Functional determinants of the quorum-sensing non-coding RNAs and their roles in target regulation
2013; Springer Nature; Volume: 32; Issue: 15 Linguagem: Inglês
10.1038/emboj.2013.155
ISSN1460-2075
AutoresYi Shao, Lihui Feng, Steven T. Rutherford, Kai Papenfort, Bonnie L. Bassler,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoArticle9 July 2013Open Access Functional determinants of the quorum-sensing non-coding RNAs and their roles in target regulation Yi Shao Yi Shao Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Lihui Feng Lihui Feng Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Steven T Rutherford Steven T Rutherford Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Kai Papenfort Kai Papenfort Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Bonnie L Bassler Corresponding Author Bonnie L Bassler Department of Molecular Biology, Princeton University, Princeton, NJ, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Yi Shao Yi Shao Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Lihui Feng Lihui Feng Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Steven T Rutherford Steven T Rutherford Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Kai Papenfort Kai Papenfort Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Bonnie L Bassler Corresponding Author Bonnie L Bassler Department of Molecular Biology, Princeton University, Princeton, NJ, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Author Information Yi Shao1,‡, Lihui Feng1,‡, Steven T Rutherford1, Kai Papenfort1 and Bonnie L Bassler 1,2 1Department of Molecular Biology, Princeton University, Princeton, NJ, USA 2Howard Hughes Medical Institute, Chevy Chase, MD, USA ‡These authors contributed equally to this work. *Corresponding author. Department of Molecular Biology, Princeton University, 329 Lewis Thomas Labs, Princeton, NJ 08544, USA. Tel.:+1 609 258 2857; Fax:+1 609 258 2957; E-mail: [email protected] The EMBO Journal (2013)32:2158-2171https://doi.org/10.1038/emboj.2013.155 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1–5, control quorum-sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem-loops: the first stem-loop is crucial for base pairing with a subset of targets. This stem-loop also protects the Qrr sRNAs from RNase E-mediated degradation. The second stem-loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem-loop plays an accessory role in base pairing and stability. The fourth stem-loop functions as a rho-independent terminator. In the quorum-sensing regulon, Qrr sRNAs-controlled genes are the most rapid to respond to quorum-sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing. Introduction Quorum sensing is a cell-to-cell communication process that bacteria use to monitor changes in cell-population density. By producing, releasing, and detecting extracellular signal molecules called autoinducers, bacteria transition between individual and group behaviours. Quorum sensing ensures that bacteria execute collective behaviours such as bioluminescence, biofilm formation, and virulence factor production only at appropriate cell densities (Waters and Bassler, 2005; Ng and Bassler, 2009; Rutherford and Bassler, 2012). In the model bacterium Vibrio harveyi, three quorum-sensing pathways function in parallel (Henke and Bassler, 2004). At low cell density (LCD), the concentrations of the three autoinducers AI-1, AI-2, and CAI-1 are low. Under this condition, the cognate receptors LuxN, LuxPQ, and CqsS act as kinases, and they phosphorylate the phosphotransfer protein LuxU (Freeman and Bassler, 1999b; Neiditch et al, 2006; Swem et al, 2008; Wei et al, 2012). LuxU∼P passes its phosphate to the response regulator LuxO (Freeman and Bassler, 1999a, 1999b). LuxO∼P, together with σ54, activates the transcription of genes encoding five non-coding quorum-regulated small RNAs (sRNAs) called Qrr1–5 (Lilley and Bassler, 2000; Lenz et al, 2004; Tu and Bassler, 2007). The Qrr sRNAs activate the translation of the LCD master regulator AphA and repress the translation of the high cell density (HCD) master