# Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli

Ye Jin, Rory M Watt, Antoine Danchin, Jian-Dong Huang

► To cite this version:

Ye Jin, Rory M Watt, Antoine Danchin, Jian-Dong Huang. Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli. BMC Genomics, 2009, 10 (1), pp.165. 10.1186/1471-2164-10-165. doi:10.1186/1471-2164-10-165

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# Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli

Ye Jin, Rory M Watt, Antoine Danchin, Jian-Dong Huang

► To cite this version:

Ye Jin, Rory M Watt, Antoine Danchin, Jian-Dong Huang. Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli. BMC Genomics, 2009, 10 (1), pp.165. 10.1186/1471-2164-10-165. doi:10.1186/1471-2164-10-165

HAL Id: hal-04581684

https://hal.science/hal-04581684v1

Submitted on 21 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Research article

Open Access

# Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli

Ye Jin¹, Rory M Watt², Antoine Danchin³ and Jian-dong Huang*¹

Address: ¹Department of Biochemistry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pok Fu Lam, Hong Kong SAR, PR China, ²Oral Biosciences, Faculty of Dentistry, University of Hong Kong, Pok Fu Lam, Hong Kong SAR, PR China and ³Genetics of Bacterial Genomes, CNRS URA2171, Institut Pasteur, 28, rue du Docteur Roux, Paris Cedex 15, 75724, France

Email: Ye Jin - yejin@hkucc.hku.hk; Rory M Watt - rmwatt@hku.hk; Antoine Danchin - antoine.danchin@normalesup.org; Jian-dong Huang* - jdhuang@hkucc.hku.hk

Published: 20 April 2009

Received: 26 January 2009

BMC Genomics 2009, 10:165 doi:10.1186/1471-2164-10-165

Accepted: 20 April 2009

This article is available from: http://www.biomedcentral.com/1471-2164/10/165

© 2009 Jin et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## Abstract

**Background:** The low pH environment of the human stomach is lethal for most microorganisms; but not *Escherichia coli*, which can tolerate extreme acid stress. Acid resistance in *E. coli* is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).

**Results:** In this study, we individually deleted seventy-nine sncRNA genes from the *E. coli* K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in *E. coli*. We demonstrate that GcvB enhances the ability of *E. coli* to survive low pH by upregulating the levels of the alternate sigma factor RpoS.

**Conclusion:** GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in *E. coli*.

## Background

The ability to survive the extremely low pH environment present within the stomach (pH values < 2.5) is essential for colonization of the intestine by commensals and for the pathogenesis of enteric bacteria. Three distinct acid resistance (AR) systems are responsible for protecting bacterial cells from acid shock [1-3]. AR system 1 is a glucose-repressed system and does not require any external amino acids to function at low pH [1]. Unidentified components of this system are controlled by the alternate sigma factor RpoS, with the underlying mechanism remaining unclear [1]. In contrast, AR systems 2 and 3 provide AR by consuming intracellular protons via the glutamate (AR2) and

arginine (AR3) decarboxylation reactions [2,3]. AR2 is the most efficient AR system. It involves two isozymes of glutamate decarboxylase, encoded by genes *gadA* and *gadB*, and a membrane-associated antiporter (GadC) that exchanges external glutamate for the intracellular decarboxylation product γ-aminobutyric acid (GABA) [2].

AR2 is hierarchically regulated by multiple regulators, which include small noncoding RNAs (sncRNA) DsrA and possibly GadY. DsrA activates the expression of *rpoS* [4] and several AR genes including *hdeAB*, *gadAX*, and *gadBC* [5]. GadY, whose effects on AR have yet to be determined, controls the synthesis of GadX and GadW, which are reg-

<footnote>* Corresponding author</footnote>
Research article

Open Access

# Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli

Ye Jin¹, Rory M Watt², Antoine Danchin³ and Jian-dong Huang*¹

Address: ¹Department of Biochemistry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pok Fu Lam, Hong Kong SAR, PR China, ²Oral Biosciences, Faculty of Dentistry, University of Hong Kong, Pok Fu Lam, Hong Kong SAR, PR China and ³Genetics of Bacterial Genomes, CNRS URA2171, Institut Pasteur, 28, rue du Docteur Roux, Paris Cedex 15, 75724, France

Email: Ye Jin - yejin@hkucc.hku.hk; Rory M Watt - rmwatt@hku.hk; Antoine Danchin - antoine.danchin@normalesup.org; Jian-dong Huang* - jdhuang@hkucc.hku.hk

Published: 20 April 2009

Received: 26 January 2009

BMC Genomics 2009, 10:165 doi:10.1186/1471-2164-10-165

Accepted: 20 April 2009

This article is available from: http://www.biomedcentral.com/1471-2164/10/165

© 2009 Jin et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## Abstract

**Background:** The low pH environment of the human stomach is lethal for most microorganisms; but not *Escherichia coli*, which can tolerate extreme acid stress. Acid resistance in *E. coli* is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).

**Results:** In this study, we individually deleted seventy-nine sncRNA genes from the *E. coli* K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in *E. coli*. We demonstrate that GcvB enhances the ability of *E. coli* to survive low pH by upregulating the levels of the alternate sigma factor RpoS.

**Conclusion:** GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in *E. coli*.

## Background

The ability to survive the extremely low pH environment present within the stomach (pH values < 2.5) is essential for colonization of the intestine by commensals and for the pathogenesis of enteric bacteria. Three distinct acid resistance (AR) systems are responsible for protecting bacterial cells from acid shock [1-3]. AR system 1 is a glucose-repressed system and does not require any external amino acids to function at low pH [1]. Unidentified components of this system are controlled by the alternate sigma factor RpoS, with the underlying mechanism remaining unclear [1]. In contrast, AR systems 2 and 3 provide AR by consuming intracellular protons via the glutamate (AR2) and

arginine (AR3) decarboxylation reactions [2,3]. AR2 is the most efficient AR system. It involves two isozymes of glutamate decarboxylase, encoded by genes *gadA* and *gadB*, and a membrane-associated antiporter (GadC) that exchanges external glutamate for the intracellular decarboxylation product γ-aminobutyric acid (GABA) [2].

AR2 is hierarchically regulated by multiple regulators, which include small noncoding RNAs (sncRNA) DsrA and possibly GadY. DsrA activates the expression of *rpoS* [4] and several AR genes including *hdeAB*, *gadAX*, and *gadBC* [5]. GadY, whose effects on AR have yet to be determined, controls the synthesis of GadX and GadW, which are reg-

<footnote>* Corresponding author</footnote>
ulators of the GadA and GadB glutamate decarboxylases [6,7]. In recent years, an increasing number of sncRNAs have been identified, but the functions of the vast majority of these are unknown. This prompted us to investigate whether sncRNAs in addition to DsrA and GadY, were involved in AR; and if so, to establish how they exerted their regulatory actions.

