Article Figures & Data
Figures
- FIG 1
Csr system in V. cholerae. (A) The two-component system VarS and VarA regulates the activity of CsrA. The inner membrane protein VarS is a sensor kinase that activates VarA via phosphorylation. Activated VarA binds to the promoters of three small RNAs, namely, CsrB, CsrC, and CsrD (CsrB/C/D, or the Csr sRNAs), to induce their transcription; the portion of the Csr sRNA promoters indicated in green represents the VarA recognition sequence. When expressed, CsrB/C/D act to sequester CsrA, antagonizing the activity of CsrA. The data determined in the present study indicate that CsrA directly binds to varA mRNA to positively regulate protein expression, introducing a regulatory feedback loop in the Var/Csr pathway in V. cholerae. (B) The RNA folding program mFold was used to generate the secondary structures of CsrB/C/D. The GGA CsrA-binding sites are highlighted by the red lettering.
- FIG 2
The Csr sRNA levels, but not the CsrA protein levels, change in response to NRES. (A) The wild-type strain, N16961, harboring a V5 epitope tagged CsrA, was grown in minimal medium with and without NRES, and whole-cell proteins were resolved and immunoblotted with anti-V5 antiserum. (B) The total abundance, shown in arbitrary units (A.U.), of each Csr sRNA was determined by the use of standard curves. #, <1 A.U. of CsrC in minimal medium. The P values were determined by an unpaired, two-tailed Student's t test from the A.U. values (*, P < 0.05; **, P < 0.01). The normalized means and standard deviations (error bars) were calculated from three biological replicates. (C) Relative proportions of CsrB/C/D present when the wild-type strain was grown in the absence and presence of NRES. The amount of Csr sRNA present in the experiment whose results are presented in panel B was converted to a percentage. (D) The rate of decay for CsrB, CsrC, and CsrD was measured by qRT-PCR from wild-type cells grown to mid-log phase in minimal medium supplemented with NRES. Rifampin was added to stop transcription, and cells were harvested immediately after rifampin addition (t = 0) and at 4, 8, 16, and 64 min after rifampin addition. The half-lives, along with the 95% confidence intervals (C.I.), were determined using one-phase decay analysis (a nonlinear regression method) and GraphPad Prism 7 software. The extra sum-of-squares F test was used to compare the decay rate constants determined for the sample sets (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
- FIG 3
NcsrC-only has increased CsrA activity in minimal medium. (A) N16961, NcsrB-only, NcsrC-only, and NcsrD-only were grown in minimal medium with and without NRES. Whole-cell proteins were resolved and immunoblotted with anti-ToxR antiserum (top panel) or stained with Coomassie blue (bottom panel). The proteins in both the immunoblot and Coomassie-stained gel were from the same samples and are representative of results from at least three biological replicates. (B to D) CsrB (B), CsrC (C), and CsrD (D) expression levels were examined by qRT-PCR in strain N16961 and strains NcsrB-only, NcsrC-only, and NcsrD-only, respectively. All strains were grown as described for panel A. The levels of CsrB/C/D were normalized to the internal control secA level. In each panel, the wild-type (WT) level seen in the absence of NRES is set to a value of 1. The P values were determined by an unpaired, two-tailed Student's t test from the ΔCT values (*, P < 0.05), comparing N16961 to the indicated mutant and comparing the levels of growth in minimal medium with or without NRES. Differences that are statistically significant are indicated on the figures. The relative means and standard deviations (error bars) were calculated from three biological replicates.
- FIG 4
CsrA positively affects the expression of the csr sRNAs. CsrB (A), CsrC (B), and CsrD (C) expression levels were examined by qRT-PCR in N16961 (WT), NcsrA.R6H (csrA mutant), and the complemented strain NcsrA.R6H/pFcsrA (csrA mutant/pFcsrA). All strains were grown in minimal medium with and without NRES. The levels of CsrB/C/D were normalized to the internal control secA level. In each panel, the WT level seen in the absence of NRES is set to a value of 1. The P values were determined by an unpaired, two-tailed Student's t test from the ΔCT values (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The relative means and standard deviations (error bars) were calculated from four biological replicates.
