A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria

Abstract

Small regulatory RNAs (sRNAs) are crucial components of many stress response systems. The envelope stress response (ESR) of Gram-negative bacteria is a paradigm for sRNA-mediated stress management and involves, among other factors, the alternative sigma factor E (σ^E ) and one or more sRNAs. In this study, we identified the MicV sRNA as a new member of the σ^E regulon in Vibrio cholerae. We show that MicV acts redundantly with another sRNA, VrrA, and that both sRNAs share a conserved seed-pairing domain allowing them to regulate multiple target mRNAs. V. cholerae lacking σ^E displayed increased sensitivity toward antimicrobials, and over-expression of either of the sRNAs suppressed this phenotype. Laboratory selection experiments using a library of synthetic sRNA regulators revealed that the seed-pairing domain of σ^E -dependent sRNAs is strongly enriched among sRNAs identified under membrane-damaging conditions and that repression of OmpA is crucial for sRNA-mediated stress relief. Together, our work shows that MicV and VrrA act as global regulators in the ESR of V. cholerae and provides evidence that bacterial sRNAs can be functionally annotated by their seed-pairing sequences.

Keywords: Hfq; MicV; sRNA; seed pairing; sigma E.

Figure 1. Transcriptional regulation of micV

1. A

   Alignment of micV sequences, including the promoter regions, from various Vibrio species. The ‐35 box, ‐10 box, the TSS, the highly conserved seed region, and the rho‐independent terminator are indicated. Lower part: consensus motif of E. coli σ^E‐dependent promoters.

2. B

   Vibrio cholerae wild‐type and ΔrpoE strains carrying the indicated plasmids were grown in LB medium to early stationary phase (OD₆₀₀ of 1.5) and induced with L‐arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.

3. C

   Vibrio cholerae wild‐type, ΔvchM, and ΔvchM ΔrpoE strains harboring the PmicV::mKate2 plasmid were grown in M9 minimal medium. Samples were collected at various stages of growth and analyzed for fluorescence.

4. D, E

   Vibrio cholerae wild‐type and ΔrpoE strains carrying PmicV::mKate2 (D) or PvrrA::mKate2 (E) plasmids were cultivated in LB medium to exponential phase (OD₆₀₀ of 0.4) and treated with ethanol (3.5% final conc.) or water. Fluorescence was determined 180 min after ethanol treatment, and mKate2 levels of the mock‐treated samples were set to 1. Corresponding Northern blot analyses of MicV and VrrA expression are shown at the bottom. 5S rRNA served as loading control.

5. F

   Vibrio cholerae wild‐type, ΔmicV, ΔvrrA, or ΔvrrA ΔmicV strains were grown in LB medium to OD₆₀₀ of 0.2 and treated with ethanol (3.5% final conc.). After 5 h of treatment, serial dilutions were prepared, recovered on agar plates, and CFU/ml were determined.

Data information: In (C–E), data are presented as mean ± SD, n = 3. In (F), the box plots indicate the median, 75^th and 25^th percentiles (boxes), and 90^th and 10^th percentiles (whiskers), n = 8. Statistical significance was determined using one‐way ANOVA and post hoc Holm–Sidak test.Source data are available online for this figure.

Figure EV1. Genomic context and conserved transcriptional control of σ^E‐dependent sRNAs in V. cholerae (related to Fig 1)

1. Gene synteny analysis between the genomic loci encoding micV in various Vibrio strains. Homologous genes are indicated by the same colors.

2. Vibrio cholerae wild‐type and ΔvchM strains carrying empty vector control plasmids (pCtr) were grown in LB medium. At the indicated time points, RNA samples were collected and tested for micV and vrrA expression by Northern blot analysis. A size marker is provided on the left (M), and 5S rRNA was used as loading control.

3. Vibrio cholerae wild‐type (control) and hfq::hfq‐3xFLAG (Hfq‐FLAG) strains were grown to stationary phase (OD₆₀₀ of 2), lysed, and subjected to immunoprecipitation using the anti‐FLAG antibody. RNA samples of lysate (total RNA) and co‐immunoprecipitated fractions were analyzed on Northern blots. 5S rRNA served as loading control.

4. Vibrio cholerae wild‐type and Δhfq strains were cultivated in LB medium to an OD₆₀₀ of 1.0. Cells were treated with rifampicin to terminate transcription. Total RNA samples were collected at the indicated time points, and MicV or VrrA transcript levels were monitored on Northern blots.

5. Vibrio cholerae wild‐type, ΔvchM, and ΔvchM ΔrpoE strains harboring PvrrA::mKate2 plasmids were grown in M9 minimal medium. Samples were collected at various stages of growth and analyzed for fluorescence.

