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Keywords:

  • Acid resistance;
  • rpoS;
  • gadB;
  • gadC;
  • Escherichia coli;
  • Glutamate decarboxylase

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

Strains of Escherichia coli K-12, O157:H7, and Shigella flexneri grown to stationary phase in complex unbuffered media can survive for several hours at pH 2.5. This stationary-phase acid resistance phenotype is dependent upon the alternate sigma factor σs and the supplementation of either glutamate or glutamine in the acidified media used for acid challenge. Acid resistance under these defined conditions can be inhibited by the glutamate analog l-trans-pyrrolidine-2,4-dicarboxylic acid which blocks uptake of glutamate/glutamine by selective inhibition. The gadC gene, encoding an inner membrane antiporter essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriacae.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

It has long been recognized that different enteric pathogens require different infective doses in order to cause disease. There is a strong correlation between the infective dose of enteric pathogens and their ability to survive the acidic conditions of the mammalian stomach [1]. Two pathogens which have been demonstrated to have extremely low infective doses of between 10 and 500 organisms are Shigella flexneri and Escherichia coli O157:H7 [2,3]. These closely related species remain viable for several hours at pH 2.5 when grown to stationary phase [1,4,5]. This stationary-phase acid resistance appears to be a unique phenotype associated with these species, although other enteric pathogens such as Salmonella can be pre-adapted to survive at pH 3.0 [6].

In earlier studies with E. coli, three distinct low-pH-induced acid survival systems were identified [12]. One system is expressed by oxidatively metabolizing bacteria grown in complex media but will protect cells in minimal media to pH 2.5. This system is not apparent in fermentatively metabolizing cells. However, two other systems of acid survival become evident under these conditions. These two systems will also protect against pH 2.5 in minimal medium but only if the medium is supplemented with arginine or glutamate.

The most effective E. coli acid resistance system is dependent upon glutamate and involves two isoforms of glutamate decarboxylase (GAD) encoded by gadA and gadB that convert intracellular glutamate to γ-amino butyrate (GABA) consuming one intracellular proton in the reaction. gadB is transcribed in an operon with gadC which encodes an inner membrane antiporter that is proposed to import glutamate inside the cell while simultaneously exporting GABA to the periplasm [5,7–9]. GadC is required for the expression of acid resistance in S. flexneri and E. coli in defined media supplemented with glutamate [5,7,10].

The stationary-phase acid resistance of E. coli and S. flexneri requires the expression of the alternate sigma factor, σs, encoded by rpoS[5,11]. σs regulates the expression of the gadA, gadB, gadC and hdeAB genes which are essential for the expression of this phenotype [5,7]. The hdeA-encoded protein has been proposed to have a chaperone-like function preventing the aggregation of periplasmic proteins under extremely acidic conditions [13]. hdeB, located downstream of hdeA, is predicted to encode a structural homolog of HdeA and to form heterodimers with HdeA [13].

gadC mutants exhibit considerable GAD activity [10] indicating that the ability to transport glutamate across the inner membrane via GadC is crucial for survival under extreme acid conditions. It is also speculated that the export of GABA out of the cell contributes to the neutralization of protons and alkalinization of the extracellular environment [10]. Consequently, blocking the antiport of these substrates should prevent the neutralization of protons which diffuse into the cell during acid stress and the export of GABA to the external media.

In this study we have concerned ourself only with the stationary-phase acid resistance phenotype expressed by cells grown in complex unbuffered media at neutral pH. We examined whether S. flexneri, E. coli K-12 and O157:H7 strains possess identical glutamate-dependent, σs-regulated stationary-phase acid resistance when grown under these conditions. We attempted to inhibit glutamate-dependent acid resistance by using a synthetic analog of glutamate as a competing substrate in defined media. We have also investigated the distribution of the gadC gene amongst other family members of the Enterobacteriacae.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

2.1Bacterial strains, growth conditions and chemicals used in this study

The bacterial strains used in this study are listed in Table 1. Minimal E salts glucose (EG) was prepared as described by Vogel and Bonner [14]. Cultures were grown at 37°C in Luria–Bertani (LB) broth with aeration for 24 h. The glutamate analog l-trans-pyrrolidine-2,4-dicarboxylic acid (l-PDC) was purchased from Sigma Chemical Co. and was used at a concentration of 1.5 mM. Amino acids were purchased from Sigma and were used at a concentration of 120 μg ml−1.

