Haycocks, J. R. J. | bioRxiv Science

bioRxiv2025

A 3UTR-derived small RNA modulates the life-cycle of the cholera toxin-encoding filamentous phage, CTXphi

Bacteriophages (phages) are well known to be one of the major driving forces in bacterial evolution. This also applies to virulent microorganisms, such as the major human pathogen Vibrio cholerae, whose pathogenic potential and epidemic proliferation largely depends on the interaction with environmental phages. Specifically, integration of the CTX{phi} phage genome into the first chromosome of V. cholerae also introduced the ctxAB genes, encoding the primary toxin responsible for the severe acute diarrheal disease, cholera. Whereas the mechanisms underlying CTX{phi}-associated horizontal gene transfer and transcriptional control of the ctxAB genes have been intensively studied over the past years, post-transcriptional regulation affecting the CTX{phi} life-cycle has not been documented. Here, we report the discovery and characterization of the CisR small RNA (sRNA) that is produced from the 3UTR (untranslated region) of the prtV gene and inhibits the expression of the CTX{phi}-encoded cep mRNA. CisR-mediated repression of cep involves Hfq-assisted base-pairing of the two transcripts and results in reduced CTX{phi} production under stress conditions. We further demonstrate that transcription of prtV-cisR requires both the master quorum-sensing regulator HapR and CRP, a global regulator of carbon metabolism. Taken together, our work provides evidence that V. cholerae employs sRNA-mediated post-transcriptional gene regulation to coordinate CTX{phi} activation with both cell density and nutrient availability. SIGNIFICANCE STATEMENTThe integration of the CTX{phi} phage genome, which carries the ctxAB toxin genes, is essential for cholera pathogenesis in humans. While transcriptional control of CTX{phi} and ctxAB has been well-studied, post-transcriptional mechanisms remain unexplored. Here, we identify and characterize CisR, a small RNA derived from the 3' untranslated region of prtV, which inhibits the CTX{phi}-encoded cep mRNA through Hfq-dependent base-pairing. CisR-mediated regulation limits phage production under stress conditions and is co-regulated by the quorum-sensing factor HapR and the metabolic regulator CRP. Our findings reveal a new RNA-based mechanism linking CTX{phi} phage activation to cell density and nutrient status of V. cholerae.

microbiology↗

bioRxiv2025

Genome-wide binding of the cyclic AMP receptor protein in enteroaggregative Escherichia coli suggests a role in modulating virulence

Bacterial pathogens use a wide array of virulence factors to colonise and subsequently elicit disease in their host. These factors are often subject to extensive regulation at the transcriptional level, to ensure that their expression is timely. Although many pathogens use bespoke transcription factors that primarily target virulence genes, global transcription factors also sometimes play a role in controlling these genes. Enteroaggregative Escherichia coli (EAEC) is a significant cause of watery and mucoid diarrhoea globally. The organism colonises the small intestine before producing toxins that elicit disease, using a multitude of virulence factors that are encoded both chromosomally and on virulence plasmids. In this work, we have studied the cAMP Receptor Protein (CRP), a well-characterised bacterial global transcription factor, focusing on its role in pathogenicity of the prototype EAEC strain 042. We show that, although most functional CRP binding sites on the chromosome are conserved between E. coli K-12 and 042, CRP has been co-opted to couple the expression of some virulence genes to the nutritional state of the cell. We report novel mechanisms for CRP-dependent regulation of genes, whose products contribute to adhesion, production of a bacterial antibiotic, and export of a polysaccharide capsule.

microbiology↗