isolated frog spinal cord. 2. The size of a depolarization induced by L-glutamate (3 mM) was inversely related to the extracellular Ca2+ concentration, but was reduced in a Ca2+-free medium containing EGTA (0.3 mM). 3. L-Glutamate caused a marked depolarization in both ventral and dorsal roots, even in a NaCl-deficient medium (Ca2+, 2.0 mM). The size of the depolarization was attenuated by a prolonged or repeated application of L-glutamate. Ca2+ can be replaced by Sr2+ or Mg2+. 4. Concanavalin A (1 microM) prevents the development of desensitization to L-glutamate. 5. Present results suggest that Ca2+ plays the role of a charge carrier for L-glutamate-induced depolarization and of a regulator of modulator for L-glutamate-receptor sensitivity. The roles are exaggerated in NaCl-free medium.     

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 β-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH₀. In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E. coli at pH₀ 7·0 ; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.     

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.     

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.     

Modulation of neuronal responses tol-glutamate inAplysia

Potentiation of the excitatory response to L-glutamate (Glu) by L-aspartate (Asp), similar to that which has been described at the crustacean neuromuscular junction, is observed in Aplysia neurons which are glutamate sensitive. Potentiation of the inhibitory responses to ionophoretically applied Glu in neurons preconditioned with Asp permits experiments which serve to differentiate among four hypotheses previously proposed to explain the underlying mechanism of the phenomenon. The potentiation is inhibited by cooling (Q10 = 1.3 +/- 0.2) and is blocked in Na+-free seawater, where the response to Glu applied alone is increased in both amplitude and duration. These results are most consistent with the view that Glu is normally removed from the extracellular medium through an active reuptake process which is Na+ dependent, is slightly temperature sensitive, and may be blocked by Asp. Potentiation of the excitatory response to L-glutamate (Glu) by L-aspartate (Asp) has been previously described at the crustacean neuromuscular junction (Kravitz et al., 1970; Nistri and Constanti, 1979). This potentiation has been attributed to an Asp-induced change in conformation of the Glu receptor, thereby increasing its affinity for Glu (Shank and Freeman, 1975); suppression of the rate of desensitization of the Glu receptor induced by Asp (Dudel, 1977); blockade by Asp of a Glu reuptake process (Crawford and McBurney, 1977); and release, triggered by Asp, of a bound store of Glu (Constanti and Nistri, 1978).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between acid tolerance, cytoplasmic pH, and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis.

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo). A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi. The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established. Each pHi below 6. 6 was characterized by unique levels of ATR, H+-ATPase, and ATP. High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate.     

Induced Release of γ-Aminobutyric Acid by a Carrier-Mediated, High-Affinity Uptake of L-Glutamate in Cultured Chick Retina Cells

[3H]gamma-aminobutyric acid (GABA) was taken up by cultured embryonic retina cells during the initial stages of cell differentiation. The accumulated GABA was released in the bathing medium and a transient increase in the efflux of GABA was observed when cultures were pulse-stimulated (2 min) with 0.1 mM L-glutamate but not with D-glutamate. The EC50 for L-glutamate to evoke [3H]GABA release was approximately 15 microM. This value is close to the Km for high-affinity uptake of L-glutamate by retina cells. When Na+ ions were replaced by Li+ ions, L-glutamate-induced release of GABA was abolished. Moreover, L-[14C]glutamate uptake by retina cells was significantly reduced when NaCl was replaced by LiCl in the incubation medium. L-Glutamate elicited release of GABA was Ca2+ independent, and was observed when Ca2+ was replaced by Co2+ or when Mg2+ ions were increased to 10 mM concentration. D-Aspartate, which is taken up by the same high-affinity uptake mechanism as L-glutamate, induced an increase in [3H]GABA efflux comparable to L-glutamate. The addition of unlabeled GABA to the medium also promoted the release of accumulated [3H]GABA. However, GABA was twofold less effective than L-glutamate in eliciting [3H]GABA release. The addition of both GABA and L-glutamate to the incubation medium indicated that [3H]GABA efflux due to L-glutamate and GABA was additive. L-Aspartate also promoted an increase in the efflux of [3H]GABA accumulated by retina cells. However, L-aspartate effect was significantly decreased in the absence of Ca2+ or when Na+ ions were replaced by Li+. Our results indicate that at least three releasable pools of GABA are present in the chick embryo retina cells: (a) a GABA-promoted GABA release-homoexchange, (b) a Ca2+-dependent L-aspartate-promoted release, and (c) a Ca2+-independent, Na+-dependent L-glutamate-evoked release. In addition, our data strongly suggest that the L-glutamate-promoted GABA release is due to a process of exchange of L-glutamate with GABA, which may play a fundamental role in the fine control of the excitability of local circuits in the retina.     