In the present study, seventy-nine snRNAs identified prior to 2006 [8-10] were selected for the construction of a single-gene knockout library within *E. coli* K12-MG1655. Comparison of each mutant with the wild type strain enabled us to identify the sncRNA GcvB as a previously unknown regulator of AR in *E. coli*. We demonstrate that GcvB positively regulates AR predominantly by upregulating the levels of the RpoS transcription factor (Sigma S). Our findings provide further insight into the regulatory roles played by sncRNAs in AR, which contributes to virulence of *E. coli*.

## Results

### Construction of single-sncRNA gene knockout mutant collection in E. coli K-12 MG1655

To systematically characterize sncRNAs, we established a single-sncRNA gene knockout library within *E. coli* K12-MG1655. Recombineering techniques were used to replace the target gene with a selectable chloramphenicol-resistance cassette, which was generated by polymerase chain reaction (PCR) using primers containing homology (45 nt) to the gene-flanking regions. This homologous recombination-based approach enabled precise gene deletion with minimal interference to adjacent genes (polar effects) [11,12]. In total, seventy-nine genes encoding sncRNAs identified prior to 2006 [8-10] were deleted individually from the MG1655 chromosome (see additional file 1).

### The sncRNA GcvB is a novel regulator of AR in E. coli

We investigated the involvement of sncRNAs in the AR of *E. coli* by challenging the sncRNA mutants as well as the wild type strain by incubation in LB medium at pH 2.0 (for 15 or 30 minutes), and comparing their percent survival and/or rates of recovery after pH neutralization (termed acid recovery). Unless otherwise stated, bacterial cells were grown for 5 hrs to the early stationary phase before the acid challenge. As expected, cells lacking DsrA, which is a known AR regulator, were sensitive to the acid challenge, confirming previous findings (Fig. 1A) [4,5]. However, deleting *gadY* did not affect acid survival (Fig. 1A), suggesting that GadY is not required for AR in spite of its positive control of GadX, a transcriptional activator of the GadA and GadB glutamate decarboxylases [6,7]. Interestingly, the absence of the sncRNA GcvB resulted in a significant reduction in acid survival (Fig. 1A, B). The same AR phenotypes in the sncRNA mutants were also

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

**Figure 1** Regulatory role of the small noncoding RNA GcvB in acid resistance in *Escherichia coli*. (A) MG1655-K12 wild type strain (wt) and various deletion mutants were grown in Luria Broth broth (pH 7.0) for 5 hr at 37°C. Cells were acid shocked at pH 2.0 for 30 min, and then neutralized by adding alkalinized LB (pH 9.3). Cells were allowed to grow for an additional 3 hrs before measurement of optical density at 600 nm (OD600). Two-tailed paired t tests were employed to compare each mutant with the wt in terms of acid resistance. (B) Because the gcvB mutant displayed a lower growth rate than the wt (data not shown), percent acid survival was determined to confirm the data of acid recovery measured as OD600. Cell viability was determined by plate counts and percent acid survival was calculated as the number of cfu remaining after the 30 min-acid treatment divided by the initial cfu at time zero (quoted as a percentage). (C) A plasmid expressing GcvB under the control of its native promoter was transformed into a gcvB mutant of *E. coli* MG1655. The mutant carrying an empty vector was used as a control. Cells were incubated for 5 hrs before being acid challenged for 15 min. Error bars represent standard deviation (*P*-value < 0.05).
ulators of the GadA and GadB glutamate decarboxylases [6,7]. In recent years, an increasing number of sncRNAs have been identified, but the functions of the vast majority of these are unknown. This prompted us to investigate whether sncRNAs in addition to DsrA and GadY, were involved in AR; and if so, to establish how they exerted their regulatory actions.

In the present study, seventy-nine snRNAs identified prior to 2006 [8-10] were selected for the construction of a single-gene knockout library within *E. coli* K12-MG1655. Comparison of each mutant with the wild type strain enabled us to identify the sncRNA GcvB as a previously unknown regulator of AR in *E. coli*. We demonstrate that GcvB positively regulates AR predominantly by upregulating the levels of the RpoS transcription factor (Sigma S). Our findings provide further insight into the regulatory roles played by sncRNAs in AR, which contributes to virulence of *E. coli*.

## Results

### Construction of single-sncRNA gene knockout mutant collection in E. coli K-12 MG1655

To systematically characterize sncRNAs, we established a single-sncRNA gene knockout library within *E. coli* K12-MG1655. Recombineering techniques were used to replace the target gene with a selectable chloramphenicol-resistance cassette, which was generated by polymerase chain reaction (PCR) using primers containing homology (45 nt) to the gene-flanking regions. This homologous recombination-based approach enabled precise gene deletion with minimal interference to adjacent genes (polar effects) [11,12]. In total, seventy-nine genes encoding sncRNAs identified prior to 2006 [8-10] were deleted individually from the MG1655 chromosome (see additional file 1).

### The sncRNA GcvB is a novel regulator of AR in E. coli

We investigated the involvement of sncRNAs in the AR of *E. coli* by challenging the sncRNA mutants as well as the wild type strain by incubation in LB medium at pH 2.0 (for 15 or 30 minutes), and comparing their percent survival and/or rates of recovery after pH neutralization (termed acid recovery). Unless otherwise stated, bacterial cells were grown for 5 hrs to the early stationary phase before the acid challenge. As expected, cells lacking DsrA, which is a known AR regulator, were sensitive to the acid challenge, confirming previous findings (Fig. 1A) [4,5]. However, deleting *gadY* did not affect acid survival (Fig. 1A), suggesting that GadY is not required for AR in spite of its positive control of GadX, a transcriptional activator of the GadA and GadB glutamate decarboxylases [6,7]. Interestingly, the absence of the sncRNA GcvB resulted in a significant reduction in acid survival (Fig. 1A, B). The same AR phenotypes in the sncRNA mutants were also