Table 1.  Bacterial strains and plasmids used in this study
  1. aATCC, American Type Culture Collection.

  2. bCSDH, California State Department of Health, Berkeley, CA, USA.

  3. cSH, Stanford Hospital.

  4. dMDH, Marcus Daly Hospital, Hamilton, MT, USA.

StrainRelevant genotypeSource/reference
E. coli K-12  
MC4100FaraD139Δ(argF-lac)U169 rpsL150 relA1 flb5301 deoC1 ptsF24 rbsR[11]
JLS9300MC4100 rpoS::Tn10[11]
JLS9311MC4100 gadC::Tn10[8]
E. coli O157:H7  
PS2Natural rpoS isolate[4]
PS2 (rpoS+)PS2 containing pPS4.4 (rpoS+)[4]
PS5Wild-type[4]
Shigella flexneri  
3136Wild-type[11]
M25-8ACured of large plasmid[5]
W422M25-8A ΔrpoS[5]
W430W422 hdeA::lacZ (pPS4.4)[5]
W431W422 gadC::lacZ (pPS4.4)[5]
Other species  
Citrobacter freundii 8090 ATCCa
Edwardsiella tarda CSDHb
Enterobacter cloacae 13047 ATCC
Klebsiella pneumoniae SHc
Proteus vulgaris MDHd
Pseudomonas aeruginosa MDH
Serratia marcesans 13880 ATCC
Salmonella typhi ISP1820 [26]
Salmonella typhimurium 91A 3428 CSDH
Yersinia enterocolitica [27]
Plasmids  
pPS4.4pACYC184 with 4.4-kb ClaI fragment with rpoS, CmR[11]
pSRW200pHC79 cosmid clone containing gadC, ApRThis work
pSRW201pCRII with 1.8-kb PCR fragment with gadC[5]
pSRW202pCRII with 0.8-kb PCR fragment with hdeAB[5]

2.2Acid resistance assays

Acid resistance assays were performed as stated previously [5]. LB and EG were adjusted to pH 2.5 using HCl. Assays were performed for 2 h at 37°C. Values shown for percentage of survival represent the mean of at least three independent trials from overnight cultures.

2.3Southern hybridization and cosmid library construction

Southern hybridization was performed as described previously [5]. The filter was washed once under low-stringency 1× Denhardt, 2× saline sodium citrate at room temperature. A 1.8-kb PCR product containing the gadC gene was used as a probe as described previously [5]. A cosmid library was made by partially digesting chromosomal DNA from S. flexneri 3136 with Sau3AI and ligating into pHC79 digested with BamHI. The ligation mix was then packaged into bacteriophage λ according to the manufacturers instructions (Stratagene), and individual colonies were screened by Southern hybridization using the gadC gene as a probe to identify clones harboring gadC (data not shown).

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

3.1σs regulation of glutamate/glutamine-dependent acid resistance in E. coli and S. flexneri

It had been previously demonstrated that E. coli and S. flexneri harbor a stationary-phase acid resistance phenotype that is dependent upon the presence of glutamate in the acidified media for its expression. It has also been postulated that multiple protection systems are acting simultaneously in E. coli K-12 and O157:H7 in order to protect it from acid challenge [12,15]. To ascertain if there were any other amino acids besides glutamate that could supplement the stationary-phase acid resistance phenotype of cells grown in complex unbuffered media, we used the amino acids glutamine, asparagine, and aspartic acid as substrates in acidified minimal media because of their structural homology to glutamate (Table 2). We also examined arginine and lysine because these amino acids have been implicated in the survival of S. flexneri, E. coli K-12 and O157:H7 as substrates in acid resistance phenotypes detected in moderate acidic growth conditions (pH 5.0) in complex media [1,12], suggesting that a different acid resistance system may be in operation under these conditions.

Table 2.  Glutamate- and glutamine-based acid resistance of S. flexneri
  1. aThese are typical values from several acid resistance assays. Assays were performed as described in Section 2.

  2. bl-glutamic acid (120 μg ml−1).

  3. cl-glutamine (120 μg ml−1).