regulator LuxR (Tu and Bassler, 2007; Rutherford et al, 2011; Shao and Bassler, 2012). At HCD, the concentrations of the three autoinducers are high. Under this condition, the three receptors act as phosphatases, and they initiate a reversal of phospho flow through the circuit. LuxO, when unphosphorylated, is unable to activate the transcription of qrr1–5 (Tu and Bassler, 2007). Therefore, aphA translation is not activated and luxR translation is not repressed (Tu and Bassler, 2007; Rutherford et al, 2011; Shao and Bassler, 2012). This regulatory architecture ensures that maximum AphA is produced at LCD, while maximum LuxR exists at HCD (Rutherford et al, 2011; van Kessel et al, 2012). AphA and LuxR, in turn, direct the proper LCD to HCD quorum-sensing gene expression patterns, respectively (Rutherford et al, 2011; van Kessel et al, 2012). In addition to the two quorum-sensing master regulators AphA and LuxR, at LCD, the Qrr sRNAs also repress luxO and the genes encoding the AI-1 pathway synthase/receptor luxMN (Tu et al, 2010; Teng et al, 2011). The former is crucial for controlling Qrr sRNA levels, and the latter is important for adjusting the sensitivity to different autoinducers at different cell densities (Tu et al, 2010; Teng et al, 2011). The Qrr sRNAs belong to a large group of trans-encoded regulatory sRNAs in bacteria (Waters and Storz, 2009). Typically, sRNA-mediated activation of targets occurs through base pairing with and alteration of secondary structures in the 5′ UTRs of target mRNAs. Generally, pairing reveals the ribosome-binding sites and promotes translation (Fröhlich and Vogel, 2009). Alternative activation mechanisms include generating accessible ribosome-binding sites via endonucleolytic cleavage and protection from endonucleolytic destruction (Obana et al, 2010; Ramirez-Peña et al, 2010; Papenfort et al, 2013). The canonical sRNA repression mechanism is through base pairing with the mRNA region encoding the ribosome-binding site to occlude ribosome access. This mechanism leads to degradation or sequestration of the target mRNAs; in both cases, no translation of the mRNA targets occurs (Waters and Storz, 2009). Alternative repression mechanisms include base pairing within target mRNA coding regions or within intergenic regions of polycistronic transcripts, which leads to endonucleolytic cleavage (Desnoyers et al, 2009; Pfeiffer et al, 2009). Interactions between sRNAs and their mRNA targets are often mediated by the RNA chaperone Hfq. Hfq stabilizes the sRNAs, brings together sRNAs and target mRNAs, and interacts with RNase E (Vogel and Luisi, 2011; Mackie, 2012). Hfq can also be recruited, at least by the Spot42 sRNA, to act as a direct repressor of translation (Desnoyers and Massé, 2012). In the present study, we identify 16 new mRNA targets of the Qrr sRNAs. Particular sequence differences among the Qrr sRNAs determine whether each Qrr sRNA regulates all 16 of the new targets or only a subset of them. Using the newly identified target genes coupled with mutagenesis, we pinpoint the role of each portion of the Qrr sRNAs in target regulation. The first two stem-loops are involved in base pairing with the mRNA targets. The most 5′ stem-loop also protects the Qrr sRNAs from RNase E-mediated degradation. The third stem-loop plays an accessory role in base pairing and stability. The fourth stem-loop functions as the terminator. Analyses of regulation of the newly identified targets show that, of all of the genes in the quorum-sensing regulon, those that are directly controlled by the Qrr sRNAs are the most rapid to respond when bacteria transit from HCD to LCD. We find that the Qrr sRNAs can independently regulate particular target genes, and they can also act in conjunction with AphA or LuxR to control target gene expression. Results Identification of targets of the Qrr sRNAs in V. harveyi The V. harveyi Qrr sRNAs regulate luxR, luxO, luxMN, and aphA (Tu and Bassler, 2007; Tu et al, 2010; Rutherford et al, 2011; Teng et al, 2011; Shao and Bassler, 2012). All of these targets