# Antagonistic Roles for GcvA and GcvB inhdeAB Expression in Escherichia coli

Authors: Lorraine T. Stauffer; George V. Stauffer
Journal: ISRN Microbiology
 (2012)

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## Abstract

InE. coli, the periplasmic proteins HdeA and HdeB have chaperone-like functions, suppressing aggregation of periplasmic proteins under acidic conditions.  A microarray analysis of RNA isolated from an E. coli wild type and a ΔgcvB strain grown to mid-log phase in Luria-Bertani broth indicated the hdeAB operon, encoding the HdeA and HdeB proteins, is regulated by the sRNA GcvB.  We wanted to verify that GcvB and its coregulator Hfq play a role in regulation of the hdeAB operon.  In this study, we show that GcvB positively regulates hdeA::lacZ and hdeB::lacZ translational fusions in cells grown in Luria-Bertani broth and in glucose minimal media + glycine.  Activation also requires the Hfq protein.  Although many sRNAs dependent on Hfq regulate by an antisense mechanism, GcvB regulates hdeAB either directly or indirectly at the level of transcription.  GcvA, the activator of gcvB, negatively regulates hdeAB at the level of transcription.  Although expression of gcvB is dependent on GcvA, activation of hdeAB by GcvB occurs independently of GcvA’s ability to repress the operon.  Cell survival and growth at low pH are consistent with GcvA negatively regulating and GcvB positively regulating the hdeAB operon.


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## Body

## 1. Introduction

Acid resistance is important for the ability of enteric bacteria to survive the low pH environment encountered in the gastrointestinal tract of mammalian hosts and other natural environments [1]. Enteric bacteria have five systems of acid resistance [2–7]. The first system, AR1, is least understood. When cells are grown in LB at pH 5 to stationary phase, they survive dilution into minimal medium at pH 2.5, which kills cells grown at pH 8. The stationary phase sigma factor RpoS and cyclic-AMP receptor protein are required to develop acid tolerance [2, 6]. The other four systems, AR2 AR3, AR4, and AR5, are decarboxylate/antiporter-dependent acid resistance systems that require glutamate, arginine, lysine, and ornithine, respectively [2, 4–10]. Additional acid protection comes from the periplasmic proteins HdeA and HdeB that have chaperone-like functions, suppressing aggregation of periplasmic proteins under extreme acidic conditions [11–13]. Both hdeA and hdeB mutants show reduced viability upon acid stress and HdeA/HdeB expressing plasmids restore viability close to wild type, suggesting both proteins are necessary for protection of the bacterial periplasm against acid stress [14]. Regulation of the hdeAB operon is complex. The hdeAB operon in E. coli is acid inducible and regulation involves GadE, RpoD, RpoS, H-NS, MarA, and several other regulators [6, 7, 15–18].TheE. coli  gcvB gene encodes a sRNA of 206 nucleotides [19]. Expression of gcvB is activated by the GcvA protein when cellular glycine is high and repressed by GcvA and GcvR when glycine is limiting [19]. In both E. coli and Salmonella entericaserovar Typhimurium, GcvB regulates genes involved in the transport of small peptides and polar and branched amino acids [19–24]. Recently, it was shown GcvB enhances the ability of E. coli to survive low pH by upregulating RpoS [25]. In addition, microarray data suggested the hdeAB operon is positively regulated by GcvB [22]. Results from this study establish a role for GcvA in repressing the hdeAB operon and GcvB in activating the operon. Hfq, an RNA chaperone required for GcvB regulation of known target genes [20, 22, 23, 26], is also required for activation. However, the results suggest GcvB and Hfq do not function as an antisense RNA system to upregulate hdeAB translation, but act at the level of transcription. The results also suggest GcvA, the activator for gcvB, negatively regulates hdeAB at the level of transcription.


## 2. Materials and Methods

### 2.1. Bacterial Strains, Plasmids, and Phage

TheE. coli strains, plasmids, and phage used are listed in Table 1 or described in the text.Table 1
Strains, plasmids, and phage.
Strain, plasmid, or phageRelevant genotypeSourceStrain*GS162WTThis labGS776cycA30::Tn10This labGS854argA81::Tn10This labGS998GS162gcvA[27, 28]GS1132GS162 Δ(gcvAgcvB)::ΩaadA (referred to as ΔgcvAB)[19]GS1144GS162 ΔgcvB::ΩCMR (referred to as ΔgcvB)[21]GS1148GS162hfq-1::ΩCMR (referred to as Δhfq)[23]PlasmidpGS554Single-copy vector + constitutivegcvB (pgcvB2+)[19]pGS571Multicopy vector + WTgcvB (pgcvB3+)[29]pGS594Single-copy vector + WTgcvB (pgcvB+)This labpGS609Multi-copy vector + WThfq (phfq3+)[23]pGS611Multi-copy vector + WTgcvA (pgcvA3+)This studypGS624Multi-copy vector + WTgcvA gcvB (pgcvA3+gcvB3+)This studyPhageλgt2λ cloning vector; cI857 repressor[30]λhdeA::lacZλ vector carrying WT hdeA::lacZ fusionThis studyλhdeB::lacZλ vector carrying WT hdeB::lacZ fusionThis studyλhdeA-36::lacZλ vector carrying hdeA-36::lacZ transcriptional fusionThis studyλPBAD::hdeA::lacZλ vector carrying hdeA::lacZ fusion under control of the PBAD promoterThis study*All strains also carry thepheA905  thi  araD129  rpsL150  relA1  deoC1  flbB5301  ptsF25  rbsR mutations.