StrainpH 2.5 challenge% Acid resistancea
M25-8AEG+Glub25.64
 EG+Asp<0.001
 EG+Asn<0.001
 EG+Arg<0.001
 EG+Lys<0.001
 EG+D-Glu0.001
 EG+Glnc47.16

In agreement with previous reports [1,12], we determined that l-glutamine can also act as a suitable substrate for the expression of stationary-phase acid resistance for S. flexneri, E. coli K-12 and O157:H7 grown in unbuffered LB (Tables 2, 4 and 5). This was not surprising as glutamate and glutamine are very similar in structure. Both the glutamate- and glutamine-dependent systems required the expression of σs as well as the σs-dependent genes gadC and hdeAB (Tables 3–5). Presumably glutamine is also transported across the inner membrane by the glutamate transporter GadC. It has been suggested that glutamine might be converted to glutamate intracellularly and then to GABA via GAD [10]. If GadC is not present GABA accumulates intracellularly and may be responsible for extreme acid sensitivity [10]. We were unable to detect any resistance to killing by acid when the amino acids arginine or lysine were used as supplements in this assay (Table 2).

Table 4.  σs regulation of a glutamate/glutamine-based acid resistance phenotype of E. coli K-12
  1. aThese are typical values from several acid resistance assays. Assays were performed as described in Section 2.

  2. bl-PDC, l-trans-pyrrolidine-2,4-dicarboxylic acid (1.5 mM).

  3. cpPS4.4 harbors the rpoS gene.

  4. dpSRW200 is a cosmid clone harboring the gadC gene.

StrainpH 2.5 challenge% Acid resistancea
MC4100LB23.19
 EG+Glu24.45
 EG+Gln21.16
 EG+Glu+l-PDCb<0.001
 EG+Gln+l-PDC<0.001
 EG0.45
 EG+Arg0.43
 EG+Lys0.28
MC4100 rpoS::Tn10LB<0.002
 EG+Glu<0.002
 EG+Gln<0.002
 EG<0.002
 EG+Arg<0.001
 EG+Lys<0.001
MC4100 rpoS::Tn10/pPS4.4cLB52.90
 EG+Glu17.30
 EG+Gln21.52
 EG0.92
MC4100 gadC::Tn10LB<0.002
 EG<0.002
 EG+Glu<0.002
 EG+Gln<0.002
MC4100 gadC::Tn10/pSRW200dLB17.6
 EG0.43
 EG+Glu19.4
 EG+Gln22.0
Table 5.  σs regulation of a glutamate/glutamine-based acid resistance phenotype of E. coli O157:H7
  1. aThese are typical values from several acid resistance assays. Assays were performed as described in Section 2.

  2. bpPS4.4 harbors the rpoS gene.

  3. cl-PDC, l-trans-pyrrolidine-2,4-dicarboxylic acid (1.5 mM).

StrainpH 2.5 challenge% Acid resistancea
E. coli O157:H7 PS2 (rpoS)LB<0.001
 EG+Glu<0.007
 EG+Gln<0.007
 EG<0.001
E. coli O157:H7 PS2/pPS4.4bLB62.18
 EG+Glu32.75
 EG+Gln43.32
 EG+Glu+l-PDCc<0.002
 EG+Gln+l-PDC<0.002
 EG0.73
 EG+Arg0.45
 EG+Lys0.39
Table 3.  σs regulation of a glutamate/glutamine-dependent acid resistance phenotype of S. flexneri
  1. aThese are typical values from several acid resistance assays. Assays were performed as described in Section 2.

  2. bl-PDC, l-trans-pyrrolidine-2,4-dicarboxylic acid (1.5 mM).

  3. cpPS4.4 harbors the rpoS gene.

  4. dpSRW201 harbors a 1.8-kb fragment containing gadC.

  5. epSRW202 harbors a 0.8-kb fragment containing hdeAB.

StrainpH 2.5 challenge% Acid resistancea
M25-8AEG+Glu30.69
 EG+Gln43.05
 EG+Glu+l-PDCb0.005
 EG+Gln+l-PDC<0.001
M25-8A ΔrpoS)EG+Gln<0.001
M25-8A ΔrpoS/pPS4.4cEG+Gln12.20
M25-8A gadC::lacZEG+Gln<0.006
M25-8A gadC::lacZ/pSRW201dEG+Gln15.57
M25-8A hdeA::lacZEG+Gln<0.005
M25-8A hdeA::lacZ/pSRW202eEG+Gln21.31

3.2Effect of the glutamate analog l-PDC on acid resistance of E. coli and S. flexneri in defined media