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

**Figure 1** Regulatory role of the small noncoding RNA GcvB in acid resistance in *Escherichia coli*. (A) MG1655-K12 wild type strain (wt) and various deletion mutants were grown in Luria Broth broth (pH 7.0) for 5 hr at 37°C. Cells were acid shocked at pH 2.0 for 30 min, and then neutralized by adding alkalinized LB (pH 9.3). Cells were allowed to grow for an additional 3 hrs before measurement of optical density at 600 nm (OD600). Two-tailed paired t tests were employed to compare each mutant with the wt in terms of acid resistance. (B) Because the gcvB mutant displayed a lower growth rate than the wt (data not shown), percent acid survival was determined to confirm the data of acid recovery measured as OD600. Cell viability was determined by plate counts and percent acid survival was calculated as the number of cfu remaining after the 30 min-acid treatment divided by the initial cfu at time zero (quoted as a percentage). (C) A plasmid expressing GcvB under the control of its native promoter was transformed into a gcvB mutant of *E. coli* MG1655. The mutant carrying an empty vector was used as a control. Cells were incubated for 5 hrs before being acid challenged for 15 min. Error bars represent standard deviation (*P*-value < 0.05).
observed during the exponential growth phase (data not shown), suggesting that GcvB exerts constant AR regulatory functions throughout the various growth phases. We also individually deleted *gcvA* and *ygdl* which are the two genes immediately adjacent to *gcvB*. Both mutants displayed comparable AR as the wild type (Fig. 1A). This shows that the observed acid vulnerability in the *gcvB* mutant is not due to the disruption of the activities of its adjacent genes.

Next, we cloned the gene encoding GcvB (together with its native promoter) into plasmid pET32a (+) and overexpressed the sncRNA in the *E. coli* MG1655 *gcvB* null mutant. The *gcvB* mutant carrying an empty vector served as a negative control. We found that overproducing GcvB restored the ability of the *gcvB* mutant to survive at low pH (Fig 1C). These observations together with the knockout data revealed that GcvB acts as a positive regulator of AR.

**The known GcvB target OppA plays a minor, if any, role in GcvB-mediated AR**

GcvB has been demonstrated to repress the translation of *oppA* and *dppA* in *E. coli* by masking the ribosome binding sites of their mRNAs, which encode periplasmic substrate-binding proteins of ABC uptake systems for amino acids and peptides [13-15]. To determine whether these two proteins played a role in GcvB-mediated AR, we individually deleted their encoding genes, and examined the ability of the resultant mutants to survive at low pH. We found that deleting *oppA* slightly increased AR, as revealed by their acid recovery (OD600) and percent acid survival (cfu) levels, while the absence of *dppA* had little effect on AR (Fig. 2A, B). Thus DppA is not involved in GcvB-mediated AR, and OppA plays a minor, if any, role in this process. We reasoned that the GcvB sncRNA may be exerting its effects on AR via a previously unidentified molecular target.

**GcvB mediates AR by upregulating RpoS expression**

RpoS is the central regulator of glutamate-dependent AR2 genes such as *gadA*, *gadBC* [1], *gadX* [16], *gadE* [17], and *gadY* [6,7]. It is also required for the AR1 system [1,18]. We therefore set out to determine whether GcvB regulated AR by modulating RpoS expression levels. We found that deleting *gcvB* did not lead to a further reduction in acid survival levels in an *rpoS* mutant background (Fig. 3A). More importantly, the absence of *rpoS* completely abolished AR elicited by the plasmid-encoded GcvB (Fig. 3B), suggesting that GcvB-mediated AR is dependent on RpoS. Next, an *rpoS-lacZ* translational fusion was constructed to examine whether the presence or absence of the GcvB sncRNA significantly affected *rpoS* expression levels. Deleting *gcvB* reduced the expression levels of the *rpoS-lacZ* fusion by 50% (Fig. 4A), whereas *gcvB* overexpression increased it 3-fold (Fig. 4B). Furthermore, deleting *oppA*,

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 2**
**Effects of individually deleting oppA and dppA on acid resistance in Escherichia coli.** The acid-stress survival abilities of two individual deletion mutant strains lacking previously identified targets of GcvB were compared with those of the MG1655 wild type (wt) strain. The abilities of strains to survive a 30 minute acid shock were quantified by two methods: (A) acid recovery measured as optical density at 600 nm (OD600). (B) Percent survival determined by colony forming units (cfu). Error bars represent standard deviation (*P*-value < 0.05).</figcaption></figure>

whose absence had previously been shown to slightly increase AR levels, had no effect on *rpoS-lacZ* expression (Fig. 4C). This indicates that the GcvB-mediated regulation of RpoS expression does not involve OppA.

We then investigated whether GcvB indirectly controls RpoS production via an upstream regulator. In the literature, there are numerous reports outlining mechanisms by which the synthesis of RpoS is regulated at the transcriptional [19-22], translational and posttranslational levels [23-25]. Of these regulators of RpoS expression, Crp [20], H-NS [23], GadX [22], and a hybrid sensor kinase called BarA [21] have previously been associated with AR. Although BarA acts as a positive regulator of *rpoS* transcription, its effect is restricted to the exponential phase. It
observed during the exponential growth phase (data not shown), suggesting that GcvB exerts constant AR regulatory functions throughout the various growth phases. We also individually deleted *gcvA* and *ygdl* which are the two genes immediately adjacent to *gcvB*. Both mutants displayed comparable AR as the wild type (Fig. 1A). This shows that the observed acid vulnerability in the *gcvB* mutant is not due to the disruption of the activities of its adjacent genes.

Next, we cloned the gene encoding GcvB (together with its native promoter) into plasmid pET32a (+) and overexpressed the sncRNA in the *E. coli* MG1655 *gcvB* null mutant. The *gcvB* mutant carrying an empty vector served as a negative control. We found that overproducing GcvB restored the ability of the *gcvB* mutant to survive at low pH (Fig 1C). These observations together with the knockout data revealed that GcvB acts as a positive regulator of AR.

**The known GcvB target OppA plays a minor, if any, role in GcvB-mediated AR**

GcvB has been demonstrated to repress the translation of *oppA* and *dppA* in *E. coli* by masking the ribosome binding sites of their mRNAs, which encode periplasmic substrate-binding proteins of ABC uptake systems for amino acids and peptides [13-15]. To determine whether these two proteins played a role in GcvB-mediated AR, we individually deleted their encoding genes, and examined the ability of the resultant mutants to survive at low pH. We found that deleting *oppA* slightly increased AR, as revealed by their acid recovery (OD600) and percent acid survival (cfu) levels, while the absence of *dppA* had little effect on AR (Fig. 2A, B). Thus DppA is not involved in GcvB-mediated AR, and OppA plays a minor, if any, role in this process. We reasoned that the GcvB sncRNA may be exerting its effects on AR via a previously unidentified molecular target.