are members of the quorum-sensing regulatory circuit. Thus, to date, the only known role of the Qrr sRNAs is to regulate quorum-sensing regulators. We wondered whether the Qrr sRNAs control targets in addition to those in the quorum-sensing cascade. To explore this possibility, we constructed a plasmid containing qrr4 under an arabinose-inducible promoter and mobilized the plasmid into a V. harveyi Δqrr1–5 strain. We chose Qrr4 for this analysis because it is the most highly expressed Qrr at LCD and thus the most likely to be capable of controlling additional targets (Tu and Bassler, 2007). Qrr4 production was induced for 15 min, and global mRNA changes were measured by microarray and compared to the same strain in the absence of arabinose (see Materials and methods, Supplementary Figure S1). The microarray revealed 30 genes that changed expression more than two-fold (Supplementary Table S1). This set of genes includes the expected quorum-sensing regulators luxR, luxMN, and aphA. The level of luxO transcript did not change following Qrr4 induction, likely because Qrr control of luxO occurs via sequestration rather than degradation (Tu et al, 2010). Thus, 26 new genes were identified. In order to eliminate genes that are induced by arabinose, we performed qRT–PCR analysis on the putative targets following arabinose induction of the empty vector in V. harveyi. Of the 26 new genes, four in the gal operon are induced by arabinose but do not require Qrr4. One other target, vibhar_03460, is located directly downstream of luxR and has only a short transcript. This gene is likely co-transcribed with luxR, so we did not investigate it further. The remaining 21 genes are located in 18 operons: 16 are repressed and two are activated by Qrr4. To confirm the microarray results, we measured mRNA changes for all 18 operons by qRT–PCR. Figure 1A shows that, indeed, all of these genes are regulated following qrr4 induction. Figure 1.Regulation of target genes by Qrr4. (A) Regulation of genes identified from the microarray following a pulse of production of Qrr4 was confirmed by qRT–PCR. Target mRNA expression levels were compared at mid-logarithmic phase in a V. harveyi Δqrr1–5 strain (KT282) harbouring a plasmid with an arabinose-inducible qrr4 (pLF575) without (white bars) or with (black bars) addition of 0.2% arabinose for 15 min. Mean and s.e.m. values of triplicate cultures are shown. (B) Fluorescence from E. coli carrying IPTG-inducible translational GFP fusions to potential Qrr targets was measured in the presence of an empty vector (pLF253, white bars) or a plasmid carrying tetracycline-inducible qrr4 (pLF127, black bars). GFP levels were normalized to the vector control for each target. Mean and s.e.m. values of triplicate samples are shown. Download figure Download PowerPoint Qrr4 regulates target mRNAs through direct base pairing The above experiment, using a pulse of expression of qrr4, was designed to reveal direct Qrr4 targets. However, it is possible that, within the 15 min of induction, Qrr4 could regulate a factor that, in turn, controls some or all of the newly identified targets. To define which of the 18 target mRNAs are directly controlled by Qrr4, we measured their regulation by Qrr4 in E. coli in the absence of other V. harveyi components. To do this, we constructed translational GFP fusions to each of the 18 targets on plasmids and measured GFP fluorescence in the presence and absence of qrr4 expression (Supplementary Figure S2). Fourteen of the 18 targets exhibited altered production when Qrr4 was induced (Figure 1B). Four targets (vibhar_00986, vibhar_05213, vibhar_05384, and vibhar_06097) showed no regulation in E. coli, suggesting that these targets are not directly controlled by Qrr4 or the regions responsible for regulation are not included in the reporter constructs (vibhar_00986: −44 to +171, vibhar_05213: −78 to +60, vibhar_05384: −156 to +42, vibhar_06097: −56 to +51). Interestingly, one activated target vibhar_02446 did not follow the expected pattern. The vibhar_02446 mRNA increased