Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing
2009; Springer Nature; Volume: 28; Issue: 4 Linguagem: Inglês
10.1038/emboj.2008.300
ISSN1460-2075
AutoresSine Lo Svenningsen, Kimberly C. Tu, Bonnie L. Bassler,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle22 January 2009Open Access Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing Sine L Svenningsen Sine L Svenningsen Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Kimberly C Tu Kimberly C Tu 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 Sine L Svenningsen Sine L Svenningsen Department of Molecular Biology, Princeton University, Princeton, NJ, USA Search for more papers by this author Kimberly C Tu Kimberly C Tu 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 Sine L Svenningsen1, Kimberly C Tu1 and Bonnie L Bassler 1,2 1Department of Molecular Biology, Princeton University, Princeton, NJ, USA 2Howard Hughes Medical Institute, Chevy Chase, MD, USA *Corresponding author. Department of Molecular Biology, HHMI and Princeton University, 329 Lewis Thomas Labs, Princeton, NJ 08544-1014, USA. Tel.: +609 258 2857; Fax: +609 258 2957; E-mail: [email protected] The EMBO Journal (2009)28:429-439https://doi.org/10.1038/emboj.2008.300 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Quorum sensing is a mechanism of cell-to-cell communication that allows bacteria to coordinately regulate gene expression in response to changes in cell-population density. At the core of the Vibrio cholerae quorum-sensing signal transduction pathway reside four homologous small RNAs (sRNAs), named the quorum regulatory RNAs 1–4 (Qrr1–4). The four Qrr sRNAs are functionally redundant. That is, expression of any one of them is sufficient for wild-type quorum-sensing behaviour. Here, we show that the combined action of two feedback loops, one involving the sRNA-activator LuxO and one involving the sRNA-target HapR, promotes gene dosage compensation between the four qrr genes. Gene dosage compensation adjusts the total Qrr1–4 sRNA pool and provides the molecular mechanism underlying sRNA redundancy. The dosage compensation mechanism is exquisitely sensitive to small perturbations in Qrr levels. Precisely maintained Qrr levels are required to direct the proper timing and correct patterns of expression of quorum-sensing-regulated target genes. Introduction Chemical communication allows groups of bacteria to monitor and synchronously alter gene expression in response to changes in cell number and species-composition of the surrounding bacterial community. Communication is accomplished through the synthesis, secretion, and subsequent detection of signalling molecules called auto-inducers (AIs). This process, known as quorum sensing, is used by many bacterial species to coordinately control a battery of behaviours (Waters and Bassler, 2005; Bassler and Losick, 2006). In the human pathogen Vibrio cholerae, quorum sensing regulates progression through the infectious cycle, controls genes encoding virulence factors, and regulates biofilm formation (Zhu et al, 2002; Hammer and Bassler, 2003; Zhu and Mekalanos, 2003). Vibrio cholerae makes and responds to two AIs that function synergistically to control group behaviours (Miller et al, 2002). At low cell-population density (LCD), when the extracellular AI concentration is low, membrane-bound AI-receptors function as kinases and phosphorylate a shared phosphotransfer protein called LuxU, which subsequently transfers the phosphate to the response regulator LuxO. LuxO-P, together with the alternative sigma factor σ54, activates transcription of four genes encoding small non-coding RNAs (sRNAs), called the quorum regulatory RNAs (Qrr1–4) (Figure 1, and Miller et al, 2002; Lenz et al, 2004). When transcribed, the Qrr sRNAs function together with the RNA chaperone Hfq to control translation of target mRNAs. One target mRNA, which is destabilized by the Qrrs at LCD, encodes the major quorum-sensing transcription factor, HapR. Figure 1.Model of the core of the V. cholerae quorum-sensing circuit. The backbone of the quorum-sensing signalling pathway is depicted in black. Auto-inducer inputs