**GcvB mediates AR by upregulating RpoS expression**

RpoS is the central regulator of glutamate-dependent AR2 genes such as *gadA*, *gadBC* [1], *gadX* [16], *gadE* [17], and *gadY* [6,7]. It is also required for the AR1 system [1,18]. We therefore set out to determine whether GcvB regulated AR by modulating RpoS expression levels. We found that deleting *gcvB* did not lead to a further reduction in acid survival levels in an *rpoS* mutant background (Fig. 3A). More importantly, the absence of *rpoS* completely abolished AR elicited by the plasmid-encoded GcvB (Fig. 3B), suggesting that GcvB-mediated AR is dependent on RpoS. Next, an *rpoS-lacZ* translational fusion was constructed to examine whether the presence or absence of the GcvB sncRNA significantly affected *rpoS* expression levels. Deleting *gcvB* reduced the expression levels of the *rpoS-lacZ* fusion by 50% (Fig. 4A), whereas *gcvB* overexpression increased it 3-fold (Fig. 4B). Furthermore, deleting *oppA*,

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 2**
**Effects of individually deleting oppA and dppA on acid resistance in Escherichia coli.** The acid-stress survival abilities of two individual deletion mutant strains lacking previously identified targets of GcvB were compared with those of the MG1655 wild type (wt) strain. The abilities of strains to survive a 30 minute acid shock were quantified by two methods: (A) acid recovery measured as optical density at 600 nm (OD600). (B) Percent survival determined by colony forming units (cfu). Error bars represent standard deviation (*P*-value < 0.05).</figcaption></figure>

whose absence had previously been shown to slightly increase AR levels, had no effect on *rpoS-lacZ* expression (Fig. 4C). This indicates that the GcvB-mediated regulation of RpoS expression does not involve OppA.

We then investigated whether GcvB indirectly controls RpoS production via an upstream regulator. In the literature, there are numerous reports outlining mechanisms by which the synthesis of RpoS is regulated at the transcriptional [19-22], translational and posttranslational levels [23-25]. Of these regulators of RpoS expression, Crp [20], H-NS [23], GadX [22], and a hybrid sensor kinase called BarA [21] have previously been associated with AR. Although BarA acts as a positive regulator of *rpoS* transcription, its effect is restricted to the exponential phase. It
<figure><img src="image_7.png" /></figure>

**Figure 3**
Effects of deleting rpoS on GcvB-mediated acid resistance. (A) Acid resistance in an rpoS mutant and an rpoS gcvB double mutant. (B) Effects of GcvB overproduction on acid resistance in an rpoS mutant. Cell cultures of E. coli MG1655 mutant strains were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min. After neutralization by adding alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (*P*-value < 0.05).

is therefore unlikely that BarA is a potential target for GcvB. Consequently, we examined whether GcvB exerts its positive control of RpoS through Crp, H-NS or GadX. We found that deleting *gcvB* reduced acid survival levels in an *hns* mutant background. This was also the case for *crp* or *gadX*-deficient strains (Fig. 5). Consistent with the knockout data, *gcvB* overexpression increased AR levels in the *hns-*, *crp-* or *gadX*-deficient strains (Fig. 6). These results suggest that Hns, Crp and GadX are not involved in the GcvB-mediated regulation of *rpoS* expression.

*rpoS* transcription is driven by a major promoter *rpoSp* and two weak promoters *nlpDp1* and 2 [26]. To examine whether GcvB affected *rpoS* expression at the transcrip-

<figure><img src="image_8.png" /></figure>

**Figure 4**
Effects of GcvB sncRNA and OppA protein on the expression of RpoS in a chromosomal *rpoS-lacZ* reporter strain. (A) Effects of deleting *gcvB* on the expression of an *rpoS-lacZ* reporter gene fusion in *E. coli* MG1655. (B) Effect of GcvB overproduction on the expression of the *rpoS-lacZ* fusion. GcvB production was driven by its native promoter on a pET32a plasmid construct. (C) Effect of deleting *oppA* on the expression of the *rpoS-lacZ* reporter. Beta-galactosidase activities due to translation of chromosomal *lacZ* fusions were normalized to those of MG1655 carrying an *rpoS-lacZ* fusion with or without an empty vector (controls). Error bars represent standard deviation (*P*-value < 0.05).
<figure><img src="image_7.png" /><figcaption>**Figure 3**
Effects of deleting rpoS on GcvB-mediated acid resistance. (A) Acid resistance in an rpoS mutant and an rpoS gcvB double mutant. (B) Effects of GcvB overproduction on acid resistance in an rpoS mutant. Cell cultures of E. coli MG1655 mutant strains were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min. After neutralization by adding alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (*P*-value < 0.05).</figcaption></figure>

is therefore unlikely that BarA is a potential target for GcvB. Consequently, we examined whether GcvB exerts its positive control of RpoS through Crp, H-NS or GadX. We found that deleting *gcvB* reduced acid survival levels in an *hns* mutant background. This was also the case for *crp* or *gadX*-deficient strains (Fig. 5). Consistent with the knockout data, *gcvB* overexpression increased AR levels in the *hns-*, *crp-* or *gadX*-deficient strains (Fig. 6). These results suggest that Hns, Crp and GadX are not involved in the GcvB-mediated regulation of *rpoS* expression.

*rpoS* transcription is driven by a major promoter *rpoSp* and two weak promoters *nlpDp1* and 2 [26]. To examine whether GcvB affected *rpoS* expression at the transcrip-

<figure><img src="image_8.png" /><figcaption>**Figure 4**
Effects of GcvB sncRNA and OppA protein on the expression of RpoS in a chromosomal *rpoS-lacZ* reporter strain. (A) Effects of deleting *gcvB* on the expression of an *rpoS-lacZ* reporter gene fusion in *E. coli* MG1655. (B) Effect of GcvB overproduction on the expression of the *rpoS-lacZ* fusion. GcvB production was driven by its native promoter on a pET32a plasmid construct. (C) Effect of deleting *oppA* on the expression of the *rpoS-lacZ* reporter. Beta-galactosidase activities due to translation of chromosomal *lacZ* fusions were normalized to those of MG1655 carrying an *rpoS-lacZ* fusion with or without an empty vector (controls). Error bars represent standard deviation (*P*-value < 0.05).</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 5**
**Effects of deleting gcvB on acid resistance in mutants lacking hns, crp or gadX.** Cell cultures of *E. coli* MG1655 mutant strains containing single deletions of the *hns*, *crp* or *gadX* genes were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min (*gadX* mutant) or 30 min (*hns* and *crp* mutants). After neutralization by adding alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (*P*-value < 0.05).</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 6**
**Effects of GcvB overexpression on acid resistance in mutants lacking hns, crp, gadX or native promoters of rpoS.** The mutant lacking the native promoters of *rpoS* was named *d*<sub>567-1342</sub>. Cell cultures of *E. coli* MG1655 strains containing single deletions were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min (*gadX* mutant) or 30 min (*hns* and *crp* mutants and *d*<sub>567-1342</sub>). After neutralization by alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (*P*-value < 0.05).</figcaption></figure>