To determine if glutamate and glutamine are actively transported into the cell under acidic conditions, we investigated whether the glutamic acid analog l-PDC could act as an inhibitor of glutamate transport in defined media. l-PDC is one of the most potent glutamate blockers which can selectively inhibit high-affinity transport of [3H]l-glutamic acid in synaptosomes by outcompeting l-glutamic acid [16]. l-PDC delineates a specific structural/conformational preference for binding to the uptake system. Addition of l-PDC to acidified minimal media supplemented with either glutamate or glutamine could selectively inhibit the glutamate-dependent acid resistance of S. flexneri, E. coli K-12 and O157:H7 (Tables 3–5). This again confirms the dependence of the expression of stationary-phase acid resistance phenotype on these two amino acids and suggests that this sensitivity to killing by acid is due to the inhibition of glutamate/glutamine uptake by the GadC antiporter caused by blocking transport with the analog l-PDC. Cells treated with with l-PDC for 2 h in phosphate-buffered saline did not lose viability, demonstrating that l-PDC is not toxic to these bacteria (data not shown).

Interestingly, E. coli K-12 and O157:H7 strains exhibit a low level of acid resistance when challenged in unsupplemented minimal media which is not detected in S. flexneri (Tables 3–5). This phenotype has been observed before [1,8] and suggests that E. coli may harbor other acid resistance systems distinct from S. flexneri, which can maintain an internal pH homeostasis under extreme acid conditions but which are not as efficient as the glutamate/glutamine-dependent system. This acid resistance in unsupplemented media was still dependent upon GadC and σs (Tables 3–5).

We had previously shown that E. coli O157:H7 expresses the same σs-dependent stationary-phase acid resistance phenotype as S. flexneri[4]. In contrast, a glutamate-dependent acid resistance system of E. coli O157:H7 had been reported that was apparently not significantly affected by rpoS[1]. Here we have demonstrated that these phenotypes are in fact the same and can also utilize glutamine as a substrate and that the O157:H7 acid resistance is not unique to this pathogen (Table 5).

3.3Distribution of gadC amongst enteric bacterial species

As the glutamate/glutamine acid resistance phenotype has been demonstrated to be present in E. coli and Shigella strains and is dependent upon the gadC gene, we performed a Southern hybridization against a number of enteric species to determine if any other enteric bacteria contained the antiporter gene necessary to express this phenotype. We found that only the E. coli and Shigella strains tested hybridized with the gadC probe (Fig. 1), suggesting that these species are the only ones capable of expressing glutamate-dependent acid resistance. The gadC gene of S. flexneri M25-8A shares 99% and 98% sequence identity with the gadC genes of E. coli O157:H7 EDL933 and E. coli K-12 MG1655 respectively [17]. This reflects the observations of others that show that GAD activity is only detected in E. coli and Shigella strains [18,19] and that only these species contain the gadA/B genes required for the expression of this enzyme [20].

image

Figure 1. Southern blot of 1 μg of chromosomal DNA isolated from several enteric bacterial species digested with EcoRI. The filter was probed using a [32P]-labelled 1.8-kb PCR fragment containing gadC. The filter was washed under low-stringency conditions. Size standards are in kilobases.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

We had previously described a stationary-phase acid resistance phenotype of E. coli and S. flexneri strains grown in complex unbuffered media that was dependent upon σs and the supplementation of glutamate. In this study we show that the phenotype is expressed in both commensal and pathogenic E. coli as well as S. flexneri and can also utilize the amino acid glutamine for its expression.

It has been previously shown that σs regulates the expression of the genes gadA, gadB, gadC and hdeAB which are essential for the expression of acid resistance in Shigella and E. coli[5,7,9]. Strains carrying mutations in these genes are unable to utilize the glutamate-dependent acid resistance system. Here we show that these strains are also unable to utilize a glutamine-dependent system. The utilization of glutamine as a substrate for the expression of acid resistance in E. coli has been reported previously [1,12]. It has been suggested that exogenous glutamine might be transported into the cell and converted intracellularly to glutamate via glutaminase and then to GABA via GAD [10].