are ultimately transmitted to LuxO. At LCD, LuxO-P functions together with σ54 to activate transcription of the genes encoding the four Qrr sRNAs. The Qrr sRNAs, in conjunction with Hfq, repress translation of hapR mRNA. When hapR translation is derepressed, HapR controls downstream target genes. The previously defined feedback loops are shown in blue. HapR and LuxO auto-repress the hapR and luxO promoters, respectively (see discussion for details on the LuxO auto-repression loop). HapR also enhances qrr transcription through an unknown factor, denoted by 'X'. The feedback loop between the Qrr sRNAs and LuxO identified in this work is shown in red. Arrows indicate positive interactions, T-bars indicate negative interactions. Download figure Download PowerPoint At high cell-population density (HCD), AIs accumulate extracellularly and bind their respective receptors. This event switches the receptors' enzymatic activity from kinase to phosphatase, ultimately resulting in dephosphorylation of LuxO-P. Dephosphorylated LuxO cannot activate qrr transcription. Existing sRNAs are rapidly turned over, as Hfq-dependent sRNAs are degraded stoichiometrically with their target mRNAs (Masse et al, 2003). In the absence of Qrr sRNAs, hapR mRNA is translated and HapR protein accumulates and activates or represses its target genes. In summary, V. cholerae cells at LCD are characterized by the presence of Qrr sRNAs and the absence of HapR, whereas V. cholerae cells at HCD are characterized by the absence of Qrr sRNAs and the presence of HapR. Small RNAs are widely used as key regulators of stress responses, virulence, and central metabolic pathways in bacteria (Romeo, 1998; Gottesman, 2004; Majdalani et al, 2005; Storz et al, 2005). In many cases, multiple homologous sRNAs exist, and often they appear to carry out identical functions (Weilbacher et al, 2003; Wilderman et al, 2004; Guillier and Gottesman, 2006). In the case of V. cholerae quorum sensing, the Qrr sRNAs are encoded by four unlinked loci. They are ∼80% identical in sequence and predicted to have similar secondary structures (Lenz et al, 2004). Previous analyses of single, double, triple, and quadruple qrr deletions in V. cholerae showed that the four Qrr sRNAs function redundantly to control quorum sensing (Lenz et al, 2004). That is, if any one of the four Qrr sRNAs is present, V. cholerae expresses quorum-sensing target genes in a density-dependent manner similar to the wild-type strain. By contrast, in the closely related bacterium Vibrio harveyi, the analogous multiple Qrr sRNAs contribute additively to control quorum sensing (Tu and Bassler, 2007). Two feedback loops have been described in the V. cholerae regulatory network, which appear, first, to ensure the network's exquisite responsiveness to changes in extracellular AI concentrations, and second, to set the AI concentration thresholds at which quorum-sensing-regulated behaviours are initiated or terminated. The two feedback loops are as follows. HapR auto-repression loop The HapR protein binds to a site immediately downstream of the hapR transcriptional start site and, in this capacity, represses its own transcription (Figure 1, HapR auto-repression loop) (Lin et al, 2005). At HCD, HapR accumulates to a level sufficient to regulate its target genes, but because it also binds to its own promoter, it prevents additional hapR transcription, and thereby prevents excessive accumulation of HapR. HapR auto-repression is essential for the proper timing of the quorum-sensing response because the HapR pool must be maintained at a low enough level that HapR can be efficiently eliminated when V. cholerae switches from the HCD to the LCD gene expression pattern (Lin et al, 2005; Svenningsen et al, 2008). HapR-Qrr feedback loop HapR enhances transcription of the four qrr genes (Figure 1, HapR-Qrr feedback loop). However, because there is also an absolute requirement for LuxO-P to initiate qrr transcription, the HapR-Qrr feedback loop only functions when V. cholerae cells shift from the HCD to the LCD condition (Svenningsen et al, 2008). At this transition, the HapR-Qrr feedback provides a