tional level, we deleted the region spanning the *rpoSp*, *nlpDp1* and 2 promoter sites (Fig. 4). As removing these promoters will eliminate *rpoS* expression, a constitutive promoter encoded in the knockout cassette (described in Materials and Methods) was used to drive *rpoS* transcription in the absence of *rpoSp*, *nlpDp1* and 2. The resulting mutant was referred to as *d*<sub>567-1342</sub>. As shown in Fig. 6, replacing the native promoters of *rpoS* with this heterologous promoter had little effect on GcvB-induced AR. These results indicate that GcvB does not regulate *rpoS* expression at the transcriptional level.

## Discussion

In this study, we constructed a library comprising single-gene knockout mutants of seventy-nine sncRNA genes in *E. coli*. We used this mutant collection to identify sncRNAs involved in acid resistance (AR) within this bacterium. Consequently, we were able to identify the sncRNA GcvB as a novel regulator of AR.

DppA and OppA, which were previously identified as targets of GcvB, were shown not to be responsible for GcvB-mediated AR. While the *dppA* deletion did not affect AR, the absence of OppA slightly improved the ability of *E. coli* to survive under extreme acid conditions. Since GcvB-mediated inhibition of *oppA* and *dppA* only weakly increased AR, we reasoned that the GcvB sncRNA may be regulating AR via some other mechanistic pathway.

Upon *rpoS* deletion, GcvB had no influence on AR. Furthermore, we found that GcvB positively regulated *rpoS* expression. Deleting *oppA* had no effect on the levels of RpoS, providing evidence that the GcvB-mediated control of RpoS does not involve the OppA protein. Also, GcvB does not regulate RpoS via H-NS, Crp, or GadX; as GcvB-mediated AR was unaffected when these genes were singularly deleted. The region spanning from -567 to -1342 nt harbors all three of the *rpoS* promoters (*rpoSp*, *nlpDp1* and 2). Replacing these native promoters with the constitutive promoter from a Cm knockout cassette had no affect on GcvB-mediated AR. This suggests that GcvB does not regulate *rpoS* expression at the transcriptional level.

Like GcvB, the sncRNAs DsrA and RprA also act as positive regulators of AR in *E. coli*. DsrA and RprA bind to the same region of the 5' leader of *rpoS* mRNA. A sequence within this region was previously predicted to form a self-inhibitory stem loop structure that probably occludes the Shine-Dalgarno ribosome binding site and prevents translation [24,27]. DsrA [24,27] and RprA [28,29] bind to this stem-loop structure, free the ribosome binding site, and thereby mediate *rpoS* translation. This region also contains RNase III cleavage sites that result in rapid decay of the *rpoS* transcript [30]. The base-pairing of DsrA within this region stabilizes *rpoS* mRNA by creating an alternative RNase III cleavage site [30]. As for GcvB, it regulates *rpoS* expression
<figure><img src="image_9.png" /><figcaption>**Figure 5**
Effects of deleting gcvB on acid resistance in mutants lacking *hns*, *crp* or *gadX*. Cell cultures of *E. coli* MG1655 mutant strains containing single deletions of the *hns*, *crp* or *gadX* genes were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min (*gadX* mutant) or 30 min (*hns* and *crp* mutants). After neutralization by adding alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (*P*-value < 0.05).</figcaption></figure>

tional level, we deleted the region spanning the *rpoSp*, *nlpDp1* and 2 promoter sites (Fig. 4). As removing these promoters will eliminate *rpoS* expression, a constitutive promoter encoded in the knockout cassette (described in Materials and Methods) was used to drive *rpoS* transcription in the absence of *rpoSp*, *nlpDp1* and 2. The resulting mutant was referred to as *d567-1342*. As shown in Fig. 6, replacing the native promoters of *rpoS* with this heterologous promoter had little effect on GcvB-induced AR. These results indicate that GcvB does not regulate *rpoS* expression at the transcriptional level.

## Discussion

In this study, we constructed a library comprising single-gene knockout mutants of seventy-nine sncRNA genes in *E. coli*. We used this mutant collection to identify sncRNAs involved in acid resistance (AR) within this bacterium. Consequently, we were able to identify the sncRNA GcvB as a novel regulator of AR.

DppA and OppA, which were previously identified as targets of GcvB, were shown not to be responsible for GcvB-mediated AR. While the *dppA* deletion did not affect AR, the absence of OppA slightly improved the ability of *E. coli* to survive under extreme acid conditions. Since GcvB-mediated inhibition of *oppA* and *dppA* only weakly increased AR, we reasoned that the GcvB sncRNA may be regulating AR via some other mechanistic pathway.

<figure><img src="image_10.png" /><figcaption>**Figure 6**
Effects of GcvB overexpression on acid resistance in mutants lacking *hns*, *crp*, *gadX* or native promoters of *rpoS*. The mutant lacking the native promoters of *rpoS* was named *d567-1342*. Cell cultures of *E. coli* MG1655 strains containing single deletions were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min (*gadX* mutant) or 30 min (*hns* and *crp* mutants and *d567-1342*). After neutralization by alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (*P*-value < 0.05).</figcaption></figure>

Upon *rpoS* deletion, GcvB had no influence on AR. Furthermore, we found that GcvB positively regulated *rpoS* expression. Deleting *oppA* had no effect on the levels of RpoS, providing evidence that the GcvB-mediated control of RpoS does not involve the OppA protein. Also, GcvB does not regulate RpoS via H-NS, Crp, or GadX; as GcvB-mediated AR was unaffected when these genes were singularly deleted. The region spanning from -567 to -1342 nt harbors all three of the *rpoS* promoters (*rpoSp*, *nlpDp1* and 2). Replacing these native promoters with the constitutive promoter from a Cm knockout cassette had no affect on GcvB-mediated AR. This suggests that GcvB does not regulate *rpoS* expression at the transcriptional level.