6. Escherichia coli BW25113 wild‐type and ΔrpoE strains carrying PmicV::gfp plasmids and either empty vector control (pBAD‐Ctr) or plasmids expressing rpoE of E. coli (pBAD‐rpoE (E.c)) or of V. cholerae (pBAD‐rpoE (V.c)) were grown in LB medium, supplemented with L‐arabinose (0.2% final conc.). Samples were collected at various stages of growth and analyzed for fluorescence.

Data information: In (D–F), data are presented as mean ± SD, n = 3.Source data are available online for this figure.

Figure 2. Target profiles of MicV and VrrA

1. A

   Vibrio cholerae ΔvrrA ΔmicV strains carrying pBAD‐micV, pBAD‐vrrA, or an empty vector control (pCtr) were cultivated to early stationary phase (OD₆₀₀ of 1.5) in LB medium. Cells were treated with L‐arabinose (0.2% final conc.), and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV, and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild‐type strain carrying pCtr were collected during various growth phases, which indicated ˜18‐fold and ˜7‐fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).

2. B

   Venn diagram summarizing the RNA‐Seq results: RNA samples were collected from V. cholerae ΔvrrA ΔmicV strains carrying pBAD‐micV, pBAD‐vrrA, or an empty vector control. Depicted are genes displaying a fold change of ≥ 3 and FDR‐adjusted p‐value ≤ 1E‐8 obtained from MicV‐expressing conditions (blue) or vrrA‐expressing conditions (green). Genes regulated by both sRNAs (fold change ≥ 3 in one condition, fold change ≥ 2.0 in the other) are depicted in light green.

3. C–E

   Vibrio cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x‐axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid were cultivated in M9 minimal medium, and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to (B): regulated by both sRNAs (C), regulated only by MicV (D), or regulated only by VrrA (E).

Data information: In (C–E), data are presented as mean ± SD, n = 3.Source data are available online for this figure.

Figure 3. Patterns of target regulation by VrrA and MicV

1. A–C

   Predicted base‐pairings of MicV with the 5′UTR of ompT (A) and with the 5′UTR of ushA (B) or VrrA with the 5′UTR of lpp (C). Mutations tested in (D, E, F) are indicated.

2. D–F

   Vibrio cholerae ΔvrrA ΔmicV strains carrying the ompT::gfp or ompT M1*::gfp fusions (D), ushA::gfp or ushA M1*::gfp fusions (E), or lpp::gfp or lpp M2*::gfp fusions (F) and an empty vector control (pCtr), the micV expression plasmids (pMicV, pMicV M1), or the vrrA expression plasmids (pVrrA, pVrrA M2) were grown in M9 minimal medium, and GFP fluorescence was measured. M1 and M2 denote the mutations indicated in (A, B, C). Fluorescence of the control strains was set to 1.

3. G–I

   Vibrio cholerae ΔrpoE, ΔrpoE ΔvrrA, ΔrpoE ΔmicV, or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD‐rpoE or an empty vector control (pCtr) were grown in LB medium to OD₆₀₀ of 1.5, and L‐arabinose (0.2% final conc.) was added. RNA samples were collected at the indicated time points and monitored for ompT (G), ushA (H), or lpp (I) levels using qRT–PCR.

Data information: In (D–I), data are presented as mean ± SD, n = 3.Source data are available online for this figure.

Figure EV2. VrrA harbors two conserved base‐pairing regions to regulate mRNA targets (related to Fig 3)

1. Alignment of the vrrA sequences of several Vibrio species. The boxes indicate the conserved seed regions R1 and R2. Mutations used in (B, D, E) are indicated.

2. Vibrio cholerae ΔvrrA ΔmicV strains carrying pMicV, pMicV M1, pVrrA, pVrrA M1, pVrrA M2, or an empty vector control (pCtr) were grown to OD₆₀₀ of 1.0 in LB medium. RNA samples were collected and monitored for micV and vrrA expression by Northern blot analysis. 5S rRNA served as loading control.

3. Vibrio cholerae ΔrpoE, ΔvrrA ΔrpoE, ΔmicV ΔrpoE, or ΔvrrA ΔmicV ΔrpoE strains carrying pBAD‐rpoE plasmids or an empty vector control (pCtr) were grown to early stationary phase (OD₆₀₀ of 1.5), and rpoE expression was induced by treatment with L‐arabinose (0.2% final conc.). RNA samples were collected at the indicated time points and monitored for micV and vrrA expression by Northern blot analysis. 5S rRNA served as loading control.