### 2.2. Construction of Recombinant Phages and Plasmids

TheλhdeA::lacZ translational fusion was constructed by PCR synthesis of a DNA fragment using an upstream primer with an EcoRI site that hybridized to DNA beginning 223 bps upstream of the hdeA transcription start site and a downstream primer with a SmaI site that hybridized to DNA beginning at codon 7 within the hdeA gene. The PCR amplified DNA fragment was digested with EcoRI + SmaI and the 303 bp EcoRI-SmaI fragment ligated into the EcoRI-SmaI sites of plasmid pMC1403 [37], fusing the first 7 codons of the hdeA gene in frame with the 8th codon of the lacZYA genes in pMC1403 (Figure 1(a)). The cloned sequence was verified by DNA sequence analysis at the DNA Core Facility of the University of Iowa. The plasmid was designated phdeA::lacZ. A 5,574 bp EcoRI-MfeI fragment from phdeA::lacZ carrying the hdeA::lacZYA fusion was then ligated into the EcoRI site of phage λgt2 [30], generating λhdeA::lacZ. A λhdeB::lacZ fusion was constructed using the same upstream primer and a downstream primer with a SmaI site that hybridized to DNA beginning at codon 9 within the hdeB gene. The 757 bp EcoRI-SmaI fragment was then used as described above, generating plasmid phdeB::lacZ and phage λhdeB::lacZ (not shown). A λhdeA::lacZtranscriptional fusion was constructed using the same upstream primer and a downstream primer with a HindIII site and that hybridized to DNA at bp −36 relative to the hdeA translation start site (Figure 1(a)). Following digestion with EcoRI and HindIII, the DNA fragment was ligated into the EcoRI and HindIII sites of plasmid pgcvB-lacZ+50 [19], replacing the gcvB fragment with the hdeAfragment, generating plasmid phdeA-36::lacZ. The cloned sequence was verified by DNA sequence analysis. A 5,538 bp EcoRI-MfeI fragment from phdeA-36::lacZ carrying the hdeA-36::lacZYA fusion was then ligated into the EcoRI site of phage λgt2 [30], generating λhdeA-36::lacZ. The 3 fusion phages were used to lysogenize E. coli host strains as described [38]. Each lysogen was tested to ensure it carried a single-copy of the λ chromosome by infection with λcI90c17 [39]. All lysogens were grown at 30°C since all fusion phage carry the λcI857 mutation, resulting in a temperature-sensitive λcI repressor [30]. The λPBAD::hdeA::lacZ fusion, where hdeA transcription is under control of the PBAD promoter, was constructed as described in Figure 1(b).(a) ThehdeAB control region. The hdeAB promoter −35, −10 and transcription start site and the SD sequence and translation start site are indicated above the sequence [31]. The transcription start site for the divergently transcribed hdeD gene is also shown [32]. Binding sites for H-NS [16] and MarR/SoxS [15] are indicated above the sequence with arrows. The binding site for GadX/W [33] is below the sequence in blue and for GadE above the sequence in green [18, 34]. In addition, there are putative binding sites for the transcriptional regulators Lrp and TorR (not shown) [34]. The consensus GcvA binding site is T-N11-A containing a 5′-CTAAT-3′ sequence [35]. Two putative GcvA binding sites are indicated in red. The fusion sites for the λhdeA::lacZ translational fusion, the λhdeA−36::lacZ transcriptional fusion, and the λPBAD::hdeA::lacZ fusion (see below) are indicated with black, red, and green arrows, respectively. (b) Construction of a λPBAD::hdeA::lacZ promoter fusion. The WT PBAD and PhdeA promoters are shown in the top line. The transcription start sites are in red [31, 36]. Small case letters show bases added during PCR amplification of the PBAD and PhdeA promoters. The PBAD promoter was amplified with an upstream primer containing an EcoRI site at bp −272 relative to the transcription start site (not shown) and a downstream primer with a BsaI site (blue). The PhdeA promoter was amplified with an upstream primer containing a BsaI site (blue) and a downstream primer containing a SmaI site at codon 10 in the hdeA gene (not shown). The arrows indicate cut sites for BsaI. The amplified products were cut with BsaI, mixed, and ligated, generating a fusion of the PBAD promoter with the +1G residue of the PhdeA promoter. The fragment was then digested with EcoRI + SmaI and ligated into the EcoRI-SmaI sites of plasmid pMC1403, and subsequently subcloned into λgt2 as described [19]. (c) The gcvA  gcvB region of the E. coli chromosome. The regions amplified by PCR and cloned into pACYC184 to generate pGS611 (pgcvA3+) and pGS624 (pgcvA3+gcvB3+) are indicated with bars. See Section 2.2 for details.
(a)(b)(c)Plasmid pGS611 (pgcvA3+), carrying the E. coli  gcvA gene on a 1,155 bp EcoRI fragment, was constructed as follows. In a PCR reaction, an upstream primer was used containing an EcoRI site and that hybridized to a region beginning 121 bp upstream of the gcvA transcription start site and a downstream primer containing an EcoRI site and hybridized to a region beginning 44 bp downstream of the gcvA translation stop codon. The EcoRI sites were added as parts of the primers. The PCR-generated fragment was digested with EcoRI and cloned into the EcoRI site in plasmid pACYC184 [40] and verified by DNA sequence analysis (Figure 1(c)). Plasmid pGS624 (pgcvA3+gcvB3+), carrying both the gcvA and gcvB genes, was constructed in the same way except the upstream primer hybridized to DNA 51 bps after the gcvB transcription terminator and the downstream primer hybridized to DNA 44 bps after the gcvA translation stop codon, generating a 1,347 bp EcoRI fragment (Figure 1(c)).


### 2.3. Media

The complex medium used was LB [41]. Agar was added at 1.5% (w/v) to make solid media. The minimal medium used was the salts of Vogel and Bonner [42] supplemented with 0.4% (w/v) glucose (GM). Ampicillin was added at 50 and 150 μg mL−1 when strains carried single-copy and multicopy plasmids, respectively. Other supplements were added at the following concentrations (μg mL−1): phenylalanine, 50; glycine, 300; thiamine, 1; TC, 10; CM, 20; X-gal, 40.


### 2.4. DNA Manipulation

Plasmid DNA was isolated using a QIAprep Spin Miniprep Kit (Qiagen, Santa Clara, CA). Vent DNA polymerase and restriction enzymes were from New England Biolabs, Inc. (Beverly, MA). T4 DNA ligase was from Roche Diagnostics (Indianapolis, IN). Reactions were as described by the manufacturers.


### 2.5. Enzyme Assay

β-galactosidase assays were performed on mid-log phase cells (OD600 ~ 0.5) using the chloroform/SDS lysis procedure [41]. Results are the averages of two or more assays with each sample done in triplicate.


### 2.6. Acid Sensitivity Assay

WT, an isogenic ΔgcvABstrain and the two strains transformed with either plasmid pgcvB2+ (constitutively produces GcvB), pgcvA3+ or pgcvA3+gcvB3+ were grown for 24 hr at 30°C in LB and then tested for acid resistance by dilution into LB at pH 2.0. Samples of 0.2 mL were taken at 0, 1, 2, and 4 hr and diluted in 2 mL of LB at pH 7. The final pH of the diluted cultures was ~7.0. Cell viability was determined by plate counts. Percent survival is the titer of colony forming units of acid-tested cells compared to the zero-time point (Figure 2).Figure 2
Percent survival ofE. coli strains plotted against time at pH 2.0. Black, WT; gray, ΔgcvAB; green, ΔgcvAB[pgcvB2+]; blue, WT[pgcvA3+gcvB3+]; purple, ΔgcvAB[pgcvA3+gcvB3+]; red, ΔgcvAB[pgcvA3+]. See Section 2.6 for details.


### 2.7. Transductions

ThegcvB gene is linked to the argA gene and hfq is linked to the cycA gene, with predicted phage P1 cotransduction frequencies of ~78% and ~67%, respectively. P1clr phage prepared on GS854 (argA81::Tn10) was used to transduce ΔgcvB::ΩCMRλhdeA::lacZ to TCR and transductants scored on CM versus TC plates. A TCR CMS transductant was purified. P1clr prepared on GS776 (cycA::Tn10) was used to transduce Δhfq-1::ΩCMRλhdeA::lacZ to TCR resistance and transductants scored on CM versus TC plates. A TCR CMS transductant was purified.


## 2.1. Bacterial Strains, Plasmids, and Phage

TheE. coli strains, plasmids, and phage used are listed in Table 1 or described in the text.Table 1
Strains, plasmids, and phage.
Strain, plasmid, or phageRelevant genotypeSourceStrain*GS162WTThis labGS776cycA30::Tn10This labGS854argA81::Tn10This labGS998GS162gcvA[27, 28]GS1132GS162 Δ(gcvAgcvB)::ΩaadA (referred to as ΔgcvAB)[19]GS1144GS162 ΔgcvB::ΩCMR (referred to as ΔgcvB)[21]GS1148GS162hfq-1::ΩCMR (referred to as Δhfq)[23]PlasmidpGS554Single-copy vector + constitutivegcvB (pgcvB2+)[19]pGS571Multicopy vector + WTgcvB (pgcvB3+)[29]pGS594Single-copy vector + WTgcvB (pgcvB+)This labpGS609Multi-copy vector + WThfq (phfq3+)[23]pGS611Multi-copy vector + WTgcvA (pgcvA3+)This studypGS624Multi-copy vector + WTgcvA gcvB (pgcvA3+gcvB3+)This studyPhageλgt2λ cloning vector; cI857 repressor[30]λhdeA::lacZλ vector carrying WT hdeA::lacZ fusionThis studyλhdeB::lacZλ vector carrying WT hdeB::lacZ fusionThis studyλhdeA-36::lacZλ vector carrying hdeA-36::lacZ transcriptional fusionThis studyλPBAD::hdeA::lacZλ vector carrying hdeA::lacZ fusion under control of the PBAD promoterThis study*All strains also carry thepheA905  thi  araD129  rpsL150  relA1  deoC1  flbB5301  ptsF25  rbsR mutations.