Like GcvB, the sncRNAs DsrA and RprA also act as positive regulators of AR in *E. coli*. DsrA and RprA bind to the same region of the 5' leader of *rpoS* mRNA. A sequence within this region was previously predicted to form a self-inhibitory stem loop structure that probably occludes the Shine-Dalgarno ribosome binding site and prevents translation [24,27]. DsrA [24,27] and RprA [28,29] bind to this stem-loop structure, free the ribosome binding site, and thereby mediate *rpoS* translation. This region also contains RNase III cleavage sites that result in rapid decay of the *rpoS* transcript [30]. The base-pairing of DsrA within this region stabilizes *rpoS* mRNA by creating an alternative RNase III cleavage site [30]. As for GcvB, it regulates *rpoS* expression
by an unknown mechanism. Computational sequence
analyses using the RNAhybrid server [31] have indicated
that GcvB does not contain any extensive regions of
sequence complementary to the 5' leader of rpoS mRNA
(data not shown). As such, the mechanism by which GcvB
regulates rpoS expression remains a subject of further
investigation.

Conclusion

Here we reveal that the sncRNA GcvB positively regulates
the ability of *E. coli* to survive low pH conditions by upreg-
ulating the expression of RpoS. Our findings provide
insight into the control of AR by GcvB. However, further
detailed studies will be required to investigate the precise
nature of the GcvB/rpoS interaction at the molecular level.

**Methods**

Bacterial strains, plasmids, and growth conditions

E. coli K12-MG1655 was used for all gene deletion and
overexpression experiments. The *E. coli* expression vector
pET32a (+) (Novagen, Madison, WI) was used for the
overexpression of sncRNAs. All bacterial strains were
grown at 37°C, with shaking at 230 rpm, in Luria-Bertani
(LB) medium supplemented with antibiotics when
required. Bacterial growth was monitored by measuring
optical density at 600 nm (OD600). The antibiotics amp-
icillin (50 ug/ml), kanamycin (50 ug/ml), and chloram-
phenicol (12.5 ug/ml) were used for selection.

Gene deletions

Gene deletion mutations were constructed using the λ-
Red recombination system. *E. coli* MG1655 was trans-
formed with plasmid pSim6 (a gift from Dr. Donald
Court) [32] on which the expression of the λ recombina-
tion proteins is induced at 42°C. PCR fragments encom-
passing a loxP-cm-loxP (‘floxed’) chloramphenicol
resistance cassette with homology (45 nt) to the regions
immediately flanking each deletion locus were trans-
formed into MG1655 harboring pSim6. After induction of
λred, recombinants were selected for chloramphenicol
(cm) resistance, and were further verified by colony PCR.

Gene cloning

The selectable loxP-cm-loxP cassette was first inserted
immediately after the stop codon of the gene of interest
on the chromosome (as described above). We then used
recombineering to 'clone' this gene of interest preceded by
its native promoter and 'floxed' cm cassette downstream
of the T7 promoter in a pET32a expression vector. Specif-
ically, we PCR amplified the gene of interest (including its
native promoter) and adjacent 'floxed' cm cassette using
primers that contained homology (45 nt) to the plasmid
insertion site. The PCR product and expression vector
were co-transformed into DY330[12] after induction of
λred. Recombinants were selected for chloramphenicol

resistance and verified by PCR and sequencing. The native
promoter drove the gene overexpression in MG1655 with-
out induction.

Construction of chromosomal lacZ translational fusions
and beta-galactosidase assays

First, the loxP-cm-loxP selectable cassette was inserted immediately after the stop codon of the *lacZ* gene on the MG1655 chromosome using *λred* recombination (as described above). Next, the *lacZ*-loxP-cm-loxP cassette was PCR amplified and inserted (in frame) immediately prior to the stop codon of the target gene on the chromosome. The inserted *lacZ* fragment started from the 8th codon of *lacZ* gene and co-transcribed and -translated with the fused genes. The expression of gene-*lacZ* fusions was quantified using a beta-galactosidase assay kit from Thermo Fisher Scientific (Rockford, Illinois, USA).

Acid resistance assay

Overnight cultures were diluted 1:50 and incubated at
37°C for 5 hrs in LB medium. 3 volumes of acidified LB
medium (pH 1.9) was then added to 1 volume of the cell
culture so that the final pH value was approximately 2.0.
The acid challenge was maintained for 15 min for cells
that were acid sensitive, and 30 min for acid-resistant
cells. The acid challenge was stopped by adding 3 volumes
of alkalinized LB medium (pH9.3), making the final pH
value approximately 7.0. For samples that displayed sim-
ilar growth rates, the neutralized cell cultures were
allowed to grow for 3 hrs following the acid challenge,
and acid survival levels were determined by measurement
of optical density at 600 nm (OD600, termed acid recov-
ery). When samples with different growth rates were
examined, cell viability was determined by plate counts
and percentage acid survival was calculated as the number
of number of colony forming units (cfu) remaining after
the acid treatment divided by the initial cfu at time zero.

Statistical analysis

Two-tailed paired t-tests were used for the comparison of
means obtained from the beta-galactosidase and acid sur-
vival assays. *P* values of < 0.05 were considered statisti-
cally significant.

Abbreviations

AR: acid resistance; sncRNA: small noncoding RNA; UTR: untranslated region.

Authors' contributions

YJ planned and designed the experiments, performed the
experiments and the data analysis, wrote the main draft of
the paper, and generated the figures. RW conceived of the
study and helped to draft the manuscript. AD aided in the
experimental design and in editing the manuscript. JH
provided guidance over the entire project, and aided in
by an unknown mechanism. Computational sequence
analyses using the RNAhybrid server [31] have indicated
that GcvB does not contain any extensive regions of
sequence complementary to the 5' leader of rpoS mRNA
(data not shown). As such, the mechanism by which GcvB
regulates rpoS expression remains a subject of further
investigation.

Conclusion

Here we reveal that the sncRNA GcvB positively regulates
the ability of *E. coli* to survive low pH conditions by upreg-
ulating the expression of RpoS. Our findings provide
insight into the control of AR by GcvB. However, further
detailed studies will be required to investigate the precise
nature of the GcvB/rpoS interaction at the molecular level.

**Methods**

Bacterial strains, plasmids, and growth conditions

E. coli K12-MG1655 was used for all gene deletion and
overexpression experiments. The *E. coli* expression vector
pET32a (+) (Novagen, Madison, WI) was used for the
overexpression of sncRNAs. All bacterial strains were
grown at 37°C, with shaking at 230 rpm, in Luria-Bertani
(LB) medium supplemented with antibiotics when
required. Bacterial growth was monitored by measuring
optical density at 600 nm (OD600). The antibiotics amp-
icillin (50 ug/ml), kanamycin (50 ug/ml), and chloram-
phenicol (12.5 ug/ml) were used for selection.