4. Predicted base‐pairing of VrrA with the 5′UTR of ompT.

5. Vibrio cholerae ΔvrrA ΔmicV strains carrying ompT::gfp or ompT M1*::gfp fusions and an empty vector control (pCtr) or vrrA expression plasmids (pVrrA, pVrrA M1, or pVrrA M2) were grown in M9 minimal medium. GFP fluorescence was measured, and fluorescence of the control strains was set to 1.

Data information: In (E), data are presented as mean ± SD, n = 3.Source data are available online for this figure.

Figure 4. A conserved sRNA seed sequence inhibits OMP production

1. Alignment of the seed‐pairing sequences of VrrA, MicV, and RybB.

2. Vibrio cholerae ΔvrrA ΔmicV strains carrying the ompT::3XFLAG gene and pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB medium to an OD₆₀₀ of 2.0. RNA and protein samples were collected and analyzed for MicV, VrrA, and RybB expression on Northern blots. OmpT::3xFLAG production was tested on Western blots. RNAPα and 5S rRNA served as loading controls for Western and Northern blots, respectively.

3. Escherichia coli wild‐type strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were grown in LB medium to an OD₆₀₀ of 2.0. RNA and protein samples were collected and investigated on Northern blots and SDS–PAGE, respectively. For comparison, we included the E. coli insertional mutant strains ompA::kan ^R and ompC::kan ^R for specific assignment of OmpA and OmpC bands.

4. Vibrio cholerae wild‐type and ΔrpoE strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB medium to OD₆₀₀ of 0.2 and treated with ethanol (3.5% final conc.). After 5 h of treatment, serial dilutions were prepared, recovered on agar plates, and CFU/ml were determined.

Data information: In (D), the box plots indicate the median, 75^th and 25^th percentiles (boxes), and 90^th and 10^th percentiles (whiskers), n = 8. Statistical significance was determined using one‐way ANOVA and post hoc Holm–Sidak test.Source data are available online for this figure.

Figure 5. A conserved sRNA motif is enriched in laboratory selection experiments

1. Experimental strategy of the laboratory selection experiments: An sRNA library was generated using the rybB scaffold with nine randomized nucleotides at the 5′ end, cloned into a broad‐range plasmid backbone, and transferred into V. cholerae ΔrpoE cells. These colonies were pooled, grown to OD₆₀₀ of 0.2, and treated with ethanol (3.5% final conc.) for 6 h. Surviving cells were recovered on agar plates, pooled, and subjected to another round of selection (3 selections total). After each selection, the plasmids of surviving cells were analyzed using high‐throughput sequencing.

2. Vibrio cholerae wild‐type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, and Sel3) were grown in LB medium to OD₆₀₀ of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 h. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates.

3. Plasmid contents of the strains carrying the sRNA libraries before selection (input) and after consecutive ethanol treatments (Sel1, Sel2, and Sel3) were analyzed using high‐throughput sequencing. Relative library complexity (left y‐axis) was determined by counting sequence variants present in the normalized samples. To test for the enrichment of possible sequence motifs, the sequence variants present in each sample were counted and normalized for sequencing depth. The resulting data were analyzed for the enrichment of the conserved CRCUGCUUUU motif (right y‐axis).

Data information: In (C), data are presented as mean ± SD, n = 2.Source data are available online for this figure.

Figure EV3. Synthetic sRNA library composition and nucleotide contributions (related to Fig 5)

1. A, B

   A synthetic sRNA library based on a RybB scaffold with nine randomized nucleotides at the 5′ end was cloned into plasmid backbones and transferred into V. cholerae ΔrpoE. The resulting clones were pooled and treated with ethanol (3.5% final conc.) for 6 h. After treatment, the surviving cells were recovered on agar plates, pooled, and subjected to consecutive rounds of ethanol treatment for a total of three selections. After each selection, plasmid contents of surviving cells were analyzed by high‐throughput sequencing. (A) Density histogram depicting the sequence read counts of obtained sRNA variants before ethanol treatment (Input). (B) Nucleotide contributions at the randomized positions in the synthetic sRNA libraries, before ethanol treatment (Input) and after consecutive ethanol treatments (Sel1, 2, 3). A = adenine, T = thymine, C = cytosine, G = guanine.

Data information: In (B), data are presented as mean, n = 2.Source data are available online for this figure.

Figure EV4. Synthetic sRNA variants are enriched in laboratory selection experiments and mediate ethanol resistance (related to Fig 6)

1. Vibrio cholerae ΔrpoE strains carrying the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, and Sel3) were cultivated in LB medium to OD₆₀₀ of 2.0. RNA samples were collected and analyzed for omp mRNA levels using qRT–PCR.

2. Pie chart indicating the distribution of synthetic sRNA variants after three consecutive ethanol treatments (Sel3). The dashed red line indicates the fraction of the 15 most abundant sequence variants.