surge in qrr transcription, which accelerates the alterations in gene expression required for the V. cholerae LCD lifestyle. Here, we investigate the mechanism underlying Qrr redundancy and we find that the Qrr sRNAs compensate for one another. Specifically, in the absence of any one Qrr, the other Qrrs are upregulated. The combination of two feedback loops, the HapR-Qrr feedback loop described above, and a new feedback loop described in this work, the LuxO-Qrr feedback loop, underlies Qrr dosage compensation. Together, these feedback loops provide a mechanism for adjusting qrr transcription on the basis of the total activity of the Qrr sRNAs present in a cell at any given time. Remarkably, the Qrr dosage compensation mechanism is able to respond to modest, that is, physiologically relevant, alterations in Qrr levels. Calibration of the Qrr sRNA levels through dosage compensation ensures precise timing of the activation and termination of quorum-sensing-controlled behaviours. Results The four Qrr sRNAs compensate for one another Our previous results showed that all four qrr sRNAs have redundant functions in quorum sensing: any one of them is sufficient for cell-density-dependent expression of HapR-controlled target genes (Lenz et al, 2004). We wondered how any one Qrr sRNA could be sufficient for an approximately wild-type quorum-sensing response. One possibility is that, in the absence of a particular sRNA, the levels of the remaining sRNAs increase. To test this possibility, we used northern blots to measure the levels of each individual Qrr sRNA in the wild-type strain and in triple qrr deletion strains lacking the other three qrr genes (Figure 2A). Each row shows a blot probed specifically for the Qrr sRNA indicated on the right. For example, results for Qrr1 are shown in the top row. Lane 1 contains total RNA from the wild-type strain, and lane 2 contains the same amount of total RNA from the triple Δqrr2,3,4 deletion strain. It is evident that greater Qrr1 is present in the absence of the other three Qrr sRNAs, than in their presence. The same pattern holds true for Qrr2, Qrr3, and Qrr4. As a control, lane 3 of each row contains total RNA from a V. cholerae mutant deleted for only the Qrr sRNA being probed. This lane shows that the Qrr1, Qrr2, and Qrr4 probes are specific for their particular sRNAs and do not cross-hybridize. Weak cross-hybridization occurs with the Qrr3 probe; however, this low level of cross-hybridization does not affect the interpretation of the results. Figure 2.Qrr sRNA levels in wild-type and triple qrr deletion strains. (A) Northern blots showing Qrr levels in wild-type V. cholerae (lane 1), V. cholerae qrr triple deletion strains, possessing only the qrr gene encoding the sRNA indicated on the right (lane 2), and V. cholerae qrr single deletion strains, lacking only the qrr gene encoding the Qrr sRNA indicated on the right (lane 3). Total RNA was visualized with ethidium bromide as the loading control (not shown). (B) Northern blot showing Qrr4 levels in a V. cholerae qrr4 single deletion strain expressing qrr4 from the Ptac promoter (lane 1) and a V. cholerae Δqrr1–4 quadruple deletion expressing qrr4 from the Ptac promoter (lane 2). 5S RNA is shown as a loading control. Total RNA was collected from the indicated strains at OD600=0.1. Download figure Download PowerPoint Dosage compensation functions at the level of qrr transcription The increased abundance of one Qrr sRNA in the absence of the other Qrr sRNAs could be the result of increased transcription of the qrr gene in question, increased stability of the Qrr sRNA, or both. If transcription of one qrr gene increases in the absence of the other Qrr sRNAs, we reasoned that a transcriptional reporter fusion would reflect this. By contrast, regulation at the level of sRNA stability would not be manifested using a transcriptional reporter fusion. We engineered lux reporter fusions to the +1 transcriptional start sites of each qrr gene (Svenningsen et al, 2008). Expression of the lux fusions in the wild-type and the Δqrr1–4 strains was measured at OD600=0.1, the cell density at which the Qrr sRNAs