## 2.2. Construction of Recombinant Phages and Plasmids

TheλhdeA::lacZ translational fusion was constructed by PCR synthesis of a DNA fragment using an upstream primer with an EcoRI site that hybridized to DNA beginning 223 bps upstream of the hdeA transcription start site and a downstream primer with a SmaI site that hybridized to DNA beginning at codon 7 within the hdeA gene. The PCR amplified DNA fragment was digested with EcoRI + SmaI and the 303 bp EcoRI-SmaI fragment ligated into the EcoRI-SmaI sites of plasmid pMC1403 [37], fusing the first 7 codons of the hdeA gene in frame with the 8th codon of the lacZYA genes in pMC1403 (Figure 1(a)). The cloned sequence was verified by DNA sequence analysis at the DNA Core Facility of the University of Iowa. The plasmid was designated phdeA::lacZ. A 5,574 bp EcoRI-MfeI fragment from phdeA::lacZ carrying the hdeA::lacZYA fusion was then ligated into the EcoRI site of phage λgt2 [30], generating λhdeA::lacZ. A λhdeB::lacZ fusion was constructed using the same upstream primer and a downstream primer with a SmaI site that hybridized to DNA beginning at codon 9 within the hdeB gene. The 757 bp EcoRI-SmaI fragment was then used as described above, generating plasmid phdeB::lacZ and phage λhdeB::lacZ (not shown). A λhdeA::lacZtranscriptional fusion was constructed using the same upstream primer and a downstream primer with a HindIII site and that hybridized to DNA at bp −36 relative to the hdeA translation start site (Figure 1(a)). Following digestion with EcoRI and HindIII, the DNA fragment was ligated into the EcoRI and HindIII sites of plasmid pgcvB-lacZ+50 [19], replacing the gcvB fragment with the hdeAfragment, generating plasmid phdeA-36::lacZ. The cloned sequence was verified by DNA sequence analysis. A 5,538 bp EcoRI-MfeI fragment from phdeA-36::lacZ carrying the hdeA-36::lacZYA fusion was then ligated into the EcoRI site of phage λgt2 [30], generating λhdeA-36::lacZ. The 3 fusion phages were used to lysogenize E. coli host strains as described [38]. Each lysogen was tested to ensure it carried a single-copy of the λ chromosome by infection with λcI90c17 [39]. All lysogens were grown at 30°C since all fusion phage carry the λcI857 mutation, resulting in a temperature-sensitive λcI repressor [30]. The λPBAD::hdeA::lacZ fusion, where hdeA transcription is under control of the PBAD promoter, was constructed as described in Figure 1(b).(a) ThehdeAB control region. The hdeAB promoter −35, −10 and transcription start site and the SD sequence and translation start site are indicated above the sequence [31]. The transcription start site for the divergently transcribed hdeD gene is also shown [32]. Binding sites for H-NS [16] and MarR/SoxS [15] are indicated above the sequence with arrows. The binding site for GadX/W [33] is below the sequence in blue and for GadE above the sequence in green [18, 34]. In addition, there are putative binding sites for the transcriptional regulators Lrp and TorR (not shown) [34]. The consensus GcvA binding site is T-N11-A containing a 5′-CTAAT-3′ sequence [35]. Two putative GcvA binding sites are indicated in red. The fusion sites for the λhdeA::lacZ translational fusion, the λhdeA−36::lacZ transcriptional fusion, and the λPBAD::hdeA::lacZ fusion (see below) are indicated with black, red, and green arrows, respectively. (b) Construction of a λPBAD::hdeA::lacZ promoter fusion. The WT PBAD and PhdeA promoters are shown in the top line. The transcription start sites are in red [31, 36]. Small case letters show bases added during PCR amplification of the PBAD and PhdeA promoters. The PBAD promoter was amplified with an upstream primer containing an EcoRI site at bp −272 relative to the transcription start site (not shown) and a downstream primer with a BsaI site (blue). The PhdeA promoter was amplified with an upstream primer containing a BsaI site (blue) and a downstream primer containing a SmaI site at codon 10 in the hdeA gene (not shown). The arrows indicate cut sites for BsaI. The amplified products were cut with BsaI, mixed, and ligated, generating a fusion of the PBAD promoter with the +1G residue of the PhdeA promoter. The fragment was then digested with EcoRI + SmaI and ligated into the EcoRI-SmaI sites of plasmid pMC1403, and subsequently subcloned into λgt2 as described [19]. (c) The gcvA  gcvB region of the E. coli chromosome. The regions amplified by PCR and cloned into pACYC184 to generate pGS611 (pgcvA3+) and pGS624 (pgcvA3+gcvB3+) are indicated with bars. See Section 2.2 for details.
(a)(b)(c)Plasmid pGS611 (pgcvA3+), carrying the E. coli  gcvA gene on a 1,155 bp EcoRI fragment, was constructed as follows. In a PCR reaction, an upstream primer was used containing an EcoRI site and that hybridized to a region beginning 121 bp upstream of the gcvA transcription start site and a downstream primer containing an EcoRI site and hybridized to a region beginning 44 bp downstream of the gcvA translation stop codon. The EcoRI sites were added as parts of the primers. The PCR-generated fragment was digested with EcoRI and cloned into the EcoRI site in plasmid pACYC184 [40] and verified by DNA sequence analysis (Figure 1(c)). Plasmid pGS624 (pgcvA3+gcvB3+), carrying both the gcvA and gcvB genes, was constructed in the same way except the upstream primer hybridized to DNA 51 bps after the gcvB transcription terminator and the downstream primer hybridized to DNA 44 bps after the gcvA translation stop codon, generating a 1,347 bp EcoRI fragment (Figure 1(c)).


## 2.3. Media

The complex medium used was LB [41]. Agar was added at 1.5% (w/v) to make solid media. The minimal medium used was the salts of Vogel and Bonner [42] supplemented with 0.4% (w/v) glucose (GM). Ampicillin was added at 50 and 150 μg mL−1 when strains carried single-copy and multicopy plasmids, respectively. Other supplements were added at the following concentrations (μg mL−1): phenylalanine, 50; glycine, 300; thiamine, 1; TC, 10; CM, 20; X-gal, 40.


## 2.4. DNA Manipulation

Plasmid DNA was isolated using a QIAprep Spin Miniprep Kit (Qiagen, Santa Clara, CA). Vent DNA polymerase and restriction enzymes were from New England Biolabs, Inc. (Beverly, MA). T4 DNA ligase was from Roche Diagnostics (Indianapolis, IN). Reactions were as described by the manufacturers.


## 2.5. Enzyme Assay

β-galactosidase assays were performed on mid-log phase cells (OD600 ~ 0.5) using the chloroform/SDS lysis procedure [41]. Results are the averages of two or more assays with each sample done in triplicate.


## 2.6. Acid Sensitivity Assay

WT, an isogenic ΔgcvABstrain and the two strains transformed with either plasmid pgcvB2+ (constitutively produces GcvB), pgcvA3+ or pgcvA3+gcvB3+ were grown for 24 hr at 30°C in LB and then tested for acid resistance by dilution into LB at pH 2.0. Samples of 0.2 mL were taken at 0, 1, 2, and 4 hr and diluted in 2 mL of LB at pH 7. The final pH of the diluted cultures was ~7.0. Cell viability was determined by plate counts. Percent survival is the titer of colony forming units of acid-tested cells compared to the zero-time point (Figure 2).Figure 2
Percent survival ofE. coli strains plotted against time at pH 2.0. Black, WT; gray, ΔgcvAB; green, ΔgcvAB[pgcvB2+]; blue, WT[pgcvA3+gcvB3+]; purple, ΔgcvAB[pgcvA3+gcvB3+]; red, ΔgcvAB[pgcvA3+]. See Section 2.6 for details.


## 2.7. Transductions

ThegcvB gene is linked to the argA gene and hfq is linked to the cycA gene, with predicted phage P1 cotransduction frequencies of ~78% and ~67%, respectively. P1clr phage prepared on GS854 (argA81::Tn10) was used to transduce ΔgcvB::ΩCMRλhdeA::lacZ to TCR and transductants scored on CM versus TC plates. A TCR CMS transductant was purified. P1clr prepared on GS776 (cycA::Tn10) was used to transduce Δhfq-1::ΩCMRλhdeA::lacZ to TCR resistance and transductants scored on CM versus TC plates. A TCR CMS transductant was purified.