Gene deletions

Gene deletion mutations were constructed using the λ-
Red recombination system. *E. coli* MG1655 was trans-
formed with plasmid pSim6 (a gift from Dr. Donald
Court) [32] on which the expression of the λ recombina-
tion proteins is induced at 42°C. PCR fragments encom-
passing a loxP-cm-loxP (‘floxed’) chloramphenicol
resistance cassette with homology (45 nt) to the regions
immediately flanking each deletion locus were trans-
formed into MG1655 harboring pSim6. After induction of
λred, recombinants were selected for chloramphenicol
(cm) resistance, and were further verified by colony PCR.

Gene cloning

The selectable loxP-cm-loxP cassette was first inserted
immediately after the stop codon of the gene of interest
on the chromosome (as described above). We then used
recombineering to 'clone' this gene of interest preceded by
its native promoter and 'floxed' cm cassette downstream
of the T7 promoter in a pET32a expression vector. Specif-
ically, we PCR amplified the gene of interest (including its
native promoter) and adjacent 'floxed' cm cassette using
primers that contained homology (45 nt) to the plasmid
insertion site. The PCR product and expression vector
were co-transformed into DY330[12] after induction of
λred. Recombinants were selected for chloramphenicol

resistance and verified by PCR and sequencing. The native
promoter drove the gene overexpression in MG1655 with-
out induction.

Construction of chromosomal lacZ translational fusions
and beta-galactosidase assays

First, the loxP-cm-loxP selectable cassette was inserted immediately after the stop codon of the *lacZ* gene on the MG1655 chromosome using *λred* recombination (as described above). Next, the *lacZ*-loxP-cm-loxP cassette was PCR amplified and inserted (in frame) immediately prior to the stop codon of the target gene on the chromosome. The inserted *lacZ* fragment started from the 8th codon of *lacZ* gene and co-transcribed and -translated with the fused genes. The expression of gene-*lacZ* fusions was quantified using a beta-galactosidase assay kit from Thermo Fisher Scientific (Rockford, Illinois, USA).

Acid resistance assay

Overnight cultures were diluted 1:50 and incubated at
37°C for 5 hrs in LB medium. 3 volumes of acidified LB
medium (pH 1.9) was then added to 1 volume of the cell
culture so that the final pH value was approximately 2.0.
The acid challenge was maintained for 15 min for cells
that were acid sensitive, and 30 min for acid-resistant
cells. The acid challenge was stopped by adding 3 volumes
of alkalinized LB medium (pH9.3), making the final pH
value approximately 7.0. For samples that displayed sim-
ilar growth rates, the neutralized cell cultures were
allowed to grow for 3 hrs following the acid challenge,
and acid survival levels were determined by measurement
of optical density at 600 nm (OD600, termed acid recov-
ery). When samples with different growth rates were
examined, cell viability was determined by plate counts
and percentage acid survival was calculated as the number
of number of colony forming units (cfu) remaining after
the acid treatment divided by the initial cfu at time zero.

Statistical analysis

Two-tailed paired t-tests were used for the comparison of
means obtained from the beta-galactosidase and acid sur-
vival assays. *P* values of *< 0.05* were considered statisti-
cally significant.

Abbreviations

AR: acid resistance; sncRNA: small noncoding RNA; UTR: untranslated region.

Authors' contributions

YJ planned and designed the experiments, performed the
experiments and the data analysis, wrote the main draft of
the paper, and generated the figures. RW conceived of the
study and helped to draft the manuscript. AD aided in the
experimental design and in editing the manuscript. JH
provided guidance over the entire project, and aided in
data analysis and editing of the manuscript. All authors
read and approved the final manuscript.

**Additional material**

Additional file 1

Small noncoding RNAs (sncRNA) that were selected for construction of a single-sncRNA gene knockout library within E. coli K12-MG1655. This table summarizes positions and functions of the small non-coding RNAs that were included in a single-sncRNA gene knockout library within E. coli K12-MG1655.

Click here for file

[http://www.biomedcentral.com/content/supplementary/1471-2164-10-165-S1.doc]

Acknowledgements

We thank D.L. Court for providing plasmid pSim6 for the recombinering experiments. This work was supported by HKU CRCG Seed Funding Programme for Basic Research and by a grant (HKU 7485/06M) from the Research Grant Council of Hong Kong to JDH. AD acknowledges support from the PROBACTYS programme, grant CT-2006-029104 in an effort to define genes essential for the construction of a synthetic cell.

References

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2. Hersh BM, Farooq FT, Barstad DN, Blankenhorn DL, Slonczewski JL: *A glutamate-dependent acid resistance gene in Escherichia coli*. *J Bacteriol* 1996, **178**(13):3978-3981.

3. Lin J, Smith MP, Chapin KC, Baik HS, Bennett GN, Foster JW: *Mechanisms of acid resistance in enterohemorrhagic Escherichia coli*. *Appl Environ Microbiol* 1996, **62**(9):3094-3100.

4. Sledjeski DD, Gupta A, Gottesman S: The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli. EMBO J 1996, 15(15):3993-4000.

5. Lease RA, Smith D, McDonough K, Belfort M: The small noncoding DsrA RNA is an acid resistance regulator in Escherichia coli. J Bacteriol 2004, 186(18):6179-6185.

6. Opdyke JA, Kang JG, Storz G: GadY, a small-RNA regulator of acid response genes in Escherichia coli. J Bacteriol 2004, 186(20):6698-6705.

7. Tramonti A, De Canio M, De Biase D: GadX/GadW-dependent regulation of the Escherichia coli acid fitness island: transcriptional control at the gadY-gadW divergent promoters and identification of four novel 42-bp GadX/GadW-specific binding sites. *Mol Microbiol* 2008, **70**(4):965-982.

8. Griffiths-Jones S, Moxon S, Marshall M, Khanna A, Eddy SR, Bateman A: *Rfam: annotating non-coding RNAs in complete genomes*. *Nucleic Acids Res* 2005;D121-124.

9. Vallenet D, Labarre L, Rouy Z, Barbe V, Bocs S, Cruveiller S, Lajus A, Pascal G, Scarpelli C, Medigue C: MaGe: a microbial genome annotation system supported by synteny results. *Nucleic Acids Res* 2006, **34**(1):53-65.

10. Vogel J, Bartels V, Tang TH, Churakov G, Slagter-Jager JG, Huttenhofer A, Wagner EG: RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria. *Nucleic Acids Res* 2003, **31**(22):6435-6443.

11. Ellis HM, Yu D, DiTizio T, Court DL: High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci USA 2001, **98**(12):6742-6746.

12. Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL: An efficient recombination system for chromosome engineer-

ing in Escherichia coli. Proc Natl Acad Sci USA 2000, 97(11):5978-5983.

13. Urbanowski ML, Stauffer LT, Stauffer GV: The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli. Mol Microbiol 2000, 37(4):856-868.

14. Urban JH, Vogel J: Translational control and target recognition by Escherichia coli small RNAs in vivo. *Nucleic Acids Res* 2007, **35**(3):1018-1037.

15. Sharma CM, Darfeuille F, Plantinga TH, Vogel J: A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites. *Genes Dev* 2007, **21**(21):2804-2817.

16. Tramonti A, Visca P, De Canio M, Falconi M, De Biase D: Functional characterization and regulation of gadX, a gene encoding an AraC/XylS-like transcriptional activator of the Escherichia coli glutamic acid decarboxylase system. J Bacteriol 2002, 184(10):2603-2613.

17. Sayed AK, Odom C, Foster JW: The Escherichia coli AraC-fam-
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activator of glutamate-dependent acid resistance. Microbiol-
ogy 2007, 153(Pt 8):2584-2592.

18. Small P, Blankenhorn D, Welty D, Zinser E, Slonczewski JL: Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH. *J Bacteriol* 1994, **176**(6):1729-1737.

19. Mika F, Hengge R: A two-component phosphotransfer network involving ArcB, ArcA, and RssB coordinates synthesis and proteolysis of sigmaS (RpoS) in E. coli. *Genes Dev* 2005, 19(22):2770-2781.

20. Lange R, Hengge-Aronis R: The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability. *Genes Dev* 1994, **8**(13):1600-1612.

21. Mukhopadhyay S, Audia JP, Roy RN, Schellhorn HE: Transcriptional induction of the conserved alternative sigma factor RpoS in Escherichia coli is dependent on BarA, a probable two-component regulator. Mol Microbiol 2000, 37(2):371-381.

22. Hommais F, Krin E, Coppee JY, Lacroix C, Yeramian E, Danchin A, Bertin P: GadE (YhiE): a novel activator involved in the response to acid environment in Escherichia coli. Microbiology 2004, 150(Pt 1):61-72.

23. Brescia CC, Kaw MK, Sledjeski DD: The DNA binding protein HNS binds to and alters the stability of RNA in vitro and in vivo. J Mol Biol 2004, 339(3):505-514.

24. Lease RA, Cusick ME, Belfort M: Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci. Proc Natl Acad Sci USA 1998, 95(21):12456-12461.

25. Zhang A, Altuvia S, Tiwari A, Argaman L, Hengge-Aronis R, Storz G: The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein. EMBO J 1998, 17(20):6061-6068.

26. Lange R, Fischer D, Hengge-Aronis R: Identification of transcriptional start sites and the role of ppGpp in the expression of rpoS, the structural gene for the sigma S subunit of RNA polymerase in Escherichia coli. J Bacteriol 1995, 177(16):4676-4680.

27. Majdalani N, Cunning C, Sledjeski D, Elliott T, Gottesman S: DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription. Proc Natl Acad Sci USA 1998, 95(21):12462-12467.

28. Majdalani N, Hernandez D, Gottesman S: Regulation and mode of action of the second small RNA activator of RpoS translation, RprA. Mol Microbiol 2002, 46(3):813-826.

29. Updegrove T, Wilf N, Sun X, Wartell RM: Effect of Hfq on RprA-rpoS mRNA pairing: Hfq-RNA binding and the influence of the 5' rpoS mRNA leader region. Biochemistry 2008, 47(43):11184-11195.

30. Resch A, Afonyushkin T, Lombo TB, McDowall KJ, Blasi U, Kaberdin VR: Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5'-leader. RNA 2008, 14(3):454-459.

31. Kruger J, Rehmsmeier M: RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Res 2006;W451-454.

32. Datta S, Costantino N, Court DL: A set of recombineering plasmids for gram-negative bacteria. Gene 2006, 379:109-115.
data analysis and editing of the manuscript. All authors
read and approved the final manuscript.

**Additional material**

Additional file 1

Small noncoding RNAs (sncRNA) that were selected for construction of a single-sncRNA gene knockout library within E. coli K12-MG1655. This table summarizes positions and functions of the small non-coding RNAs that were included in a single-sncRNA gene knockout library within E. coli K12-MG1655.

Click here for file

[http://www.biomedcentral.com/content/supplementary/1471-2164-10-165-S1.doc]

Acknowledgements

We thank D.L. Court for providing plasmid pSim6 for the recombinering experiments. This work was supported by HKU CRCG Seed Funding Programme for Basic Research and by a grant (HKU 7485/06M) from the Research Grant Council of Hong Kong to JDH. AD acknowledges support from the PROBACTYS programme, grant CT-2006-029104 in an effort to define genes essential for the construction of a synthetic cell.

References

1. Castanie-Cornet MP, Penfound TA, Smith D, Elliott JF, Foster JW: *Control of acid resistance in Escherichia coli*. *J Bacteriol* 1999, **181**(11):3525-3535.

2. Hersh BM, Farooq FT, Barstad DN, Blankenhorn DL, Slonczewski JL: *A glutamate-dependent acid resistance gene in Escherichia coli*. *J Bacteriol* 1996, **178**(13):3978-3981.

3. Lin J, Smith MP, Chapin KC, Baik HS, Bennett GN, Foster JW: *Mechanisms of acid resistance in enterohemorrhagic Escherichia coli*. *Appl Environ Microbiol* 1996, **62**(9):3094-3100.

4. Sledjeski DD, Gupta A, Gottesman S: The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli. EMBO J 1996, 15(15):3993-4000.

5. Lease RA, Smith D, McDonough K, Belfort M: The small noncoding DsrA RNA is an acid resistance regulator in Escherichia coli. J Bacteriol 2004, 186(18):6179-6185.

6. Opdyke JA, Kang JG, Storz G: GadY, a small-RNA regulator of acid response genes in Escherichia coli. J Bacteriol 2004, 186(20):6698-6705.

7. Tramonti A, De Canio M, De Biase D: GadX/GadW-dependent regulation of the Escherichia coli acid fitness island: transcriptional control at the gadY-gadW divergent promoters and identification of four novel 42-bp GadX/GadW-specific binding sites. *Mol Microbiol* 2008, **70**(4):965-982.

8. Griffiths-Jones S, Moxon S, Marshall M, Khanna A, Eddy SR, Bateman A: *Rfam: annotating non-coding RNAs in complete genomes*. *Nucleic Acids Res* 2005;D121-124.

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