are maximally produced (Svenningsen et al, 2008), and the results are shown in Table I. Expression of each qrr gene is higher in the Δqrr1–4 mutant than in the wild type. Thus, dosage compensation occurs at the level of qrr transcription. We note that dosage compensation affects the four qrr promoters to different extents (see 'Fold Repression', Table I). We return to this point later. Table 1. Dosage compensation acts at the level of transcription of the qrr genes qrr1-luxa qrr2-luxa qrr3-luxa qrr4-luxa Wild type 56 (3) 140 (20) 7 (3) 74 (15) Δqrr1-4 149 (3) 373 (28) 159 (5) 378 (26) Fold repressionb 2.7 (0.05) 2.7 (0.16) 24 (0.39) 5.1 (0.22) ΔhapR 28 (1) 154 (28) 6 (1) 47 (30) ΔhapR Δqrr1–4 34 (1) 226 (55) 29 (10) 179 (52) Fold repressionb 1.2 (0.04) 1.5 (0.30) 4.8 (0.35) 3.8 (0.69) a Light production from the indicated qrr–lux construct was measured at OD600=0.1 in the indicated V. cholerae strains. The average relative light units (RLU/108) from three independent cultures is reported. The standard error from the mean (RLU/108) is indicated in parentheses. b Fold repression is calculated as the light produced by the Δqrr1–4 mutant divided by the light produced by the isogenic qrr1–4+ strain. In addition to transcriptional control, dosage compensation could also be a consequence of regulation of sRNA stability. To examine this possibility, we needed to uncouple regulation at the transcriptional level from regulation at the post-transcriptional level. To do this, we expressed the qrr4 gene from an exogenous Ptac promoter in Δqrr4 and Δqrr1–4 strains and measured Qrr4 levels by Northern blot (Figure 2B). Qrr4 driven by the Ptac promoter accumulates to identical levels in the presence and absence of the other qrr genes, indicating that the Ptac-qrr4 construct is not sensitive to alterations in sRNA levels. Thus, we conclude that, at least for qrr4, and presume for the other qrr genes, dosage compensation stems from transcriptional control, and not from the regulation of sRNA stability. Dosage compensation is independent of the origin of the Qrr sRNAs We considered two possible mechanisms that could give rise to the Qrr dosage compensation observed above. First, Qrr dosage compensation could be a regulatory element wired into the quorum-sensing network, that is, a Qrr-responsive negative feedback loop that represses the qrr promoters could exist. In this scenario, any shortage in Qrr sRNAs would result in reduced repression of the qrr promoters, leading to a compensatory increase in Qrr sRNA production. Second, dosage compensation could be an incidental consequence of titration of a transcription factor(s) required for expression of the qrr promoters. In this scenario, in the absence of one or more qrr genes, increased levels of this putative transcription factor(s) would be available to bind and activate the expression of the remaining qrr promoters. In the first case, an exogenously provided source of Qrr sRNA would cause repression of qrr transcription. In the second case, only Qrr sRNAs made from endogenous qrr promoters would cause repression of qrr transcription. To test which mechanism is correct, we measured light production from the qrr–lux promoter fusions in the absence of Qrr sRNAs (Figure 3, white bars), in the presence of Qrr sRNAs produced from their endogenous promoters (Figure 3, black bars), Qrr4 sRNA produced from a plasmid-borne endogenous qrr4 promoter (Figure 3, striped bars), and Qrr4 sRNA produced from a plasmid carrying the exogenous Ptac promoter, which, besides core RNA polymerase, shares no transcription factors with those required for native qrr expression (Figure 3, dotted bars). The figure shows that Qrr sRNAs produced from any source cause repression of the qrr–lux promoter fusions. Thus, dosage compensation must be a result of negative feedback control of qrr expression by the Qrr sRNAs themselves, and not due to titration of factors required for qrr transcription. Figure 3.Dosage compensation is insensitive to the origin of the Qrr sRNAs. Light production from the indicated qrr–lux constructs was measured at OD600=0.1 in a V. cholerae Δqrr1–4 mutant