By examining the effect of the glutamate analog l-PDC on the expression of acid resistance in defined in vitro assays, we were able to demonstrate that the glutamate/glutamine-dependent acid resistance system does not function if there is competition between either of these substrates and the synthetic glutamate analog. This infers that the GadC antiporter plays a role in transporting glutamate/glutamine across the inner membrane where these amino acids are decarboxylated by GAD isoforms in the cytoplasm to neutralize diffusing protons. Recent work has provided conclusive evidence associating GadC with the export of GABA by detecting excreted GABA from Salmonella typhimurium carrying the gadBC genes [7]. Strategies designed to inhibit GadC antiporter activity in vivo may have a detrimental effect on cell survival in a number of acidic environments [10]. This can be explained if the GABA generated by the decarboxylation of glutamate cannot be exported outside of the cell. GABA accumulation has been demonstrated to be responsible for extreme acid sensitivity in E. coli[10]. Blocking glutamate transport is therefore a possible strategy to subvert acid resistance and could play a potential role in protection from infection by ingestion of low infective doses of pathogenic E. coli. To our knowledge this is the first demonstration that glutamate-dependent acid resistance can be inhibited by a synthetic glutamate analog.

GAD activity, though characteristic of mammalian cells, is less common among bacterial species and it has been found only in strains of E. coli and Shigella[18,19] and more recently Lactococcus lactis[21] and Listeria monocytogenes[22]. Additionally the genes encoding both subunits of this enzyme have been touted as useful genetic probes for the identification of E. coli and Shigella species [20]. Here we have demonstrated that the gadC gene, just downstream from gadB, has a similarly restricted distribution. These observations were confirmed by searching the microbial genome sequences contained in the TIGR Homepage for the gadC sequence [23]. This again demonstrates the strong link between the possession of these genes and the expression of stationary-phase acid resistance. This correlates with the epidemiological data that associates these species with having a lower infective dose compared to other enteric pathogens and confirms the close evolutionary relationship between E. coli and Shigella amongst the Enterobacteriacae.

We have shown that strains of E. coli can express a low level of survival in unsupplemented acidified minimal media. This has been reported previously for E. coli K-12 grown in unbuffered [8] and buffered [1,12] complex media. This acid resistance is absent in an rpoS mutant of K-12 [1]. As this acid resistance system is still σs dependent, a possible explanation is that these strains of E. coli are capable of generating internal pools of glutamate/glutamine in their cytoplasm which can be utilized by the σs-dependent GAD to neutralize protons, enabling a small percentage of cells in the population to survive under extreme acid conditions. It has been suggested that there are additional genes involved in glutamate-dependent acid resistance [10]. Some reports have also indicated that the gad decarboxylase/antiport system would create a futile proton cycle by consuming a proton that is transported into the cell together with glutamate [10,24]. To compensate, a mechanism for generating endogenous glutamate would be required. Some recently identified genes, including a putative glutaminase encoded by ybaS, have been proposed to play such a role in E. coli acid resistance [24].

The glutamate-dependent acid resistance system of E. coli strains K-12 and O157:H7 has been demonstrated to be essential for survival in synthetic gastric juice [15,25]. The enteric pathogen L. monocytogenes also harbors a homologous GAD system responsible for a glutamate-mediated acid tolerance that has been shown to provide protection from killing in synthetic, and ex vivo, porcine gastric fluid (pH 2.5) [22]. These results imply that sufficient glutamate is present within the proteose peptone component of the gastric fluid to enable protection against killing under extreme acid conditions. These studies indicate that levels of glutamate in foods, and subsequently in stomach fluid, can directly influence gastric survival and presumably transit of these species through the mammalian stomach.

Although the gad system may play a role in the low infective dose of these pathogens it should be emphasized that most normal flora isolates of E. coli are as acid resistant as pathogenic strains. This suggests that the major role of this system might be to facilitate the colonization of the intestines by commensal strains of E. coli.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