3. The frequency of the 15 most abundant (top 15) sRNA variants was determined before ethanol treatment (Input) and after consecutive ethanol treatments (Sel1, 2, 3).

4. Vibrio cholerae wild‐type and ΔrpoE strains carrying an empty vector control (pCtr), synthetic sRNA expression plasmids (psRNA1‐15), rybB expression plasmids (pRybB), or expression plasmids containing a rybB variant with deletion of nine nucleotides at the 5′ end (pRybBΔ9) were grown to OD₆₀₀ of 0.2. Cells were treated with ethanol (3.5% final conc.) for 5 h. After treatment, the strains were serially diluted (1:10 steps) and spotted onto agar plates.

Data information: In (A, C), data are presented as mean ± SD, n = 2.Source data are available online for this figure.

Figure EV5. Base‐pairing of enriched sRNA variants to ompA mRNA is sufficient to mediate ethanol resistance (related to Fig 6)

1. Sequence alignment of the 15 most abundant (top 15) sRNA variants.

2. Consensus motif for the top 15 sRNA variants.

3. Secondary structure model of ompA mRNA including the predicted base‐pairing interactions of the top 15 sRNA variants. Straight lines indicate pairing bases, and bulges indicate non‐pairing bases. Pairing bases corresponding to the variable region of the variants are depicted in color, and pairing bases corresponding to the backbone are depicted in black. MicV and VrrA are shown in gray. Numbers indicate the position on the ompA mRNA relative to the AUG start codon. The predicted position of the 30S ribosomal subunit and the ompA scr mutation are indicated.

4. Vibrio cholerae wild‐type ompA scr and ΔrpoE ompA scr strains carrying an empty vector control (pCtr) or synthetic sRNA expression plasmids (psRNA1‐15) were grown to OD₆₀₀ of 0.2. Cells were treated with ethanol (3.5% final conc.) for 5 h. After treatment, the strains were serially diluted (1:10 steps) and spotted onto agar plates.

5. Vibrio cholerae wild‐type and ompA scr mutant strains carrying empty vector controls (pCtr) were grown to OD₆₀₀ of 0.2 and challenged with ethanol (3.5% final conc.). After 5 h of treatment, serial dilutions were prepared, recovered on agar plates, and CFU/ml were determined.

Data information: In (E), data are presented as mean ± SD, n = 4. Statistical significance was determined using a two‐tailed, unpaired Student's t‐test.Source data are available online for this figure.

Figure 6. Enriched sRNA variants mediate ethanol resistance by OmpA repression

1. Vibrio cholerae wild‐type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, and Sel3) were cultivated in LB medium to OD₆₀₀ of 2.0. Membrane fractions were identified by SDS–PAGE. The indicated bands were identified by mass spectrometry.

2. Vibrio cholerae ΔvrrA ΔmicV cells expressing the ompA::3xFLAG gene and carrying an empty vector control (pCtr), or plasmids producing the 15 most highly enriched sRNA variants (sRNA variants 1–15) were grown in LB medium to an OD₆₀₀ of 2.0. RNA and protein samples were collected and tested for sRNA and OmpA::3xFLAG expression on Northern and Western blots, respectively (with 5S rRNA and RNAPα as loading controls).

3. Vibrio cholerae ΔvrrA ΔmicV strains carrying the ompA::gfp fusion and an empty vector control (pCtr) or the enriched sRNA expression plasmids were grown in M9 minimal medium, and GFP fluorescence was measured. Fluorescence of the control strains was set to 1.

4. Vibrio cholerae wild‐type, ΔrpoE, ΔompA, or ΔrpoE ΔompA strains were grown in LB medium to OD₆₀₀ of 0.2 and treated with ethanol (3.5% final conc.). After 5 h of treatment, serial dilutions were prepared, recovered on agar plates, and CFU/ml were determined.

Data information: In (C), data are presented as mean ± SD, n = 3. In (D), the box plots indicate the median, 75^th and 25^th percentiles (boxes), and 90^th and 10^th percentiles (whiskers), n = 8. Statistical significance was determined using one‐way ANOVA and post hoc Holm–Sidak test.Source data are available online for this figure.

Figure 7. Conserved seed sequences control envelope homeostasis in V. cholerae

Misfolded OMPs activate an intra‐membrane proteolysis cascade resulting in the release of σ^E from its anti‐σ factor RseA. Free σ^E activates the the expression of at least 73 transcripts in V. cholerae, including the rpoE‐rseABC operon and the MicV and VrrA sRNAs. MicV and VrrA employ the conserved base‐pairing region R1 to repress omp mRNAs, restoring membrane homeostasis, and the rpoE‐rseABC operon. VrrA specifically downregulates pal and lpp, encoding two major lipoproteins, via the base‐pairing region R2.