carrying the vector (white bars), V. cholerae wild type carrying the vector (black bars), a V. cholerae Δqrr1–4 mutant expressing qrr4 under control of the endogenous qrr4 promoter on the vector (striped bars), and a Δqrr1–4 mutant expressing qrr4 under control of the Ptac promoter on the same vector (dotted bars). Each bar shows the average light production from three independent cultures. Error bars indicate one standard deviation from the mean. RLU: relative light units. Download figure Download PowerPoint The HapR-Qrr feedback loop is partially responsible for Qrr dosage compensation On the basis of the above results, we hypothesize that the Qrr sRNAs compensate for one another by controlling the translation of a transcription factor, which in turn feeds back to regulate qrr gene expression. As described in the Introduction, one obvious candidate is the HapR-Qrr feedback loop identified previously (Svenningsen et al, 2008, Figure 1; HapR-Qrr feedback loop). We reason that if there is a shortage of Qrr sRNAs, increased HapR could be produced, which in turn could feed back to increase synthesis of Qrr sRNAs, resulting in Qrr dosage compensation. To test if the HapR-Qrr feedback loop is required for Qrr dosage compensation, we compared qrr–lux light production in a ΔhapR V. cholerae strain with that in a ΔhapR, Δqrr1–4 strain (Table I). Our rationale is that if the HapR-Qrr feedback loop is responsible for Qrr dosage compensation, dosage compensation will not occur in the ΔhapR strains because any feedback loop requiring HapR will not be functioning in the ΔhapR strains. Indeed, when compared with the wild-type strain background, the extent of dosage compensation is reduced for all four qrr promoters in the ΔhapR strain backgrounds (Table I, 'Fold Repression'), suggesting that the HapR-Qrr feedback loop is involved in dosage compensation. However, whereas removal of the HapR-Qrr feedback loop nearly eliminated dosage compensation for qrr1 and qrr2, dosage compensation at qrr3 and qrr4 continued to occur in the ΔhapR strains. Thus, qrr1 and qrr2, which are the least subject to dosage compensation in wild-type V. cholerae (Table I), require the HapR-Qrr feedback loop for dosage compensation. By contrast, qrr3 and qrr4, which show a greater degree of dosage compensation, although obviously regulated by the HapR-Qrr feedback loop, must also respond to an additional regulatory component(s) for dosage compensation. luxOU mRNA is a target of Qrr sRNA regulation To identify the additional regulatory component involved in qrr3 and qrr4 dosage compensation, we relied on our findings in V. harveyi, which is closely related to V. cholerae and has a similar quorum-sensing circuit. In V. harveyi, the Qrr sRNAs repress translation of LuxO (Tu et al, manuscript in preparation). Thus, we wondered if the Qrr sRNAs might feed back to regulate luxO translation as part of the dosage compensation mechanism in V. cholerae. Alignment of the 5′-untranslated region (5′-UTR) of V. cholerae luxO and the two known targets of Qrr1–4, hapR and vca0939, showed that the 5′-UTR of the poly-cistronic luxOU mRNA contains a region of complementarity to the Qrr sRNAs similar to that predicted in the hapR and vca0939 5′-UTRs (Figure 4A, and Tu et al, manuscript in preparation, Lenz et al, 2004; Hammer and Bassler, 2007). Figure 4.luxOU mRNA is a target of Qrr sRNA translational repression. (A) Alignment of the reverse complement of the conserved pairing region of the Qrr sRNAs with the 5′-UTR of two known target mRNAs, hapR and vca0939, and the 5′-UTR of luxOU mRNA. The region of the Qrr sRNAs that pair with the hapR and vca0939 5′-UTRs is completely conserved among the four Qrrs. Nucleotides in the target mRNAs that are complementary to the Qrr sRNAs are highlighted in white on black background. The underlined sequence (UAGG) of luxOU mRNA is mutated to AUCC in the luxOAUCC mutant. (B) Degradation of the luxOU mRNA was measured by northern blot in V. cholerae wild-type, Δqrr1–4 and Δqrr1–4, Ptac-qrr4 following transcription termination. The indicated times are seconds after addition of rifampicin. 