## 3. Results and Discussion

### 3.1. GcvA/GcvB Role in Acid Sensitivity

Microarray data suggested thehdeAand hdeB mRNAs are 1.9- and 2.7-fold higher in WT than a ΔgcvB strain grown in LB, respectively [22]. These genes were not reported to be regulated by GcvB in that study because they fell below the 3-fold cut-off level used for GcvB-regulated genes. Since HdeA and HdeB are necessary for protection of the bacterial periplasm against acid stress [14, 17], we tested if GcvB plays a role in cellular acid resistance. WT and an isogenic ΔgcvABstrain were grown for 24 hr at 30°C in LB and tested for acid resistance by dilution into LB at pH 2.0 [43]. The WT was killed significantly more readily at pH 2 than the ΔgcvAB strain (Figure 2, compare black and gray lines). However, when the ΔgcvAB strain was transformed with pgcvB2+ that constitutively expresses GcvB [19], we did not see complementation that restored acid sensitivity (Figure 2, green line). When transformed with the multi-copy plasmid pgcvA3+gcvB3+, both the WT and the ΔgcvAB transformants were more acid sensitive (Figure 2, compare the black and blue lines and the gray and purple lines). Plasmid pgcvA3+, which carries only the gcvA gene, also complemented the ΔgcvAB mutation, increasing acid sensitivity (Figure 2, compare the gray and red lines). The results suggest it is the absence of GcvA that is responsible for increased acid resistance in the ΔgcvAB strain. It was reported previously that GcvB plays a positive role in acid resistance [25]. Our failure to observe a significant effect on acid resistance is possibly due to the assay conditions. We tested for acid resistance after 24 hours of growth in LB, whereas in the earlier study acid resistance was tested after 5 hr of growth in LB [25]. Although the precise stage of growth was not stated in the earlier study, it is possible cells were still in log phase. In E. coli and Salmonella grown in LB, GcvB was only detected through early stationary phase, with the highest levels observed at the mid-exponential phase [20, 26]. Thus, GcvB regulation of target genes involved in acid resistance is likely during log phase and if GcvB plays a role in stationary phase, it is its absence that is important for allowing an appropriate regulatory response.


### 3.2. Effects of GcvB onλhdeA::lacZ Expression in LB Grown Cells

Although GcvB had no effect in the acid sensitivity assay, we made and tested expression ofλhdeA::lacZ and λhdeB::lacZtranslational fusions. Expression of the hdeA::lacZ fusion was 2.7- and 4-fold higher in WT grown in LB compared to ΔgcvB and Δhfq strains (Figure 3(a), compare lanes 1, 2 and 3). Activation was partially restored in the ΔgcvB[pgcvB+] and Δhfq[phfq3+] complemented strains (Figure 3(a), compare lanes 2 and 4 and lanes 3 and 5). It is unknown why the plasmids fail to fully complement the ΔgcvB and Δhfq mutations. Nevertheless, the results agree with microarray data and suggest GcvB and Hfq positively regulate hdeA::lacZ.Effect of GcvB, Hfq, and GcvA onλhdeA::lacZ and λhdeB::lacZ expression. λhdeA::lacZ lysogens were grown in (a) LB or (b) GM + glycine to mid-log phase and assayed for β-galactosidase activity. λhdeB::lacZ lysogens were grown in (c) LB or (d) GM + glycine to mid-log phase and assayed for β-galactosidase activity.
(a)(b)(c)(d)


### 3.3. ReducedhdeA::lacZ Expression in ΔgcvB and Δhfq Strains Is due to the Absence of GcvB and Hfq

Due to the failure of pgcvB+ and phfq3+ to fully complement the gcvB and hfq mutations (Figure 3), we wanted to verify the reduced levels of hdeA-lacZ expression are due to the absence of GcvB and Hfq. We transduced the ΔgcvB and Δhfq lysogens with WT alleles using linked Tn10 markers. The gcvB+ and hfq+ transductants showed about the same levels of expression as the WT lysogen (Figure 3(a), compare lanes 1, 12 and 13). Thus, despite the failure of pgcvB+ and phfq3+ to fully complement, the results support the reduced levels of expression are due to the absence of GcvB and Hfq.


### 3.4. Effects of GcvA onλhdeA::lacZ Expression in LB Grown Cells

The acid sensitivity assay showed GcvA plays a role in acid resistance (Figure2). In addition, putative GcvA binding sites can be identified in the hdeA promoter region (Figure 1(a)). Thus, we tested the effects of a spontaneous gcvA mutation in strain GS1198 (which is phenotypically GcvB− [19]), on hdeA::lacZ expression. Expression of hdeA::lacZ was ~1.5-fold higher in WT than in the gcvA mutant (Figure 3, lanes 1 and 6). However, expression was 2-fold higher in the gcvA lysogen than in the ΔgcvB lysogen (Figure 3(a), compare lanes 2 and 6). The results could be explained if GcvA, in addition to activating expression of gcvB, which encodes a positive regulator for hdeA, also has a negative role to keep HdeAB levels low. The intermediate level of expression would result from the absence of GcvB to upregulate the hdeA::lacZ fusion and the absence of GcvA to negatively regulate the fusion. To test this hypothesis, we transformed the gcvA mutant with multi-copy pgcvA3+. In the gcvA[pgcvA3+] lysogen, one would expect high GcvA levels, but GcvB would also be produced. Expression of hdeA::lacZ was 2.5-fold higher in WT than in the gcvA[pgcvA3+] transformant (Figure 3(a), compare lanes 1 and 8). In addition, hdeA::lacZ expression was reduced 2-fold compared to the nontransformed gcvA strain (Figure 3(a), lanes 6 and 8). We then transformed the ΔgcvAB lysogen with pgcvA3+. In the ΔgcvAB[pgcvA3+] lysogen, there would be high GcvA levels and no GcvB, and repression of hdeA::lacZ should be greatest. In the ΔgcvAB[pgcvA3+] lysogen there was a 5.8-fold reduction of hdeA-lacZ expression compared to WT and a 2.4-fold reduction compared to the ΔgcvAB lysogen (Figure 3(a), compare lanes 1, 7 and 9). The results support a role for GcvA in negatively regulating hdeA::lacZ expression.


### 3.5. GcvB Positively RegulateshdeA::lacZ Independent of GcvA

If GcvA plays a negative role inhdeA::lacZ expression, GcvB could function to prevent the GcvA effect. Alternatively, GcvB could function independent of GcvA to activate hdeA::lacZ. To test these two possibilities, we transformed the ΔgcvABλhdeA::lacZ lysogen with pgcvB2+, which makes GcvB constitutively [19]. If GcvB’s role is to block GcvA’s ability to repress hdeA::lacZ expression, we hypothesized there would be no effect of GcvB in a ΔgcvABλhdeA::lacZ lysogen without GcvA. Alternatively, if GcvB positively regulates hdeA::lacZ, we hypothesized expression of gcvB would increase hdeA::lacZ expression. In the ΔgcvAB[pgcvB2+] lysogen, hdeA::lacZ expression increased 1.7-fold compared to the non-transformed lysogen, almost to the WT level (Figure 3(a), compare lanes 1, 7 and 10). We also transformed the ΔgcvABλhdeA::lacZ lysogen with pgcvA3+gcvB3+, which overproduces both GcvA and GcvB. Repression of hdeA::lacZ was restored, but not as low as in the pgcvA3+ transformant (Figure 3(a), compare lanes 9 and 11). It is likely that the high GcvB levels partially negate the effect of high GcvA levels. The results suggest GcvB plays a role in activating hdeA::lacZ independent of GcvA.


### 3.6. Effect of GcvA, GcvB, and Hfq onhdeA::lacZ Expression in GM + Glycine

InE. coli, GcvB represses dppA::lacZ, oppA::phoA, cycA::lacZ, and sstT::lacZ fusions when cells are grown in LB, but does not significantly repress these fusions when grown in GM + glycine [19, 22, 23]. However, gcvB is differentially regulated over a 25-fold range in GM supplemented with inosine versus glycine [19]. We hypothesize some genes respond to GcvB levels in GM media. Microarray data suggested the hdeAmRNA is 1.6-fold higher in WT than a ΔgcvB strain grown in GM + glycine [22]. In GM + glycine, hdeA::lacZ expression was significantly higher than for cells grown in LB (Figure 3, compare a and b). In addition, although there are small differences in fold regulation for individual strains, there was a similar regulatory pattern in GM + glycine as observed in LB. The results suggest GcvB positively regulates hdeA in LB and GM + glycine. The results are important since they confirm GcvB does regulate in GM + glycine. In addition, acid resistance mechanisms are most active in the stationary phase in rich media [6, 7, 44, 45]. Our results suggest GcvA and GcvB could play important roles in acid resistance during the log phase of growth in both rich and minimal medium.


