Identification of the promoter regions and σ^s-dependent regulation of the gadA and gadBC genes associated with glutamate-dependent acid resistance in Shigella flexneri

Abstract

Resistance to killing by low pH is a common feature of both Escherichia coli and Shigella flexneri. The most effective E. coli acid resistance system utilizes two isoforms of glutamate decarboxylase encoded by gadA and gadB, and a putative glutamate/γ-amino butyric acid antiporter encoded by gadC. Expression of the gad system is dependent upon the alternate sigma factor, σ^s. We confirm that gadA, gadB, and gadC are also all dependent upon σ^s for their expression in S. flexneri. −10 sequences similar to the σ^s−10 consensus sequence were identified by primer extension in the upstream promoters of all three genes and the transcriptional start points were identical in both E. coli and S. flexneri.

Acid resistance, RpoS, gadC, Glutamate decarboxylase, Shigella flexneri

1 Introduction

Pathogenic and non-pathogenic strains of Escherichia coli and Shigella flexneri must survive transit through the acidic conditions of the stomach, where the pH is normally between 2 and 3, before they can colonize the intestines of a mammalian host. These two species can tolerate conditions of extreme acidity in vitro when grown to the stationary phase [1]. This acid resistance is considered to be responsible for the low infective dose (10–100 organisms) required by these pathogens to cause disease [2,3].

A glutamate decarboxylase (GAD) system has been shown to play a major role in the acid resistance of E. coli[4,5]. This involves two isoforms of GAD encoded by gadA and gadB that convert intracellular glutamate to γ-amino butyrate (GABA) consuming one intracellular proton in the reaction. Directly downstream of gadB is gadC which encodes an inner membrane antiporter that is proposed to import glutamate inside the cell while simultaneously exporting GABA. GadC is required for the expression of acid resistance in S. flexneri and E. coli in defined media supplemented with glutamate [4,6,7]. The expression of all of the gad genes in E. coli is dependent upon the alternate sigma factor, σ^s, encoded by rpoS[5], and repressed by the cAMP receptor protein [8] and H-NS [5]. Only one of the two glutamate decarboxylases is necessary for protection at pH 2.5; but survival at pH 2.0, however, requires both GAD isozymes [4].

It has recently been established that the expression of the gad system in E. coli is mediated by the GadX protein, a member of the AraC/XylS family of transcriptional regulators, encoded by gadX (formerly named yhiX), located downstream of gadA[9,10]. GadX has been shown to play a central role in the H-NS control of genes required in glutamate-dependent acid resistance and bind to the gadA and gadBC promoters [9,10]. GadW, another AraC-like regulator, mediates control of the gadA and gadBC genes directly in rich medium at pH 8 in stationary phase [11].

Despite this complex regulatory network, only one promoter for each gad gene appears to be involved in their expression in E. coli regardless of the induction conditions [8]. In recent years there has been much interest in the phylogenetic relationship between E. coli and Shigella. The sporadic phylogenetic distribution of pathogenic organisms has long suggested that bacterial virulence results from the independent acquisition of genes that are absent from avirulent forms. However, in Shigella, virulence also depends upon the absence of resident genes that diminish pathogenic potential. Shigella strains lack catabolic pathways otherwise widely present in E. coli. Shigella has deletions in the region containing the cadA gene, which encodes lysine decarboxylase. When the cadA gene from a benign strain of E. coli was introduced into S. flexneri, the resulting strain could no longer induce the fluid secretion associated with infection [12]. Shigella strains do not have a single evolutionary origin indicating convergent evolution of Shigella phenotypic properties [13]. One can speculate that Shigella strains, which reside inside epithelial cells, do not need the range of catabolic pathways that generally characterize E. coli as a species.

Because of the importance of GAD expression to glutamate-dependent acid resistance, we investigated the transcriptional expression of the gad genes and their promoter sequences in the enteric pathogen S. flexneri to establish if there were any apparent differences with E. coli. The recent completion of the S. flexneri genome confirms that this species also harbors the gadA gene as well as the regulatory genes gadX and gadW in an identical location to E. coli[14]. Any distinct differences in gad expression in S. flexneri may have occurred as a result of its adaptation to a pathogenic lifestyle. We also constructed a gadB mutant in S. flexneri to confirm the association between glutamate decarboxylase activity and glutamate-dependent acid resistance in this species.