We wish to thank Joan Slonczweski for providing us with JLS9311 and Renato Morona for critically reviewing this paper.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References
  • [1]
    Lin, J., Smith, M.P., Chapin, K.C., Baik, H.S., Bennett, G.N., Foster, J.W. (1996) Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. Appl. Environ. Microbiol. 62, 30943100.
  • [2]
    Dupont, H.L., Levine, M.M.L., Hornick, R.B., Formal, S.B. (1989) Inoculum size in Shigellosis and implications for expected mode of transmission. J. Infect. Dis. 159, 11261128.
  • [3]
    Griffin, P.M., Tauxe, R.V. (1991) The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13, 6098.
  • [4]
    Waterman, S.R., Small, P.L.C. (1996) Characterization of the acid resistance phenotype and rpoS alleles of shiga-like toxin-producing Escherichia coli. Infect. Immun. 64, 28082811.
  • [5]
    Waterman, S.R., Small, P.L.C. (1996) Identification of σs-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri. Mol. Microbiol. 21, 925940.
  • [6]
    Foster, J.W., Hall, H.K. (1990) Adaptive acidification tolerance response of Salmonella typhimurium. J. Bacteriol. 172, 771778.
  • [7]
    De Biase, D., Tramonti, A., Bossa, F., Visca, P. (1999) The response to stationary-phase stress conditions in Escherichia coli: role and regulation of the glutamic acid decarboxylase system. Mol. Microbiol. 32, 11981211.
  • [8]
    Hersh, B.D., Farooq, F.T., Barstad, D.N., Blnkenhorn, D.L., Slonczewski, J.L. (1996) A glutamate-dependent acid resistance gene in Escherichia coli. J. Bacteriol. 178, 39783981.
  • [9]
    Small, P.L.C., Waterman, S.R. (1998) Acid stress, anaerobiosis and gadCB: lessons from Lactococcus lactis and Escherichia coli. Trends Microbiol. 6, 214216.
  • [10]
    Castanie-Cornet, M.P., Penfound, T.A., Smith, D., Elliot, J.F., Foster, J.W. (1999) Control of acid resistance in Escherichia coli. J. Bacteriol. 181, 35253535.
  • [11]
    Small, P., Blankenhorn, D., Welty, D., Zinser, E., Slonczewski, J.L. (1994) Acid and base resistance in Escherichia coli and Shigella flexneri: Role of rpoS and growth pH. J. Bacteriol. 176, 17291737.
  • [12]
    Lin, J., Lee, I-S., Frey, J., Slonczewski, J.L., Foster, J.W. (1995) Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J. Bacteriol. 177, 40974104.
  • [13]
    Gajiwala, K.S., Burley, S.K. (2000) HDEA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria. J. Mol. Biol. 295, 605612.
  • [14]
    Vogel, H.J., Bonner, D.M. (1956) Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218, 97106.
  • [15]
    Arnold, K.W., Kaspar, C.W. (1995) Starvation-and stationary-phase induced acid tolerance in Escherichia coli O157:H7. Appl. Environ. Microbiol. 61, 20372039.
  • [16]
    Bridges, R.J., Stanley, M.S., Anderson, M.W., Cotman, C.W., Chamberlin, A.R. (1991) Conformationally defined neurotransmitter analogues. Selective inhibition of glutamate uptake by one pyrrolidine-2, 4-dicarboxylate diastereomer. J. Med. Chem. 34, 717725.
  • [17]
    NCBI BLAST Homepage (online). Available from URL: http://www.ncbi.nlm.nih.gov/BLAST/.
  • [18]
    Rice, E.W., Johnson, C.H., Dunnigan, M.E., Reasoner, D.J. (1993) Rapid glutamate decarboxylase assay for detection of Escherichia coli. Appl. Environ. Microbiol. 59, 43474349.
  • [19]
    Schubert, R., Esanu, J.G., Schafer, V. (1988) The glutamic acid decarboxylase disc test: an approach to a faster and more simple detection of E. coli. Zentralbl. Bakteriol. Hyg. B187, 107111.
  • [20]
    McDaniels, A.E., Rice, E.W., Reyes, A.L., Johnson, C.H., Haugland, R.A., Stelma, G.N.Jr. (1996) Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and β-glucuronidase. Appl. Environ. Microbiol. 62, 33503354.
  • [21]
    Sanders, J.W., Leenhouts, K., Burghoorn, J., Brands, J.R., Venema, G., Kok, J. (1998) A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation. Mol. Microbiol. 27, 299310.
  • [22]
    Cotter, P.D., Gahan, C.G.M., Hill, C. (2001) A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Mol. Microbiol. 40, 465475.
  • [23]
    TIGR Homepage (online). Available from URL: http://www.tigr.org.
  • [24]
    Tucker, D.L., Tucker, N., Conway, T. (2002) Gene expression profiling of the pH response in Escherichia coli. J. Bacteriol. 184, 65516558.
  • [25]
    Cui, S., Meng, J., Bhagwat, A.A. (2001) Availability of glutamate and arginine during acid challenge determines cell density-dependent survival phenotype of Escherichia coli strains. Appl. Environ. Microbiol. 67, 49144918.
  • [26]
    Hone, D.M., Harris, A.M., Chatfield, S., Dougan, G., Levine, M.M. (1991) Construction of genetically defined double aro mutants of Salmonella typhi. Vaccine 9, 810816.
  • [27]
    Portnoy, D.A., Moseley, S.L., Falkow, S. (1981) Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis. Infect. Immun. 31, 775782.