### 3.7. Effect of GcvA, GcvB, and Hfq onhdeB::lacZ Expression

ThehdeB gene is the second gene in the hdeAB operon. We tested if hdeB is regulated in a manner similar to the hdeA gene. There were small differences in the levels of hdeB::lacZ expression in response to GcvB, GcvA, and Hfq compared to hdeA::lacZ in both LB and GM + glycine (compare Figures 3(a) and 3(b) with Figures 3(c) and 3(d)). Qualitatively, however, the λhdeB::lacZ fusion showed essentially an identical pattern of expression compared to the hdeA::lacZ fusion, suggesting both genes of the operon are regulated in a similar manner by GcvA, GcvB, and Hfq.It is worth noting that Δhfq lysogens consistently showed lower levels of hdeA::lacZ and hdeB::lacZ expression than ΔgcvB lysogens in both LB and GM + glycine (Figure 3). Two other sRNAs, DsrA, and GadY, are known to play roles in regulation of acid-resistance genes [46, 47]. Since both sRNAs require Hfq, it is not surprising the absence of Hfq has a more dramatic effect on hdeAB expression than the absence of GcvB.


### 3.8. High Levels of GcvA, GcvB, and Hfq in WT AltershdeA::lacZ and hdeB::lacZ Expression

To verify GcvA negatively regulates and GcvB and Hfq positively regulate thehdeAB operon, we transformed WTλhdeA::lacZ, and WTλhdeB::lacZ lysogens with plasmids carrying gcvA,gcvB, both gcvA+ gcvB, or hfq. We hypothesized high GcvB and Hfq would increase expression and high GcvA would repress expression. The lysogens were grown in LB and assayed for β-galactosidase. The presence of pgcvB3+ resulted in a small increase in hdeA::lacZ expression and about a 2-fold increase in hdeB::lacZ (Figures 4(a) and 4(b), lines 1 and 2). The presence of phfq3+ resulted in a 2-fold increase in both hdeA::lacZ and hdeB::lacZ expression (Figures 4(a) and 4(b), line 3). The presence of pgcvA3+ resulted in a 3.5-fold and a 2.2-fold reduction in hdeA::lacZ and hdeB::lacZ expression, respectively (Figures 4(a) and 4(b), line 4). The presence of plasmid pgcvA3+gcvB3+ reduced hdeA::lacZ and hdeB::lacZ expression, but not to the levels of the pgcvA3+ plasmid (Figures 4(a) and 4(b), line 5), suggesting high GcvB antagonize the GcvA effect.WTλhdeA::lacZ and WTλhdeB::lacZ lysogens with the indicated plasmids were grown in LB (black) or GM + glycine (blue) to mid-log phase and assayed for β-galactosidase activity.
(a)(b)The lysogens were also grown in GM + glycine. The pattern of regulation was similar to the LB grown lysogens with one exception. The pgcvB3+ transformant did not show increased expression of hdeB::lacZ as in LB (Figure 4(b), compare lines 1 and 2 with lines 6 and 7). It is possible that in WT grown in GM + glycine GcvB is already in excess for regulation. Nevertheless, the results are in agreement with GcvB and Hfq positively regulating the hdeAB operon and GcvA negatively regulating the operon.


### 3.9. GcvA, GcvB, and Hfq RegulatehdeA::lacZ at the Level of Transcription

GcvA binds DNA and functions to either activate or repress transcription [27, 35, 48], whereas sRNAs that require Hfq usually regulate posttranscriptionally [19, 21–23]. To determine at what step in regulation of hdeA GcvA, GcvB and Hfq function, we constructed a λPBAD::hdeA::lacZ fusion where transcription from the PBAD promoter begins at the +1 start site of the hdeA gene (Figure 1(b)). We initially lysogenized a WT strain with the fusion, the lysogen was grown in LB + arabinose (0.0 to 0.2% concentrations) and assayed for β-galactosidase. There was a 379-fold induction (2.3 units versus 872 units of activity) at 0.0% and 0.05% arabinose, respectively. This is similar to the level observed from the λhdeA::lacZ lysogen grown in LB (Figure 3) and confirmed the fusion is inducible by arabinose. We then lysogenized WT, ΔgcvB, ΔgcvAB, and Δhfq strains. The WT lysogen was also transformed with the plasmids indicated in Figure 5(a). The strains were grown in LB + 0.05% arabinose and assayed for β-galactosidase. If GcvA, GcvB, and Hfq regulate at the transcriptional level, we expected they would no longer have an effect on the PBAD::hdeA::lacZ fusion. Alternatively, if any of the factors regulates posttranscriptionally, we expected it would still regulate the fusion, as the mRNA is identical to the WTλhdeA::lacZ mRNA transcript. There was no significant difference in PBAD::hdeA::lacZ expression in the WT, WT[pgcvA3+] and WT[pgcvA3+gcvB3+] transformants (Figure 5(a), compare lane 1 with lanes 5 and 6). The results show GcvA regulates hdeAB at the level of transcription. Furthermore, there was no significant difference between WT and the ΔgcvB, ΔgcvAB, and Δhfq lysogens and the WT[pgcvB3+] and WT[phfq3+] transformants (Figure 5(a), compare line 1 with 2, 3, 4, 7 and 8). The results show GcvB and Hfq also regulate at the level of transcription.(a) PBAD::hdeA::lacZ  and (b) hdeA−36::lacZ lysogens were grown in LB to mid-log phase and assayed for β-galactosidase activity.
(a)(b)As a complement to the above experiment, we constructed a transcriptional fusion of thehdeA promoter 36 bps upstream of the hdeA translation start site to a promoterless lacZYA operon (Figure 1(a)). Expression of hdeA-36::lacZ was 3.3- and 5.6-fold higher in WT grown in LB compared to ΔgcvB and Δhfq strains (Figure 5(b), lanes 1–3) and activation was partially restored in the ΔgcvB[pgcvB+] and Δhfq[phfq3+] complemented strains (Figure 5(b), compare lanes 2 and 4 and 3 and 5). The results show GcvB and Hfq still regulate the fusion. Expression in the WT was ~2-fold higher than in the ΔgcvA and ΔgcvAB lysogens (Figure 5(b), lanes 1, 6 and 7). In the ΔgcvA[pgcvA3+] and ΔgcvAB[pgcvA3+] lysogens, there was a 2.4- and 3.9-fold reduction of hdeA−36::lacZ expression compared to WT (Figure 5(b), compare lanes 1, 8 and 9), showing GcvA negatively regulates the fusion. We also transformed the WT lysogen with plasmids phfq3+, pgcvA3+, and pgcvA3+gcvB3+. The presence of phfq3+ resulted in a 1.4-fold increase in expression (Figure 5(b), lanes 1 and 10), showing Hfq does positively regulate the fusion. The presence of plasmid pgcvA3+ resulted in a 2.7-fold decrease in expression (Figure 5(b), lanes 1 and 11), consistent with GcvA negatively regulating the fusion. The presence of pgcvA3+gcvB3+ reduced expression 1.3-fold (Figure 5(b), lanes 1 and 12), suggesting GcvB partially overcome the GcvA effect. The results are consistent with GcvA negatively regulating hdeAB at the transcriptional level. Our results also show GcvB and Hfq function during log phase to positively regulate hdeAB at the transcriptional level, counterbalancing the negative effect of GcvA on downregulating these genes. GcvB is known to bind Hfq [49]. It is possible GcvB binds to and sequesters Hfq during exponential growth, and the effects observed are due to decreased levels of Hfq to alter regulation of genes such as rpoS or the activity of sRNAs such as DsrA and GadY that play roles in acid resistance. Additional studies will verify if GcvA directly binds the hdeAB promoter region and how GcvB and Hfq activate the operon.