2 Materials and methods

2.1 Bacterial strains and plasmids

The S. flexneri strain background used was M25-8A [6]. The E. coli K-12 strain used was MC4100 [7]. Overnight (o/n) cultures were grown at 37°C in Luria–Bertani (LB) broth with aeration for 24 h unless otherwise stated. Minimal E salts glucose (EG) was prepared as described by Vogel and Bonner [15]. l-Glutamic acid was purchased from Sigma Chemical Co. and used at 120 µg ml⁻¹.

2.2 Restriction enzyme and DNA manipulations

Chromosomal DNA was isolated as described previously [6]. Restriction enzyme digests, gel electrophoresis, and nick translation were performed as described elsewhere [16]. Cloning of PCR products was performed by ligation into the TA cloning vector pCRII (Invitrogen). The ligation mix was transformed into INVαF′ and plated onto LB agar containing X-Gal and kanamycin.

2.3 Polymerase chain reaction (PCR) amplification

PCR amplification was carried out as described previously [6]. Primers used for generating the 1.0-kb internal fragment of gadB were 5′-TCC GCT GCA CGA AAT GCG CGA CGA TGT CGC A-3′ and 5′-AAC CTG GTA AGA GGC GTT CTG TAC TTT GGT-3′. Primers used for generating the 1.2-kb internal fragment of gadC were 5′-TCT GGG TCC GAG ATG GGG ATT TGC AGC GAT-3′ and 5′-TGG TGA ACG TCG ACG CGG GTG CAG GAA GAA-3′. A 1.8-kb fragment containing the entire gadB gene used for complementation studies was generated by PCR using the primers 5′-ACC GGA GTA TTA TTG CCA AAA TAA TAA CAG CCC GTC A-3′ and 5′-TCC CCC TAA AAC GGT ATT CCT GTC GGA ACC GCA-3′ which reside in regions which flank gadB and was cloned as described above.

2.4 Acid resistance assay

Acid resistance assays were performed as stated previously [6]. 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 o/n cultures.

2.5 Construction of a S. flexneri gadB mutant

Disruption of gadB was performed using the suicide vector pCVD442 [17] containing the internal PCR fragment of gadB cloned by the flanking XbaI and SstI sites from pCRII. The resulting plasmid, pSRW203, was electroporated into S17-1λpir and mobilized into S. flexneri M25-8A containing pACYC184 (Cm^R) by conjugation at 37°C. Exconjugants were selected on ampicillin (100 µg ml⁻¹)/chloramphenicol (50 µg ml⁻¹) plates to select for recombination of the plasmid into the S. flexneri chromosome. Exconjugants representing a single homologous recombination were isolated and screened by Southern hybridization to determine the position of the disruption caused by pSRW203 in the chromosome.

2.6 RNA isolation, Northern hybridization, and primer extension analysis

Total RNA was prepared from cultures grown in LB at various stages of the growth cycle using TRIzol™ reagent as described by the manufacturers (Gibco) and DNase-treated for 1 h (Boehringer-Mannheim). For Northern hybridization, RNA samples (30 µg) were heated for 5 min at 65°C and fractionated on 1.2% formaldehyde-agarose gels [16] and blotted onto nylon membranes (Amersham). Membranes were hybridized with the internal fragments of gadB and gadC, respectively, nick-translated with [α-³²P]dNTP (Amersham). Primer extension analysis was performed as described previously [18] using oligonucleotides complementary to the 5′ end of the coding region of gadB 5′-TCC GAC CTT AAA TCC GTT ACT TGC TTC TTA-3′ and gadC 5′-GGC AAA GAA TCC GAG TAA TGT GAG CTG CTT AGC-3′. Primers were labelled at the 5′ end with [γ-³²P]ATP (Amersham) using T4-polynucleotide kinase (New England Biolabs). The ³²P-labelled primers were hybridized with 50 µg of total RNA. The cDNA was extended at 45°C for 1 h with reverse transcriptase AMV (Boehringer-Mannheim). The migration of the primer extension products was compared to dideoxy double-stranded sequencing reactions generated from the same oligonucleotides used in the primer extension reactions and measured in a DNA sequencing gel.