following overexpression of Qrr4 in V. harveyi (Figure 1A). However, the VIBHAR_02446-GFP translational fusion was repressed by Qrr4 in E. coli (Figure 1B). One possibility is that base pairing between Qrr4 and vibhar_02446 mRNA prevents the mRNA from being degraded in V. harveyi, but in E. coli, blocking the ribosome-binding site prevents the protein from being translated. The 14 direct targets include three metabolic enzymes (vibhar_00417/prephenate dehydratase, vibhar_03626/deacetylase DA1, and vibhar_04936/glutathione-dependent formaldehyde-activating-like protein), two potential transcription factors (vibhar_00504 within operon vibhar_00506-vibhar_00504 and vibhar_05763), one hemagglutinin/protease (vibhar_02509), one RTX toxin transporter operon (vibhar_06455-vibhar_06452), one methyl-accepting chemotaxis protein (vibhar_05691), and one operon potentially involved in polysaccharide export (vibhar_06665–vibhar_06667) (Table I). Table 1. Novel Qrr sRNA target genes Gene number Predicted function qRT–PCR FACS VIBHAR_00417 Prephenate dehydratase −3.62 −13.22 VIBHAR_00504 RNA polymerase ECF-type sigma factor −3.33 −2.66 VIBHAR_00505 Chromosome segregation ATPase VIBHAR_02446 Hypothetical protein 1.68 −4.79 VIBHAR_02474 Virulence factor, aerolysin/hemolysin/leukocidin toxin −1.59 −5.67 VIBHAR_02509 Hemagglutinin/protease −3.38 −7.19 VIBHAR_03626 Deacetylase DA1 −6.81 −7.61 VIBHAR_04936 Glutathione-dependent formaldehyde-activating-like protein −3.13 −5.89 VIBHAR_05020 Hypothetical protein −3.15 −1.43 VIBHAR_05691 Histidine kinase −10.84 −10.94 VIBHAR_05763 Hypothetical protein −2.73 −11.09 VIBHAR_06448 Hemolysin A −2.61 −2.20 VIBHAR_06453a Putative toxin transport protein −2.27 −2.51 VIBHAR_06665 Polysaccharide export outer membrane protein −1.22 −10.08 VIBHAR_06666 Phosphatase VIBHAR_06667 Tyrosine-protein kinase VIBHAR_06888 Hypothetical protein −5.50 −2.86 VIBHAR_06930 Hypothetical protein 4.14 6.89 VIBHAR_06931 GGDEF family protein VIBHAR_p08221 Hypothetical protein −1.61 −2.50 VIBHAR_p08222 Isoprenoid biosynthesis protein with amidotransferase-like domain VIBHAR_p08223 Hypothetical protein qRT–PCR results in Figure 1A and FACS assay results in Figure 1B are shown for confirmed Qrr sRNA targets identified by microarray analysis. +, activation; −, repression. a VIBHAR_06453 is predicted to be in the same operon with VIBHAR_06454 and VIBHAR_06455. To demonstrate that the response of the 14 new targets was due to base pairing with Qrr4, we engineered mutations disrupting the putative pairing regions in Qrr4. We also constructed compensatory mutations in the targets to restore pairing. We show our analysis for two representative targets vibhar_05691 and vibhar_06930 that are repressed and activated, respectively (Figure 1, Figures 2A, B, and Table I). Computational prediction of the interaction between the 5′ UTR of the repressed vibhar_05691 mRNA and Qrr4 suggests pairing between −10 to −2 and −32 to −26 relative to the vibhar_05691 translation start site. Mutating AGCC to UCGG at nucleotides 13–16 of Qrr4 (Qrr4mut1) substantially reduced the ∼50-fold repression exhibited by wild-type Qrr4. By contrast, mutating CAACU to GUUGA between nucleotides 31–35 of Qrr4 (Qrr4mut2) only modestly affected repression. Consistent with these findings, altering GGCU to CCGA at −9 to −6 in vibhar_05691 (vibhar_05691-MutI) abolished regulation by wild-type Qrr4. However, this mutation restored regulation by Qrr4mut1 (Figure 2A). We suspect that the low basal expression of vibhar_05691MutI is due to a weakened ribosome-binding site (Figure 2A). Mutating AGUUG to UCAAC at −31 to −27 in vibhar_05691 (vibhar_05691MutII) somewhat impaired repression by wild-type Qrr4. Qrr4mut2 containing the compensatory changes fully restored regulation, whereas the Qrr4mut1 changes did not (Figure 2A). Together, these data suggest that pairing of the Qrr4 nucleotides 9–17 with the vibhar_05691 translation initiation region is most critical for regulation, however, the second pairing site (nucleotides −32 to −26 in vibhar_05691 and 