### 3.10. Effect of pH ongcvB Expression

Our results suggest GcvB plays a role in acid resistance during log phase of growth in rich and minimal media. Therefore, we tested if pH plays a role in regulatinggcvB expression. A WTλgcvB::lacZ fusion was grown to mid-log phase in LB at different pH values from 5.0 to 9.0 and assayed for β-galactosidase. There was no significant effect from pH 7 to pH 9 on gcvB::lacZ expression (Figure 6). However, there was a 3-fold increase as the pH was lowered from pH 7 to pH 5 (Figure 6). Since GcvB activates hdeAB, an increase in gcvB expression at low pH is likely to play a role in final HdeAB levels and in controlling acid resistance.Figure 6
A WTλgcvB::lacZ lysogen was grown in LB buffered at different pH values to mid-log phase of growth and assayed for β-galactosidase activity.


### 3.11. Effect of GcvA and GcvB on Cell Growth at Low pH

We carried out studies to show the effects of high GcvA and GcvB levels on growth at low pH. In a ΔgcvAB strain transformation with pgcvA3+ or pgcvA3+gcvB3+ did not significantly alter generation times (GTs) in LB at pH 7 (Table 2). At pH 4.5, GTs of both the WT and ΔgcvBstrains were significantly increased (Table 2, compare rows 1 and 2, pH 7.0 versus pH 4.5). In addition, in the WT[pgcvA3+] strain, with high GcvA and low GcvB, there was a significant increase in the GT compared to the non-transformed WT strain (Table 2, compare rows 1 and 3, pH 4.5). In the gcvAB[pgcvA3+] transformant, with high GcvA and no GcvB, there was an additional increase in the GT (Table 2, compare rows 3 and 4, pH 4.5 column). In the WT[pgcvA3+gcvB3+] and ΔgcvAB[pgcvA3+gcvB3+] strains, with high GcvA and GcvB, the GTs were not significantly different than in the non-transformed strains (Table 2, compare rows 1 and 2 with rows 5 and 6, pH 4.5). The results are consistent with GcvA negatively regulating acid resistance genes and GcvB overcoming the negative effect of GcvA. The results also show GcvA and GcvB affect acid resistance in log phase cells and could play important roles in the ability of enteric organisms to colonize the GI tract.Table 2
Effect of GcvA and GcvB on growth at low pH.
StrainGT (min) grown in LB atpH 7.0pH 4.5*(1) WT52±5133±2(2) ΔgcvAB56±2160±6(3) WT[pgcvA3+]67±7306±9(4) ΔgcvAB[pgcvA3+]60±6437±45(5) WT[pgcvA3+gcvB3+]67±7157±4(6) ΔgcvAB[pgcvA3+gcvB3+]62±1150±5*Cultures were tested at the end of the experiment to verify the pH had not changed.


### 3.12. Role of GcvB in Cell Physiology

InE. coli, GcvB negatively regulates SstT, CycA, OppA, and DppA levels, the serine transporter, glycine transporter and the oligopeptide, and dipeptide periplasmic binding proteins, respectively [19, 22, 23]. These proteins not only transport amino acids and peptides to provide nutrients, but possibly toxins and antibiotics [50, 51]. If conditions that favor relatively high levels of amino acids and small peptides also favor the presence of small toxic compounds, the decreased expression of transport systems for these small molecules by GcvB could prevent transport of toxic compounds into the cell [22]. Our results show that GcvB also positively regulates genes involved in acid resistance. In addition, GcvA, the activator for gcvB expression, negatively regulates genes involved in acid resistance. These findings suggest GcvB and GcvA play important roles in the ability of E. coli to survive low pH conditions. Recently, in a screen of a sRNA gene knockout library, GcvB was shown to enhance E. coli survival at low pH [25]. Thus, GcvB likely allows E. coli to respond to and survive two stress conditions, the presence of toxic compounds and low pH environments. Both of these conditions are encountered as E. coli moves from an external environment into the GI tract. Understanding the biological roles of GcvB and GcvA in acid resistance and their mechanism(s) of regulation will provide insights as to how cells respond to environmental challenges to infect host organisms.


## 3.1. GcvA/GcvB Role in Acid Sensitivity

Microarray data suggested thehdeAand hdeB mRNAs are 1.9- and 2.7-fold higher in WT than a ΔgcvB strain grown in LB, respectively [22]. These genes were not reported to be regulated by GcvB in that study because they fell below the 3-fold cut-off level used for GcvB-regulated genes. Since HdeA and HdeB are necessary for protection of the bacterial periplasm against acid stress [14, 17], we tested if GcvB plays a role in cellular acid resistance. WT and an isogenic ΔgcvABstrain were grown for 24 hr at 30°C in LB and tested for acid resistance by dilution into LB at pH 2.0 [43]. The WT was killed significantly more readily at pH 2 than the ΔgcvAB strain (Figure 2, compare black and gray lines). However, when the ΔgcvAB strain was transformed with pgcvB2+ that constitutively expresses GcvB [19], we did not see complementation that restored acid sensitivity (Figure 2, green line). When transformed with the multi-copy plasmid pgcvA3+gcvB3+, both the WT and the ΔgcvAB transformants were more acid sensitive (Figure 2, compare the black and blue lines and the gray and purple lines). Plasmid pgcvA3+, which carries only the gcvA gene, also complemented the ΔgcvAB mutation, increasing acid sensitivity (Figure 2, compare the gray and red lines). The results suggest it is the absence of GcvA that is responsible for increased acid resistance in the ΔgcvAB strain. It was reported previously that GcvB plays a positive role in acid resistance [25]. Our failure to observe a significant effect on acid resistance is possibly due to the assay conditions. We tested for acid resistance after 24 hours of growth in LB, whereas in the earlier study acid resistance was tested after 5 hr of growth in LB [25]. Although the precise stage of growth was not stated in the earlier study, it is possible cells were still in log phase. In E. coli and Salmonella grown in LB, GcvB was only detected through early stationary phase, with the highest levels observed at the mid-exponential phase [20, 26]. Thus, GcvB regulation of target genes involved in acid resistance is likely during log phase and if GcvB plays a role in stationary phase, it is its absence that is important for allowing an appropriate regulatory response.


## 3.2. Effects of GcvB onλhdeA::lacZ Expression in LB Grown Cells

Although GcvB had no effect in the acid sensitivity assay, we made and tested expression ofλhdeA::lacZ and λhdeB::lacZtranslational fusions. Expression of the hdeA::lacZ fusion was 2.7- and 4-fold higher in WT grown in LB compared to ΔgcvB and Δhfq strains (Figure 3(a), compare lanes 1, 2 and 3). Activation was partially restored in the ΔgcvB[pgcvB+] and Δhfq[phfq3+] complemented strains (Figure 3(a), compare lanes 2 and 4 and lanes 3 and 5). It is unknown why the plasmids fail to fully complement the ΔgcvB and Δhfq mutations. Nevertheless, the results agree with microarray data and suggest GcvB and Hfq positively regulate hdeA::lacZ.Effect of GcvB, Hfq, and GcvA onλhdeA::lacZ and λhdeB::lacZ expression. λhdeA::lacZ lysogens were grown in (a) LB or (b) GM + glycine to mid-log phase and assayed for β-galactosidase activity. λhdeB::lacZ lysogens were grown in (c) LB or (d) GM + glycine to mid-log phase and assayed for β-galactosidase activity.
(a)(b)(c)(d)


## 3.3. ReducedhdeA::lacZ Expression in ΔgcvB and Δhfq Strains Is due to the Absence of GcvB and Hfq

Due to the failure of pgcvB+ and phfq3+ to fully complement the gcvB and hfq mutations (Figure 3), we wanted to verify the reduced levels of hdeA-lacZ expression are due to the absence of GcvB and Hfq. We transduced the ΔgcvB and Δhfq lysogens with WT alleles using linked Tn10 markers. The gcvB+ and hfq+ transductants showed about the same levels of expression as the WT lysogen (Figure 3(a), compare lanes 1, 12 and 13). Thus, despite the failure of pgcvB+ and phfq3+ to fully complement, the results support the reduced levels of expression are due to the absence of GcvB and Hfq.