- FIG 5
CsrA positively affects the transcription of the csr sRNA genes. The activities of the full-length promoter fusions (A) and the truncated promoter fusions (B) for CsrB, CsrC, and CsrD were measured in the NlacZ::kan (WT) and NcsrA.R6H.lacZ::kan (csrA mutant) strains. The truncated fusions lack 50 nucleotides on the 5′ end encompassing the VarA-binding site, shown as the light blue segment in the schematic. The means are plotted, and the error bars represent standard deviations of data from three biological replicates. The P values were calculated using the unpaired t test (*, P < 0.05; **, P < 0.01).
- FIG 6
CsrA positively affects the level of VarA and binds directly to the varA mRNA. (A) NvarA-V5 (WT), NcsrA.R6H.varA-V5 (csrA mutant), and NcsrA.R6H.varA-V5/pFcsrA (csrA mutant/pFcsrA) were grown in minimal medium supplemented with NRES. Cells were harvested at mid-log phase, and whole-cell proteins were resolved and immunoblotted with anti-V5 antiserum. The immunoblot is representative of results from at least three biological replicates. (B) The RNA EMSA was performed with purified CsrA, the negative-control 5′ biotinylated atpI RNA, and the 5′ biotinylated varA RNA. The highest concentration of CsrA in the gradient was 40 nM, followed by 8 nM and 1.6 nM. (C) The sequence of the varA oligonucleotide used for the RNA EMSA whose results are presented in panel B. The closed arrow is positioned above the annotated start of translation UUG (61), and GGA motifs are indicated in red and underlined. (D) In vivo CsrA-RNA immunoprecipitation showing that the varA transcript was enriched ∼5-fold in the immunoprecipitated (IP) sample compared to the input RNA population. The P values were determined by an unpaired, two-tailed Student's t test from the ΔCT values (**, P < 0.01). (E) The varA transcriptional reporter, pQFvarA.TS, includes the varA promoter sequence (−301 to −1 relative to the transcriptional start site, blue bar) fused to lacZ, while the varA translational fusion, pQEvarA.TL, includes the 5′ UTR and 73 amino acids of the coding sequence (−1 to +312 relative to the start of transcription, orange bar) as indicated in the schematic below the panel. The translational fusion uses the plasmid T5 promoter. The open arrow represents the start of transcription, while the closed arrow indicates the start of translation. The activity of these reporters was measured in the NlacZ::kan (WT) and NcsrA.R6H.lacZ::kan (csrA mutant) strains. IPTG (100 μM) was present in the growth medium during testing of the translational reporter. To generate the plotted means and standard deviations (error bars), three biological replicates were used for the transcriptional reporter, and four biological replicates were used for the translational reporter. The P values were calculated using the unpaired, two-tailed Student's t test (ns, not significant; ***, P < 0.001; bd, below detection).
Supplemental Material
FIG S1
V5-tagged CsrA restores ToxR expression in NcsrA.R6H. N16961, NcsrA.R6H, NcsrA.R6H/pFcsrA, and NcsrA.R6H/pFcsrA-V5 were grown in minimal medium with and without NRES. Cells were harvested at mid-log phase and resuspended in LB sample buffer, and whole-cell proteins were resolved by 10% polyacrylamide SDS-PAGE. The gels were processed and immunoblotted with anti-ToxR antiserum. Download FIG S1, EPS file, 0.1 MB.
Copyright © 2019 Butz et al.
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FIG S2
Verification of priming efficiency in RT and qPCR. (A) Total abundance of each Csr sRNA shown in raw nanograms (ng). To ensure that the RT priming efficiencies were the same for all three sRNAs, a specific probe that binds to identical sequences in the 3′ end of all three sRNAs was used. The abundance of each was determined by standard curves. The P values were determined by an unpaired, two-tailed Student’s t test using the raw nanogram values (**, P < 0.01; *** P < 0.001). The means and standard deviations (error bars) were calculated from three biological replicates. (B) Priming efficiency for qPCR analysis of the cDNA as described in the panel A legend. The slope, R2 value, and percentage of efficiency determined for each of the primer sets, which were extrapolated from their respective standard curves, are shown. Download FIG S2, EPS file, 0.10 MB.
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FIG S3
Guide tree of the Csr sRNAs from multiple bacterial species. The DNA sequences of the csr sRNA genes from the indicated species were aligned through multiple ClustalW analyses. Download FIG S3, EPS file, 0.1 MB.
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TABLE S1
Strains and plasmids. Download Table S1, DOCX file, 0.02 MB.
Copyright © 2019 Butz et al.
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TABLE S2
Primers. Download Table S2, DOCX file, 0.02 MB.
Copyright © 2019 Butz et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