5S RNA is shown as a loading control. (C) E. coli SLS1277 carrying plasmids harbouring either LuxO–GFP (pSLS146) or LuxOAUCC–GFP (pSLS152) protein fusions were grown overnight in either LB (black bars) or LB supplemented with 0.4% arabinose (white bars). The experiment was performed in duplicate on three separate occasions. Error bars indicate one standard deviation from the mean of all six measurements. Download figure Download PowerPoint To test if the Qrr sRNAs feed back to regulate luxOU mRNA in V. cholerae, we assayed the stability of luxOU mRNA using northern blots. Rifampicin was added to LCD V. cholerae cultures to terminate transcription, after which the level of luxOU mRNA transcript was monitored over time (Figure 4B). In wild-type cells (denoted by WT), luxOU mRNA is degraded with a half-life of ∼94 s following termination of transcription. In the Δqrr1–4 strain, the stability of the luxOU mRNA is increased, (half-life=∼115 s). By contrast, in a V. cholerae strain that overexpresses Qrr4 (denoted by Δqrr1–4 Ptac-qrr4), the half-life of luxOU mRNA is reduced to ∼35 s, supporting the idea that Qrr1–4 destabilize luxOU mRNA. To measure the consequence of Qrr sRNA-mediated degradation of the luxOU mRNA on LuxO levels, we engineered a translational fusion of the luxO 5′-UTR including the first 10 amino acids of the LuxO ORF to green fluorescent protein (GFP). We introduced the plasmid-borne LuxO–GFP fusion into Escherichia coli strain SLS1277, which expresses V. cholerae qrr4 from the chromosome, under control of the PBAD promoter. Figure 4C (left bars) shows the production of LuxO–GFP in SLS1277 without or with induction of Qrr4 synthesis by the addition of arabinose. LuxO–GFP expression is repressed ∼4-fold by Qrr4, suggesting that the Qrr sRNAs repress translation of luxOU mRNA. The LuxO-Qrr feedback loop is partially responsible for Qrr dosage compensation The results presented in Figure 4 suggest that Qrr repression of luxO could aid in Qrr dosage compensation because reduced Qrr sRNA levels could lead to increased LuxO production, which in turn could result in increased Qrr sRNA production (Figure 1, LuxO-Qrr feedback loop). To explore this idea, we engineered mutations in the luxO 5′-UTR that prevent pairing between the luxOU mRNA and the Qrr sRNAs. The predicted region of pairing overlaps the ribosome binding site of luxO, so most nucleotide changes in this region alter the basal level of luxO expression (data not shown). One mutation, however, luxOAUCC, nearly eliminates Qrr-mediated repression of luxO (Figure 4C, right pair of bars), without significantly changing the basal expression level of luxO (Figure 4C, compare the two black bars). In this mutant, nucleotides −6 to −3 (TAGG) with respect to the first nucleotide in the luxO start codon were mutated to the complementary sequence (ATCC). The mutated sequence is underlined in Figure 4A. In Figure 5, we compare the extent of Qrr dosage compensation in the wild-type (black bars), the ΔhapR strain lacking the HapR-Qrr feedback loop (white bars), the luxOAUCC strain, which lacks the LuxO-Qrr feedback loop (grey bars), and the ΔhapR, luxOAUCC double mutant, which lacks both feedback loops (striped bars). Qrr dosage compensation was measured as the fold repression of each qrr–lux transcriptional fusion in the qrr1–4+ strain compared with that in the isogenic Δqrr1–4 strain. Figure 5.The LuxO-Qrr feedback loop contributes to Qrr dosage compensation. Light production from the indicated qrr–lux fusions was measured at OD600=0.1 in V. cholerae wild-type (black bars), ΔhapR (white bars), luxOAUCC (grey bars) and ΔhapR, luxOAUCC (striped bars) strains containing or lacking the four chromosomal qrr genes. In each case, Qrr dosage compensation is calculated as the light produced from the Δqrr1–4 mutant divided by the light produced from the isogenic qrr1–4+ strain. Regarding the qrr3–lux data, the two slashes indicate that the bars extend beyond the scale of the y axis. The fold-dosage-compensation value is indicated above the corresponding bar. Light production from each strain was measured in triplicate. Error bars indicate one standard