30–36 in Qrr4) is required for full target control. Figure 2.Qrr4 regulates target genes through direct base pairing. (A) Qrr4 represses vibhar_05691 through base pairing. Fluorescence from E. coli carrying a plasmid with an IPTG-inducible translational GFP fusion to wild-type vibhar_05691 (pLF767), vibhar_05691MutI (pYS256), or vibhar_05691MutII (pYS257) was measured in the presence of an empty vector (pLF253), a vector with wild-type Qrr4 (pLF127), a vector with Qrr4mut1 (pYS258), or a vector with Qrr4mut2 (pYS259). The mutations are highlighted in the sequences by over or underlines. Mean and s.e.m. values of triplicate samples are shown. (B) Qrr4 activates vibhar_06930 through base pairing. Fluorescence from E. coli carrying a plasmid with an IPTG-inducible translational GFP fusion to wild-type vibhar_06930 (pLF1285), vibhar_06930 truncation (Δ−129 to −79) (pLF1730), or vibhar_6930MutI (pLF840) was measured in the presence of an empty vector (pLF253), a vector with wild-type Qrr4 (pLF127), or a vector with Qrr4mut3 (pLF770). The mutations in the sequences are highlighted by over or underlines. Mean and s.e.m. values of triplicate samples are shown. Base pairings between the mRNA targets (vibhar_05691 and vibhar_06930) and Qrr4 were predicted by RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). Nucleotides involved in base pairing are shown in red. Translational start sites are denoted as +1. The structure of the 5′ UTR of vibhar_06930 was predicted by RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Base pairing between other mRNA targets and Qrr4 are shown in Supplementary Figure S3. Download figure Download PowerPoint In the case of the activated target vibhar_06930, the 125 nucleotide 5′ UTR is predicted to form a secondary structure that conceals the ribosome-binding site (Figure 2B). Deletion of the first 47 nucleotides (Δ−125 to −79) increased production of the VIBHAR_06930-GFP fusion by ∼12.5-fold, indicating that this region is crucial for intrinsic translation inhibition (Figure 2B). Qrr4 is predicted to base pair with this self-inhibitory loop to relieve repression (Figure 2B). Indeed, wild-type Qrr4 activated production of VIBHAR_06930-GFP by ∼7.5-fold and did not activate the truncated VIBHAR_06930-GFP fusion. Mutating GGC to CCG at −95 to −97 nucleotides in the 5′ UTR of vibhar_06930 (vibhar_06930MutI) eliminated activation by wild-type Qrr4. Mutating GCC to CGG at positions 29–31 in Qrr4 (Qrr4mut3) impaired regulation of wild-type vibhar_06930, but restored regulation to vibhar_06930MutI (Figure 2B). Basal GFP production from vibhar_06930MutI is higher than that of wild type but lower than that from the truncated construct. Likely, the self-inhibitory loop is only partially disrupted in vibhar_06930MutI whereas it is completely eliminated in the truncation mutant. Qrr2–5 regulate an identical set of target mRNAs The above experiments examined the function of Qrr4 in target mRNA regulation. Given that the other Qrr sRNAs possess similar sequences to Qrr4, we wondered whether they likewise regulate the same or other additional targets. Using an identical strategy, we performed microarray experiments following a pulse of expression of each Qrr sRNA (Supplementary Figure S1). In all cases, the genes that exhibited two-fold or higher changes in levels corresponded well to those identified in the Qrr4 experiment (Supplementary Table S1). Four additional targets (vibhar_02474, vibhar_06299, vibhar_06448, and vibhar_06895) were identified as controlled by Qrr2, Qrr3, or Qrr5. However, qRT–PCR showed that vibhar_06895 did not respond to induction of any Qrr sRNA. We conclude that vibhar_06895 was a false positive. We tested vibhar_02474, vibhar_06299, and vibhar_06448 for control by Qrr4, and these genes did in fact exhibit expression changes upon qrr4 induction (Figure 1A). We suspect that these three genes were not identified in the original Qrr4 pulse microarray experiment because they fell below the two-fold cut-off. Qrr4 also controlled GFP translational fusions to vibhar_02474 