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.     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

An extracellular stress-sensing protein is activated by heat and u.v. irradiation as well as by mild acidity, the activation producing an acid tolerance-inducing protein

During growth of Escherichia coli in broth at pH 5·0, an extracellular protein termed an extracellular induction component (EIC) appears in the medium, this component being essential for acid tolerance induction. The present study establishes that the EIC arises from an extracellular precursor which is formed during growth at pH 7·0, and that conversion of the precursor to the EIC occurs at pH 5·0 (and other mildly acidic pH values) in the absence of organisms. On the basis that this precursor is produced by non-stressed cells as well as by stressed ones, and that it is converted to the EIC (which in turn induces the tolerance response) by the stress, the precursor can be considered to be a stress sensor, the first extracellular stimulus sensor to be reported. The EIC formed at pH 5·0 was inactivated at pH 9·0. This inactivation probably involved conversion back to the precursor as EIC was reformed if the alkali-inactivated component was incubated at pH 5·0. Both mild heat treatments (exposure to 40–55 °C) and u.v. irradiation also activated the precursor; the active induction component formed by the mild heat treatments was reversibly inactivated at pH 9·0 and so it seems likely that the component formed by heat treatment is similar or identical to the EIC produced at acidic pH. In contrast, the EIC produced by u.v. irradiation was not inactivated at pH 9·0, suggesting that it is different in some way to the EICs produced from the precursor by acidity or by heat treatment. It is likely that many responses affecting stress tolerance involve the functioning of such extracellular sensors, as similar components were shown to be involved in the acid tolerance responses induced at pH 7·0 by glucose, l -aspartate and l -glutamate. Extracellular stimulus sensors may also be needed for other inducible responses.     

Regional Heterogeneity of L-Glutamate and L-Aspartate High-Affinity Uptake Systems in the Rat CNS

The high-affinity uptake of L-[3H]glutamate and L-[3H]aspartate into synaptosomes prepared from rat cerebral cortical, hippocampal, and cerebellar tissue was reduced by a number of structural analogues of L-glutamate and L-aspartate. threo-3-Hydroxy-L-aspartic acid was a more potent inhibitor of L-glutamate uptake than of L-aspartate uptake in the cerebral cortex, but not in the hippocampus or cerebellum. A similar pattern of selectivity was observed for cis-1-aminocyclobutane-1,3-dicarboxylic acid. Dihydrokainate was also more potent against L-glutamate than against L-aspartate in the cerebral cortex, but in the hippocampus, it was more potent against L-aspartate than against L-glutamate. By contrast, L-alpha-aminoadipate was significantly more potent in the cerebellum than in the cerebral cortex and hippocampus as an antagonist of both L-glutamate and L-aspartate. These results support other evidence that there is regional heterogeneity in acidic amino acid uptake sites and that the amino acids L-glutamate and L-aspartate may be taken up by a number of transport systems with overlapping substrate specificity but different inhibitor profiles.     

Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3

In the present study, we report the identification of a new gene from the Bacillus subtilis B3 strain (aatB3), which comprises 1308 bp encoding a 436 amino acid protein with a monomer molecular weight of 49.1 kDa. Phylogenetic analyses suggested that this enzyme is a member of the Ib subgroup of aspartate aminotransferases (AATs; EC 2.6.1.1), although it also has conserved active residues and thermostability characteristic of Ia-type AATs. The Asp232, Lys270 and Arg403 residues of AATB3 play a key role in transamination. The enzyme showed maximal activity at pH 8.0 and 45 °C, had relatively high activity over an alkaline pH range (pH 7.0-9.0) and was stable up to 50 °C. AATB3 catalyzed the transamination of five amino acids, with L-aspartate being the optimal substrate. The K(m) values were determined to be 6.7 mM for L-aspartate, 0.3 mM for α-ketoglutarate, 8.0 mM for L-glutamate and 0.6 mM for oxaloacetate. A 32-residue N-terminal amino acid sequence of this enzyme has 53% identity with that of Bacillus circulans AAT, although it is absent in all other AATs from different organisms. Further studies on AATB3 may confirm that it is potentially beneficial in basic research as well as various industrial applications.     

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.