deviation from the mean. The standard deviation is ±0.39 for qrr3–lux in wild type, and ±0.09 for qrr3–lux in the luxOAUCC mutant. Download figure Download PowerPoint For reference, we show again that the HapR-Qrr feedback loop is involved in dosage compensation (compare white bars with black bars). The luxOAUCC mutation partially eliminates dosage compensation for each qrr gene (compare grey bars with black bars), showing that indeed the LuxO-Qrr feedback loop contributes to Qrr dosage compensation. However, we note that the two feedback loops contribute distinctly to dosage compensation of each qrr gene. Dosage compensation in the case of qrr1 and qrr2 is largely due to the HapR-Qrr feedback loop. By contrast, the Qrr-LuxO feedback loop is the major source of dosage compensation for qrr4. In all three of these cases, simultaneous disruption of the HapR-Qrr and LuxO-Qrr feedback loops completely eliminates dosage compensation (compare striped bars with black bars). These results show that for qrr1, qrr2, and qrr4, the two feedback loops are sufficient to account for dosage compensation. Remarkably, qrr3, although clearly regulated by the two feedback loops, remains responsive to dosage compensation in the absence of both the HapR-Qrr and the LuxO-Qrr feedback loops. We interpret this to mean that an additional feedback loop, which is involved in dosage compensation, exists that has yet to be identified. This feedback loop is apparently specific to qrr3. Determining the boundaries of Qrr dosage compensation The Qrr sRNAs constitute the core of the quorum-sensing regulatory cascade, and regulation by them ultimately dictates the expression patterns of all downstream quorum-sensing target genes. Thus, we predict that keeping Qrr levels tightly constrained is a priority for this regulatory network. To investigate this idea, we examined the accuracy of Qrr dosage compensation in the quorum-sensing circuit. We made one assumption; that the four Qrr sRNAs are equally effective in pairing with their target mRNAs. If so, accurate dosage compensation should result in an identical total Qrr sRNA pool size in each of the qrr mutant strains because the loss of the contribution of a particular sRNA following deletion should be compensated for by overexpression of the remaining Qrr sRNAs. To survey a range of altered Qrr levels, we examined Qrr dosage compensation accuracy in response to a large perturbation in qrr gene dosage by deleting all combinations of three qrr genes, as well as more modest changes in gene dosage by sequentially deleting individual qrr genes. Dosage compensation is inaccurate in triple qrr mutant strains. To measure the total Qrr sRNA pool, we performed northern blots with a probe complementary to the 32 bp region that is 100% conserved among the four Qrr sRNAs. This probe binds the four Qrr sRNAs indiscriminately as confirmed using known concentrations of each Qrr transcribed in vitro (data not shown). Figure 6A shows the total Qrr sRNAs in wild type and the four qrr triple mutant strains. All the triple mutant strains, especially the qrr1+ mutant, contain markedly less total Qrr sRNAs than does the wild-type strain. This finding indicates that Qrr dosage compensation is not accurate in the qrr triple mutants. Figure 6.Qrr dosage compensation is not accurate in the qrr triple deletion mutants. (A) The total level of Qrr sRNAs in wild-type (denoted WT), Δqrr2,3,4 (denoted qrr1+), Δqrr1,3,4 (denoted qrr2+), Δqrr1,2,4 (denoted qrr3+), and Δqrr1,2,3 (denoted qrr4+) V. cholerae strains grown to OD600=0.1 was measured by northern blot with a probe for the 32-bp region that is completely conserved among Qrr1–4 sRNAs. 5S RNA is shown as a loading control. (B) The level of hapR mRNA in the same strains shown in (A) and in a Δqrr1–4 V. cholerae strain grown to OD600=0.1 was measured by RT–PCR. Error bars indicate one standard deviation from the mean of triplicate measurements. The experiment was repeated three times with similar results. Download figure Download PowerPoint We were surprised that dosage compensation is not
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