Thus, we conclude that Hfq-mediated RNA-duplex formation is pervasive in V. In summary, we found 2889 statistically significant RNA-RNA interaction candidates, supported in total by 847,939 and 493,875 chimeric cDNA reads at low and high cell density, respectively (Fig. cholerae wild-type strain lacking the 3XFLAG epitope served as a negative control in these experiments. RNA molecules in close proximity were ligated and prepared for cDNA synthesis and paired-end Illumina sequencing. Next, the Hfq::3XFLAG protein together with its associated RNA ligands was co-immunoprecipitated and RNA ends were trimmed. Specifically, we collected cells from low and high cell densities (OD 600 of 0.2 and 2.0, respectively), which we exposed to UV-crosslinking. cholerae cells producing a Hfq::3XFLAG protein from the native chromosomal location 13. cholerae, we performed RIL-seq analysis using V. To study Hfq-mediated RNA duplex formation in V. cholerae and identify the QrrX sponge sRNA as a critical regulator of QS-associated collective behaviors. Together, our findings reveal the genome-wide impact of Hfq-associated sRNAs on gene expression in V. In accordance with this regulatory scheme, lack of qrrX facilitates biofilm formation, whereas QrrX over-expression has the opposite effect. Expression of qrrX is controlled by the LysR-type regulator QrrT and together QrrX and QrrT accelerated QS transition in V. Detailed analysis of one sponge sRNA, named QrrX, showed that this regulator specifically binds to the Qrr1-4 sRNAs, which inhibits their regulatory functions. Sponge sRNAs are a class of non-coding regulators that base-pair with other sRNAs to neutralize their activities 21, 22. This analysis revealed hundreds of previously unknown sRNA-target interactions at low and high cell densities and led to the discovery of several sponge sRNAs. To close this gap, we employed RIL-seq (RNA-interaction-by-ligation-and-sequencing) 18, 19, 20 to identify sRNA-target RNA pairs bound by Hfq in V. cholerae, however, global studies addressing their regulatory roles in this major pathogen are yet missing. These examples show that Hfq-binding sRNAs are crucial for QS control and collective behavior in V. For instance, the VqmR sRNA is induced by DPO (3,5-dimethyl-pyrazin-2-ol) autoinducer and inhibits biofilm formation and virulence gene expression 14, 15, 16 whereas the VadR sRNA adjusts cell shape and biofilm formation 17. cholerae 13 and for few of them a function in QS, virulence, or biofilm formation has been established. In addition to the Qrrs, dozens of Hfq-binding sRNAs have been identified in V. cholerae cells deficient for qrr1-4 expression, or lacking the hfq gene, display strongly reduced colonization of mice and fail to produce biofilms 9, 10, 11, 12. Second, Qrr2-4 stabilize the mRNA encoding the AphA transcriptional regulator, which antagonizes HapR activity and promotes virulence and biofilm formation 7, 8. First, Qrr1-4 inhibit the expression of the hapR mRNA, which encodes a major regulator of high-cell density behaviors that represses biofilm formation and virulence genes 6. Two Qrr-target mRNA interactions are of overarching importance for QS performance in V. cholerae encodes four Qrr homologs, Qrr1-4), all Qrrs act together with the RNA chaperone Hfq to control gene expression by base-pairing with multiple target mRNAs 5. While the numbers of Qrr homologs vary among different Vibrio species ( V. In all Vibrios studied so far, QS relies on post-transcriptional gene regulation by small regulatory RNAs (sRNAs) called Qrr (quorum regulatory RNA) 4. Marine Vibrio species, including the major human pathogen Vibrio cholerae, have one of the most thoroughly studied QS systems 3. Thus, QS is a promising target for novel antimicrobial intervention strategies, however, for this concept to become a reality, we must first fully understand model QS systems 1, 2. QS allows bacteria to synchronously control processes that are only productive when undertaken in unison by the collective, including various virulence-related functions, such as biofilm formation and toxin production. The process involves the production, detection, and response to extracellular signaling molecules called autoinducers. Quorum sensing (QS) is the process of cell-cell communication in bacteria.
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