and vibhar_06448, but not vibhar_06299 in E. coli (Figure 1B). VIBHAR_06299-GFP was also not regulated by Qrr2, Qrr3, or Qrr5 in E. coli, so we did not investigate it further. The two newly identified targets are potential virulence factors: vibhar_02474 contains an aerolysin toxin motif and vibhar_06448 encodes a hemolysin A protein. This brings the total to 16 new Qrr targets (Table I). qRT–PCR following pulse induction of each Qrr sRNA was used to verify the microarray results. Thirteen of the 16 targets are regulated by Qrr2–5 (Supplementary Figure S4). As an example, we use vibhar_03626 and show that Qrr2, Qrr3, Qrr4, and Qrr5 control its expression (Figure 3A). Three targets, vibhar_02474, vibhar_02509, and vibhar_06665, are regulated by Qrr2, Qrr4, and Qrr5, but not by Qrr3 in V. harveyi. The data for vibhar_02509 are shown in Figure 3B and results for vibhar_02474 and vibhar_06665 are provided in Supplementary Figure S4. Of these three targets, vibhar_02509 showed the strongest defect. This is borne out in recombinant E. coli; Qrr2, Qrr4, and Qrr5 repress VIBHAR_02509-GFP while Qrr3 is somewhat defective (Figure 3C). The difference between the V. harveyi and E. coli results could come from the fact that Qrr targets in addition to the one we are measuring exist in V. harveyi and compete for regulation by the Qrr sRNAs. Thus, in vivo differences in the roles of the Qrr sRNAs can be revealed. Because no competition for the Qrr occurs in E. coli, even when a Qrr is defective in V. harveyi, residual regulatory capability can occur in E. coli. Figure 3.Regulation of targets by Qrr2–5. qRT–PCR of vibhar_03626 (A) and vibhar_02509 (B) without (white bars) and with (black bars) arabinose induction of the specific Qrr. Mean and s.e.m. values of replicates are shown. (C) Fluorescence from E. coli carrying a plasmid with an IPTG-inducible VIBHAR_02509-GFP fusion (pYS214) was measured in the presence of an empty vector (pLF253), a vector with Qrr2 (pLF186), Qrr3 (pLF126), Qrr4 (pLF127), or Qrr5 (pLF187). Mean and s.e.m. values of triplicate samples are shown. Download figure Download PowerPoint Sequence differences at the 5′ terminus dictate Qrr1 target selectivity Qrr1 regulates all of the target genes that are controlled by Qrr2–5 except vibhar_00505, vibhar_05691, and aphA (Figure 4B; Supplementary Figure S3 and S4) (Shao and Bassler, 2012). Qrr1 lacks nine nucleotides that are conserved in Qrr2–5 near the 5′ terminus (Figure 4A) (Tu and Bassler, 2007). This gap makes Qrr1 unable to activate aphA translation but has no effect on Qrr1 repression of luxR (Figure 4B, top graphs) (Shao and Bassler, 2012). Reintroducing the missing 9 nucleotides into Qrr1 (denoted Qrr19+) restores regulation of aphA, and does not alter luxR regulation (Figure 4B, top graphs). We deleted the corresponding 9 nucleotides from Qrr4 to 'convert' it to Qrr1. We call this construct Qrr49−. Like Qrr1, Qrr49− is impaired in activation of aphA, but it is wild type for repression of luxR (Figure 4B, top graphs). We predict that these 9 conserved nucleotides could also be important for regulating vibhar_00505 and vibhar_05691. If so, Qrr19+ should be functional at these two targets, and indeed Figure 4B shows this is the case (bottom graphs). Likewise, Qrr49−, which lacks these 9 nucleotides, cannot control vibhar_00505 and vibhar_05691 (Figure 4B, bottom graphs). Thus, we conclude that the 9 nucleotides that Qrr1 lacks are necessary for regulating a subset of the Qrr targets including aphA, vibhar_00505, and vibhar_05691. These results are consistent with the base pairing patterns we mapped through mutagenesis analysis (Figure 2A; Supplementary Figure S5) (Shao and Bassler, 2012). Figure 4.5′ sequence differences confer distinct regulatory capabilities to Qrr1. (A) RNA sequence alignment of V. harveyi Qrr1–5. The conserved 9 nucleotides that are absent in Qrr1 are shown in blue and other highly conserved sequences in the 5′ region are shown in red. Sequences corresponding to predicted stem-loops and to the terminator are indicated