## 3.4. Effects of GcvA onλhdeA::lacZ Expression in LB Grown Cells

The acid sensitivity assay showed GcvA plays a role in acid resistance (Figure2). In addition, putative GcvA binding sites can be identified in the hdeA promoter region (Figure 1(a)). Thus, we tested the effects of a spontaneous gcvA mutation in strain GS1198 (which is phenotypically GcvB− [19]), on hdeA::lacZ expression. Expression of hdeA::lacZ was ~1.5-fold higher in WT than in the gcvA mutant (Figure 3, lanes 1 and 6). However, expression was 2-fold higher in the gcvA lysogen than in the ΔgcvB lysogen (Figure 3(a), compare lanes 2 and 6). The results could be explained if GcvA, in addition to activating expression of gcvB, which encodes a positive regulator for hdeA, also has a negative role to keep HdeAB levels low. The intermediate level of expression would result from the absence of GcvB to upregulate the hdeA::lacZ fusion and the absence of GcvA to negatively regulate the fusion. To test this hypothesis, we transformed the gcvA mutant with multi-copy pgcvA3+. In the gcvA[pgcvA3+] lysogen, one would expect high GcvA levels, but GcvB would also be produced. Expression of hdeA::lacZ was 2.5-fold higher in WT than in the gcvA[pgcvA3+] transformant (Figure 3(a), compare lanes 1 and 8). In addition, hdeA::lacZ expression was reduced 2-fold compared to the nontransformed gcvA strain (Figure 3(a), lanes 6 and 8). We then transformed the ΔgcvAB lysogen with pgcvA3+. In the ΔgcvAB[pgcvA3+] lysogen, there would be high GcvA levels and no GcvB, and repression of hdeA::lacZ should be greatest. In the ΔgcvAB[pgcvA3+] lysogen there was a 5.8-fold reduction of hdeA-lacZ expression compared to WT and a 2.4-fold reduction compared to the ΔgcvAB lysogen (Figure 3(a), compare lanes 1, 7 and 9). The results support a role for GcvA in negatively regulating hdeA::lacZ expression.


## 3.5. GcvB Positively RegulateshdeA::lacZ Independent of GcvA

If GcvA plays a negative role inhdeA::lacZ expression, GcvB could function to prevent the GcvA effect. Alternatively, GcvB could function independent of GcvA to activate hdeA::lacZ. To test these two possibilities, we transformed the ΔgcvABλhdeA::lacZ lysogen with pgcvB2+, which makes GcvB constitutively [19]. If GcvB’s role is to block GcvA’s ability to repress hdeA::lacZ expression, we hypothesized there would be no effect of GcvB in a ΔgcvABλhdeA::lacZ lysogen without GcvA. Alternatively, if GcvB positively regulates hdeA::lacZ, we hypothesized expression of gcvB would increase hdeA::lacZ expression. In the ΔgcvAB[pgcvB2+] lysogen, hdeA::lacZ expression increased 1.7-fold compared to the non-transformed lysogen, almost to the WT level (Figure 3(a), compare lanes 1, 7 and 10). We also transformed the ΔgcvABλhdeA::lacZ lysogen with pgcvA3+gcvB3+, which overproduces both GcvA and GcvB. Repression of hdeA::lacZ was restored, but not as low as in the pgcvA3+ transformant (Figure 3(a), compare lanes 9 and 11). It is likely that the high GcvB levels partially negate the effect of high GcvA levels. The results suggest GcvB plays a role in activating hdeA::lacZ independent of GcvA.


## 3.6. Effect of GcvA, GcvB, and Hfq onhdeA::lacZ Expression in GM + Glycine

InE. coli, GcvB represses dppA::lacZ, oppA::phoA, cycA::lacZ, and sstT::lacZ fusions when cells are grown in LB, but does not significantly repress these fusions when grown in GM + glycine [19, 22, 23]. However, gcvB is differentially regulated over a 25-fold range in GM supplemented with inosine versus glycine [19]. We hypothesize some genes respond to GcvB levels in GM media. Microarray data suggested the hdeAmRNA is 1.6-fold higher in WT than a ΔgcvB strain grown in GM + glycine [22]. In GM + glycine, hdeA::lacZ expression was significantly higher than for cells grown in LB (Figure 3, compare a and b). In addition, although there are small differences in fold regulation for individual strains, there was a similar regulatory pattern in GM + glycine as observed in LB. The results suggest GcvB positively regulates hdeA in LB and GM + glycine. The results are important since they confirm GcvB does regulate in GM + glycine. In addition, acid resistance mechanisms are most active in the stationary phase in rich media [6, 7, 44, 45]. Our results suggest GcvA and GcvB could play important roles in acid resistance during the log phase of growth in both rich and minimal medium.


## 3.7. Effect of GcvA, GcvB, and Hfq onhdeB::lacZ Expression

ThehdeB gene is the second gene in the hdeAB operon. We tested if hdeB is regulated in a manner similar to the hdeA gene. There were small differences in the levels of hdeB::lacZ expression in response to GcvB, GcvA, and Hfq compared to hdeA::lacZ in both LB and GM + glycine (compare Figures 3(a) and 3(b) with Figures 3(c) and 3(d)). Qualitatively, however, the λhdeB::lacZ fusion showed essentially an identical pattern of expression compared to the hdeA::lacZ fusion, suggesting both genes of the operon are regulated in a similar manner by GcvA, GcvB, and Hfq.It is worth noting that Δhfq lysogens consistently showed lower levels of hdeA::lacZ and hdeB::lacZ expression than ΔgcvB lysogens in both LB and GM + glycine (Figure 3). Two other sRNAs, DsrA, and GadY, are known to play roles in regulation of acid-resistance genes [46, 47]. Since both sRNAs require Hfq, it is not surprising the absence of Hfq has a more dramatic effect on hdeAB expression than the absence of GcvB.


## 3.8. High Levels of GcvA, GcvB, and Hfq in WT AltershdeA::lacZ and hdeB::lacZ Expression

To verify GcvA negatively regulates and GcvB and Hfq positively regulate thehdeAB operon, we transformed WTλhdeA::lacZ, and WTλhdeB::lacZ lysogens with plasmids carrying gcvA,gcvB, both gcvA+ gcvB, or hfq. We hypothesized high GcvB and Hfq would increase expression and high GcvA would repress expression. The lysogens were grown in LB and assayed for β-galactosidase. The presence of pgcvB3+ resulted in a small increase in hdeA::lacZ expression and about a 2-fold increase in hdeB::lacZ (Figures 4(a) and 4(b), lines 1 and 2). The presence of phfq3+ resulted in a 2-fold increase in both hdeA::lacZ and hdeB::lacZ expression (Figures 4(a) and 4(b), line 3). The presence of pgcvA3+ resulted in a 3.5-fold and a 2.2-fold reduction in hdeA::lacZ and hdeB::lacZ expression, respectively (Figures 4(a) and 4(b), line 4). The presence of plasmid pgcvA3+gcvB3+ reduced hdeA::lacZ and hdeB::lacZ expression, but not to the levels of the pgcvA3+ plasmid (Figures 4(a) and 4(b), line 5), suggesting high GcvB antagonize the GcvA effect.WTλhdeA::lacZ and WTλhdeB::lacZ lysogens with the indicated plasmids were grown in LB (black) or GM + glycine (blue) to mid-log phase and assayed for β-galactosidase activity.
(a)(b)The lysogens were also grown in GM + glycine. The pattern of regulation was similar to the LB grown lysogens with one exception. The pgcvB3+ transformant did not show increased expression of hdeB::lacZ as in LB (Figure 4(b), compare lines 1 and 2 with lines 6 and 7). It is possible that in WT grown in GM + glycine GcvB is already in excess for regulation. Nevertheless, the results are in agreement with GcvB and Hfq positively regulating the hdeAB operon and GcvA negatively regulating the operon.


