The Vibrio cholerae virulence regulatory cascade controls glucose uptake through activation of TarA, a small regulatory RNA - PubMed (original) (raw)

The Vibrio cholerae virulence regulatory cascade controls glucose uptake through activation of TarA, a small regulatory RNA

Aimee L Richard et al. Mol Microbiol. 2010 Dec.

Abstract

Vibrio cholerae causes the severe diarrhoeal disease cholera. A cascade of regulators controls expression of virulence determinants in V. cholerae at both transcriptional and post-transcriptional levels. ToxT is the direct transcription activator of the major virulence genes in V. cholerae. Here we describe TarA, a highly conserved, small regulatory RNA, whose transcription is activated by ToxT from toxboxes present upstream of the ToxT-activated gene tcpI. TarA regulates ptsG, encoding a major glucose transporter in V. cholerae. Cells overexpressing TarA exhibit decreased steady-state levels of ptsG mRNA and grow poorly in glucose-minimal media. A mutant lacking the ubiquitous regulatory protein Hfq expresses diminished TarA levels, indicating that TarA likely interacts with Hfq to regulate gene expression. RNAhybrid analysis of TarA and the putative ptsG mRNA leader suggests potential productive base-pairing between these two RNA molecules. A V. cholerae mutant lacking TarA is compromised for infant mouse colonization in competition with wild type, suggesting a role in the in vivo fitness of V. cholerae. Although somewhat functionally analogous to SgrS of Escherichia coli, TarA does not encode a regulatory peptide, and its expression is activated by the virulence gene pathway in V. cholerae and not by glycolytic intermediates.

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Figures

Figure 1. Schematic and diagram of tcpI promoters and the surrounding region

A.) Schematic. Scale bar: 0.5 centimeters represents 200 bp of DNA. Bent arrows indicate putative transcriptional start sites, and long black arrows represent coding sequences of the known genes tcpP and tcpI. tcpI has been shortened from 1.6 kb for clarity. Toxboxes, or ToxT-recognition sequences, are indicated by paired boxes. B.) Diagram of tcpI distal promoter, tarA, and surrounding sequences. Bent arrows indicate transcriptional start sites for tcpP and tarA. Numbers indicate position relative to tarA transcriptional start site. ToxT binding sites (toxboxes) for the tcpI distal promoter are underlined with horizontal arrows. The −35 and −10 elements for tarA are indicated by thick boxes, and thin boxes represent potential base-pairing regions in putative hairpin structure.

Figure 2. TarA is produced under ToxT-inducing conditions

Wild type and Δ_toxT_ strains were grown under inducing and non-inducing conditions (LB pH 6.5, 30°C and LB pH 8.5, 37°C, respectively) and total RNA was collected at indicated timepoints. Equal amounts of RNA from each sample (as determined by OD260) were subjected to Northern blot analysis, using an oligonucleotide complementary to the putative sRNA sequence as a probe. Methylene blue staining was used to confirm load amounts (data not shown).

Figure 3. Comparison of tarA sequences among V. cholerae strains

Sequences from strains indicated in Table 2 are aligned, with bold letters indicating mutations. The box indicates a conserved potential Hfq binding site.

Figure 4. Confirmation of ptsG regulation by qRT-PCR analysis

Cultures of the listed strains were grown under ToxT-inducing conditions for 7 hours and total RNA was isolated using Trizol. cDNA was prepared from equivalent amounts of RNA for each sample, and cDNA was used for SYBR Green qRT-PCR. Numbers indicate fold change in transcript level between strains, calculated using the ΔΔCT method and recA transcript levels as an internal control.

Figure 5. Overexpression of TarA results in severely reduced glucose uptake

(A.) LB growth. Indicated strains were grown overnight in LB media, subcultured into fresh LB and grown to mid-exponential phase, then washed with PBS and diluted into fresh LB to OD = 0.04. Cultures were grown in a 96-well plate at 30°C with constant aeration, and the OD600 was measured every 20 minutes. Growth assays were performed at least three times, and a representative growth curve is shown. (B.) MOPS/glucose growth. Strains were prepared as in (A), but diluted into MOPS/0.5% glucose instead of LB and then grown as in (A). Growth assays were performed at least three times, and a representative growth curve is shown. (C.) Strains were grown overnight in LB, subcultured into LB and grown to mid-exponential phase, then washed and diluted into MOPS/0.5% glucose, and grown in flasks. At specified timepoints, supernatants were collected and the glucose concentration was tested using a tetrazolium blue reducing sugar assay. Calculated glucose concentrations were divided by the starting concentration to determine the amount of glucose remaining in the media. This experiment was performed three times, and a representative assay is shown. Error bars indicate the standard deviation among triplicate samples in the representative experiment.

Figure 6. Induction of TarA reduces ptsG transcript levels

Strains AR059 and AR060, containing an IPTG-inducible copy of TarA and an empty vector control, respectively, were grown overnight in LB. Cultures were diluted 1:200 into fresh LB, grown until OD ~ 0.4, and stimulated with IPTG. Thirty minutes after induction, cultures were washed and resuspended in fresh media lacking IPTG. RNA was collected at indicated timepoints (from the start of induction) using Trizol, and equal amounts of RNA for each sample were subjected to Northern blot analysis. Methylene blue staining was used to confirm load amounts (not shown). Blots were probed with oligonucleotides complementary to TarA (A) and to the ptsG transcript (B).

Figure 7. Alignment of TarA and ptsG upstream sequences

The program RNAhybrid (

http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/welcome.html

) was used to predict potential base-pairing between TarA and the ptsG leader sequence (defined here as 100 bp upstream of the AUG, including the +1 A). The TarA sequence is shown in green and the ptsG leader sequence in red. The putative Hfq binding site is indicated. The minimum predicted free energy for this alignment is −54.4 kcal/mol.

Figure 8. TarA is unstable in the absence of Hfq

Wild type, Δ_tarA_, and Δ_hfq_ strains were grown under inducing conditions (LB pH 6.5, 30°C) and total RNA was collected after seven hours of growth. Equal amounts of RNA from each sample (as determined by OD260) were subjected to Northern blot analysis, using an oligonucleotide complementary to TarA as a probe. Methylene blue staining was used to confirm load amounts (data not shown).

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The Vibrio cholerae virulence regulatory cascade controls glucose uptake through activation of TarA, a small regulatory RNA - PubMed (original) (raw)