with underlines. (B) Fluorescence from plasmid-encoded V. harveyi AphA-GFP (pLF255), LuxR-GFP (pLF128), VIBHAR_00505-GFP (pLF804), and VIBHAR_05691-GFP (pLF767) translational fusions were measured in E. coli carrying an empty vector (pLF253), a vector expressing a tetracycline-inducible qrr1 (pLF396), qrr1 with 9 nucleotides reintroduced (Qrr19+, pYS241), qrr4 (pLF127), or qrr4 with 9 nucleotides deleted (Qrr49−, pYS239). GFP from three independent cultures was measured for each strain and the mean and s.e.m. values are shown. All measurements were normalized to the means of the vector controls. Download figure Download PowerPoint Contribution of each stem-loop to base pairing between Qrr sRNAs and target mRNAs Based on secondary structure predictions, there exist four stem-loops in the Qrr sRNAs (Tu and Bassler, 2007). We name them, from 5′ to 3′: SL1, SL2, SL3, and SL4 (Figures 4A and 5A). Each of the Qrr sRNAs has all four stem-loops but, as mentioned, Qrr1 is the most different from the other Qrr sRNAs, because it lacks 9 nucleotides in SL1. SL4 contains the rho-independent terminator. Having a large set of Qrr sRNA targets in hand allows us to investigate the individual and combined roles of each of the stem-loops in Qrr function. We constructed a series of stem-loop deletions in Qrr4 and measured the effects on target regulation (Figure 5A, ΔSL1, ΔSL2, ΔSL3, and ΔSL1 and SL3 with deleted sequences shown as blanks). To examine the effects of these changes, we started with two well-studied targets, aphA and luxR. Deletion of SL1 eliminated aphA activation but did not affect luxR repression (Figure 5B). Deletion of SL2 eliminated regulation of both aphA and luxR. Deletion of SL3 had only a modest effect on each target (Figure 5B). Deletion of both SL1 and SL3 gave results identical to the SL1 deletion alone (Figure 5B). We used this exact strategy to test the role of each stem-loop in regulation of each of the 16 newly identified Qrr sRNA targets. The results are summarized in Figure 5C. All of the data are shown in Supplementary Figure S6. In brief, deletion of SL1 primarily affects regulation of only two targets in addition to aphA; vibhar_00505 and vibhar_05691. Deletion of SL2 abolishes regulation of all of the targets with the exception of vibhar_00505 and vibhar_05691. Deletion of SL3 affects regulation of several targets to different extents, especially vibhar_02509 and vibhar_05020. Additive effects occur in most cases when both SL1 and SL3 are deleted (Figure 5C; Supplementary Figure S6). Thus, SL2 contains conserved sequences required for base pairing with the majority of the target mRNAs, and SL1 and SL3 are crucial for base pairing with a subset of targets. In cases in which SL2 is not crucial, SL1 has increased importance (for example, see vibhar_00505 and vibhar_05691). These results are consistent with the above findings that vibhar_00505 and vibhar_05691 are regulated by Qrr4 but not by Qrr1 (Figure 4). Figure 5.Stem-loop 2 functions as the core base pairing region of the Qrr sRNAs. (A) Predicted secondary structure of Qrr4 and schematics of WT Qrr4, Qrr4 deletion mutants, chimeric Qrr4 sRNAs, and Qrr4 inversion mutants are shown. Colour codes are the same as in Figure 4, with the conserved 9 nucleotides missing in Qrr1 in blue, other 5′ highly conserved sequences in red, deleted sequences left blank, inverted sequences hatched, and RybB sequences in green. Nucleotides mutated to construct the SL1 disruption and restoration mutants are highlighted in the box. Data for those mutants are in Figure 6. (B) Fluorescence from plasmid-encoded V. harveyi AphA-GFP (pLF255) and LuxR-GFP (pLF128) translational fusions was measured in E. coli carrying an empty vector (pLF253), a vector expressing a tetracycline-inducible qrr4 (pLF127), qrr4 stem-loop 1 deletion (ΔSL1, pYS225), qrr4 stem-loop 2 deletion (ΔSL2, pYS226), qrr4 stem-loop 3 deletion (ΔSL3, pYS227), qrr4 stem-loop 1 and stem-loop 3 double deletion (ΔSL1&3, pYS229), qrr4 stem-loop 1
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