## 3.9. GcvA, GcvB, and Hfq RegulatehdeA::lacZ at the Level of Transcription

GcvA binds DNA and functions to either activate or repress transcription [27, 35, 48], whereas sRNAs that require Hfq usually regulate posttranscriptionally [19, 21–23]. To determine at what step in regulation of hdeA GcvA, GcvB and Hfq function, we constructed a λPBAD::hdeA::lacZ fusion where transcription from the PBAD promoter begins at the +1 start site of the hdeA gene (Figure 1(b)). We initially lysogenized a WT strain with the fusion, the lysogen was grown in LB + arabinose (0.0 to 0.2% concentrations) and assayed for β-galactosidase. There was a 379-fold induction (2.3 units versus 872 units of activity) at 0.0% and 0.05% arabinose, respectively. This is similar to the level observed from the λhdeA::lacZ lysogen grown in LB (Figure 3) and confirmed the fusion is inducible by arabinose. We then lysogenized WT, ΔgcvB, ΔgcvAB, and Δhfq strains. The WT lysogen was also transformed with the plasmids indicated in Figure 5(a). The strains were grown in LB + 0.05% arabinose and assayed for β-galactosidase. If GcvA, GcvB, and Hfq regulate at the transcriptional level, we expected they would no longer have an effect on the PBAD::hdeA::lacZ fusion. Alternatively, if any of the factors regulates posttranscriptionally, we expected it would still regulate the fusion, as the mRNA is identical to the WTλhdeA::lacZ mRNA transcript. There was no significant difference in PBAD::hdeA::lacZ expression in the WT, WT[pgcvA3+] and WT[pgcvA3+gcvB3+] transformants (Figure 5(a), compare lane 1 with lanes 5 and 6). The results show GcvA regulates hdeAB at the level of transcription. Furthermore, there was no significant difference between WT and the ΔgcvB, ΔgcvAB, and Δhfq lysogens and the WT[pgcvB3+] and WT[phfq3+] transformants (Figure 5(a), compare line 1 with 2, 3, 4, 7 and 8). The results show GcvB and Hfq also regulate at the level of transcription.(a) PBAD::hdeA::lacZ  and (b) hdeA−36::lacZ lysogens were grown in LB to mid-log phase and assayed for β-galactosidase activity.
(a)(b)As a complement to the above experiment, we constructed a transcriptional fusion of thehdeA promoter 36 bps upstream of the hdeA translation start site to a promoterless lacZYA operon (Figure 1(a)). Expression of hdeA-36::lacZ was 3.3- and 5.6-fold higher in WT grown in LB compared to ΔgcvB and Δhfq strains (Figure 5(b), lanes 1–3) and activation was partially restored in the ΔgcvB[pgcvB+] and Δhfq[phfq3+] complemented strains (Figure 5(b), compare lanes 2 and 4 and 3 and 5). The results show GcvB and Hfq still regulate the fusion. Expression in the WT was ~2-fold higher than in the ΔgcvA and ΔgcvAB lysogens (Figure 5(b), lanes 1, 6 and 7). In the ΔgcvA[pgcvA3+] and ΔgcvAB[pgcvA3+] lysogens, there was a 2.4- and 3.9-fold reduction of hdeA−36::lacZ expression compared to WT (Figure 5(b), compare lanes 1, 8 and 9), showing GcvA negatively regulates the fusion. We also transformed the WT lysogen with plasmids phfq3+, pgcvA3+, and pgcvA3+gcvB3+. The presence of phfq3+ resulted in a 1.4-fold increase in expression (Figure 5(b), lanes 1 and 10), showing Hfq does positively regulate the fusion. The presence of plasmid pgcvA3+ resulted in a 2.7-fold decrease in expression (Figure 5(b), lanes 1 and 11), consistent with GcvA negatively regulating the fusion. The presence of pgcvA3+gcvB3+ reduced expression 1.3-fold (Figure 5(b), lanes 1 and 12), suggesting GcvB partially overcome the GcvA effect. The results are consistent with GcvA negatively regulating hdeAB at the transcriptional level. Our results also show GcvB and Hfq function during log phase to positively regulate hdeAB at the transcriptional level, counterbalancing the negative effect of GcvA on downregulating these genes. GcvB is known to bind Hfq [49]. It is possible GcvB binds to and sequesters Hfq during exponential growth, and the effects observed are due to decreased levels of Hfq to alter regulation of genes such as rpoS or the activity of sRNAs such as DsrA and GadY that play roles in acid resistance. Additional studies will verify if GcvA directly binds the hdeAB promoter region and how GcvB and Hfq activate the operon.


## 3.10. Effect of pH ongcvB Expression

Our results suggest GcvB plays a role in acid resistance during log phase of growth in rich and minimal media. Therefore, we tested if pH plays a role in regulatinggcvB expression. A WTλgcvB::lacZ fusion was grown to mid-log phase in LB at different pH values from 5.0 to 9.0 and assayed for β-galactosidase. There was no significant effect from pH 7 to pH 9 on gcvB::lacZ expression (Figure 6). However, there was a 3-fold increase as the pH was lowered from pH 7 to pH 5 (Figure 6). Since GcvB activates hdeAB, an increase in gcvB expression at low pH is likely to play a role in final HdeAB levels and in controlling acid resistance.Figure 6
A WTλgcvB::lacZ lysogen was grown in LB buffered at different pH values to mid-log phase of growth and assayed for β-galactosidase activity.


## 3.11. Effect of GcvA and GcvB on Cell Growth at Low pH

We carried out studies to show the effects of high GcvA and GcvB levels on growth at low pH. In a ΔgcvAB strain transformation with pgcvA3+ or pgcvA3+gcvB3+ did not significantly alter generation times (GTs) in LB at pH 7 (Table 2). At pH 4.5, GTs of both the WT and ΔgcvBstrains were significantly increased (Table 2, compare rows 1 and 2, pH 7.0 versus pH 4.5). In addition, in the WT[pgcvA3+] strain, with high GcvA and low GcvB, there was a significant increase in the GT compared to the non-transformed WT strain (Table 2, compare rows 1 and 3, pH 4.5). In the gcvAB[pgcvA3+] transformant, with high GcvA and no GcvB, there was an additional increase in the GT (Table 2, compare rows 3 and 4, pH 4.5 column). In the WT[pgcvA3+gcvB3+] and ΔgcvAB[pgcvA3+gcvB3+] strains, with high GcvA and GcvB, the GTs were not significantly different than in the non-transformed strains (Table 2, compare rows 1 and 2 with rows 5 and 6, pH 4.5). The results are consistent with GcvA negatively regulating acid resistance genes and GcvB overcoming the negative effect of GcvA. The results also show GcvA and GcvB affect acid resistance in log phase cells and could play important roles in the ability of enteric organisms to colonize the GI tract.Table 2
Effect of GcvA and GcvB on growth at low pH.
StrainGT (min) grown in LB atpH 7.0pH 4.5*(1) WT52±5133±2(2) ΔgcvAB56±2160±6(3) WT[pgcvA3+]67±7306±9(4) ΔgcvAB[pgcvA3+]60±6437±45(5) WT[pgcvA3+gcvB3+]67±7157±4(6) ΔgcvAB[pgcvA3+gcvB3+]62±1150±5*Cultures were tested at the end of the experiment to verify the pH had not changed.


## 3.12. Role of GcvB in Cell Physiology

InE. coli, GcvB negatively regulates SstT, CycA, OppA, and DppA levels, the serine transporter, glycine transporter and the oligopeptide, and dipeptide periplasmic binding proteins, respectively [19, 22, 23]. These proteins not only transport amino acids and peptides to provide nutrients, but possibly toxins and antibiotics [50, 51]. If conditions that favor relatively high levels of amino acids and small peptides also favor the presence of small toxic compounds, the decreased expression of transport systems for these small molecules by GcvB could prevent transport of toxic compounds into the cell [22]. Our results show that GcvB also positively regulates genes involved in acid resistance. In addition, GcvA, the activator for gcvB expression, negatively regulates genes involved in acid resistance. These findings suggest GcvB and GcvA play important roles in the ability of E. coli to survive low pH conditions. Recently, in a screen of a sRNA gene knockout library, GcvB was shown to enhance E. coli survival at low pH [25]. Thus, GcvB likely allows E. coli to respond to and survive two stress conditions, the presence of toxic compounds and low pH environments. Both of these conditions are encountered as E. coli moves from an external environment into the GI tract. Understanding the biological roles of GcvB and GcvA in acid resistance and their mechanism(s) of regulation will provide insights as to how cells respond to environmental challenges to infect host organisms.



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*Source: 697308-2012-05-16.xml*