3 Results and discussion

3.1 Transcription of gadA and gadB in S. flexneri is dependent upon σ^s

To confirm that gadA and gadB, like gadC, were regulated by σ^s in S. flexneri we performed Northern hybridization analysis on mRNA extracted from S. flexneri M25-8A at various stages of the growth curve (A₆₀₀=0.4, 1.0, 1.5) using an internal fragment of the gadB gene as a probe (Fig. 1). Due to the high DNA sequence homology between gadB and its isoform gadA (96%) [14], the gadB probe used would also hybridize with any transcriptional product from gadA. mRNA was also isolated from the isogenic rpoS mutant of S. flexneri[6] in the same stages of the growth curve. Northern hybridization analysis was also performed using an internal fragment of gadC as a probe for a positive control of a σ^s-dependent gene (Fig. 1). No gadB or gadC transcripts were detected in the rpoS mutant strain of S. flexneri at any stage of growth. In the parental strain signals were detected with both probes at an A₆₀₀ of 1.5; a faint band was also present at an A₆₀₀ of 1.0, demonstrating that transcription of both genes was initiated in late exponential phase. These expression patterns are consistent with those observed by De Biase et al. [5] for the gad genes in E. coli. The sizes of the mRNA obtained corresponded to the sizes predicted from the ORFs of both gadB and gadC, respectively (data not shown). We also observed a higher molecular mass transcript that corresponded with the deduced length of a polycistronic gadBC message but this transcript was weaker than either the gadB or gadC transcripts (data not shown). The absence of any detectable signal in the rpoS mutant confirms that both gadA and gadB expression are also under the control of σ^s in S. flexneri.

1

Northern analysis of gadB and gadC transcription in S. flexneri M25-8A and W422 (ΔrpoS). Total RNA was extracted from cultures of both strains at the growth curve points of A₆₀₀ 0.4, 1.0, 1.5. The membrane was probed with the ³²P-labelled internal fragments of gadB (A) and gadC (B).

3.2 Mapping the transcriptional start sites in the promoter regions of gadA, gadB, and gadC in S. flexneri and E. coli.

The transcriptional start points of the gadA, gadB, and gadC transcripts were detected by primer extension in both E. coli K-12 and S. flexneri (Fig. 2). mRNA isolated from E. coli and S. flexneri in stationary phase (A₆₀₀=1.8 and 1.5, respectively) was used as a template for the primer extension reactions. The primer used for gadB was taken from the most heterogeneous region of the 5′ end of the gadB gene (70%) [19] but would still anneal to gadA transcripts under the hybridization conditions that were employed. The transcriptional start points for gadA and gadB were identical in both E. coli and S. flexneri and corresponded to a predicted −10 sequence of CTACTTT (Fig. 3) and are identical to those identified by De Biase et al. [5] located 27 nucleotides upstream of the ATG start codon.

2

Mapping of the 5′ ends of gadB and gadC mRNA. Primer extension was performed to map the transcriptional start points of gadB (A) and gadC (B). dNTP lanes are as marked and the extension reactions are from RNA extracted from E. coli MC4100 (1) and S. flexneri M25-8A (2). Primer extension was performed with RNA from LB broth grown cultures in stationary phase using ³²P-labelled primers complementary to the N-terminal regions of gadB and gadC as described in Section 2. The extension products were run in parallel with sequence ladders generated using the same primers and with the plasmids pSRW204 (gadB) and pSRW201 (gadC) as templates, respectively.

3

Upstream promoter regions of gadA, gadB, and gadC. The transcriptional start points as determined from primer extension are as indicated at +1 and marked with an (*) and an arrow showing the direction of transcription. Predicted −10 sequences are indicated in bold. The putative Shine–Dalgarno (S.D.) sequences are underlined. Inverted repeats are shown by arrows.

Primers used to map the transcriptional start point of gadC produced two signals of equal intensity in identical positions for both E. coli and S. flexneri. Analysis based on exact spacing of the −10 nucleotides from the start point revealed no sequence compatible with the σ^s consensus. However, similarity between the gadC transcriptional start point and σ^s promoter consensus sequence could be demonstrated using the ambiguous spacing method employed by Espinosa-Urgel et al. [20] (Fig. 3). This transcriptional start point is identical to that reported for gadC by De Biase et al. [5] at nucleotide 1656. We also observed a weak second 5′ termination band at nucleotide 1693 in S. flexneri as De Biase et al. [5] had reported in E. coli (data not shown). This is predicted to originate from endonucleolytic cleavage of the polycistronic gadBC mRNA at an intergenic processing site because of its location near the 3′ end of a predicted stem loop structure. The faint signals detected for the gadC 5′ termini by both groups may imply that these termini originate from intergenic processing of the polycistronic gadBC mRNA.

We previously noted that the intergenic region upstream of the gadB gene is highly AT rich (78%) [6]. The region upstream of many σ^s-dependent promoters is often AT rich and shows intrinsic DNA curvature [21]. Eσ^s preferentially recognizes the −10 hexamer (TATAC/AT), but does not require a specific sequence in the −35 region, which is normally required for promoter recognition by Eσ^s[22,23]. This sequence is almost identical to a −10 consensus sequence (CTATACT) determined for σ^s-dependent promoters based on the comparison of characteristic promoters known to be under the control of Eσ^s and those that can be recognized in vitro by both Eσ^s and Eσ⁷⁰[23].

3.3 Acid resistance in S. flexneri is dependent upon the expression of gadB

To determine whether gadA and gadB play a role in the expression of acid resistance in S. flexneri we ligated an internal fragment of gadB into the suicide vector pCVD442 in an attempt to construct mutations in each gene. Individual Ap^RS. flexneri colonies carrying potential disruptions in either gadA or gadB were randomly screened by Southern hybridization using the internal fragment of gadB as a probe. All the colonies we examined had a disruption within the smaller 1.7-kb ClaI fragment carrying the gadB gene, and a single isolate (W587) was chosen for further study. We were unable to identify an isolate carrying a mutation within the gadA gene (data not shown). The gadB mutant of S. flexneri had lost its ability to resist killing by acid, and acid resistance could be restored by complementation with the wild-type gadB gene (Table 1). Acid resistance in this complemented mutant was glutamate-dependent. GAD activity in the S. flexneri gadB mutant was examined by whole cell permeabilization using the GAD assay of Rice et al. [24]. The gadB mutant still demonstrated GAD activity which derived from its functional gadA gene (data not shown). An rpoS mutant of S. flexneri gave a negative reaction in this assay (data not shown). GAD activity was restored when this mutant was complemented by the wild-type rpoS gene. This again confirms that GAD expression is dependent upon σ^s and that only one GAD isoform is required for GAD activity in S. flexneri.

1

Effect of a gadB mutation on the acid resistance of S. flexneri M25-8A

Strain	Complementing plasmid	pH 2.5 challenge	Acid resistance
M25-8A		LB	84.65
		EG	<0.004
		EG+Glu	20.36
W587		LB	<0.004
W587	pSRW204	LB	73.62
		EG	<0.007
		EG+Glu	25.31

pSRW204 harbors the gadB gene.

Minimal E salts+glucose (0.5%).

Glu, glutamic acid (120 µg ml⁻¹).

In accordance with a previous report in E. coli[5], we have demonstrated that an insertional mutation of the gadB gene rendered S. flexneri sensitive to killing by extreme acidic conditions. In E. coli it was speculated that this sensitivity to acid was due to a polar effect on gadC. However, the gadB mutation did not cause a complete loss of acid resistance or a complete loss of gadC expression [5]. We were able to restore acid resistance in our S. flexneri gadB mutant by complementation with the wild-type gadB gene. This result implies that there is a significant level of expression of the gadC gene which is derived from its promoter located within the intergenic region downstream from gadB which we have identified by primer extension. Our inability to isolate a gadA mutant of S. flexneri was not dissimilar to that of De Biase et al. [5] who detected a single isolate with a mutation within gadA after screening 60 putative mutants in E. coli. This may have been caused by using a deletion construct derived from the gadB sequence. Their gadA mutant was fully acid resistant, presumably because of the functional gadB gene.

There appears to be no difference in the transcriptional expression of the gad genes in S. flexneri as compared to non-pathogenic E. coli. This suggests that glutamate-dependent acid resistance is a phenotype common to the ancestral E. coli species that evolved to assist commensal E. coli safe passage through the extreme acidic conditions of the mammalian stomach. This phenotype has been maintained by S. flexneri and is likely to contribute to the low infective dose displayed by this enteric pathogen.