Identification of a Chitin-Induced Small RNA That Regulates Translation of thetfoXGene, Encoding a Positive Regulator of Natural Competence in Vibrio cholerae

Abstract

The tfoX (also called sxy) gene product is the central regulator of DNA uptake in the naturally competent bacteria Haemophilus influenzae and Vibrio cholerae. However, the mechanisms regulating tfoX gene expression in both organisms are poorly understood. Our previous studies revealed that in V. cholerae, chitin disaccharide (GlcNAc) 2 is needed to activate the transcription and translation of V. cholerae tfoX (tfoX VC ) to induce natural competence. In this study, we screened a multicopy library of V. cholerae DNA fragments necessary for translational regulation of tfoX VC . A clone carrying the VC2078-VC2079 intergenic region, designated tfoR, increased the expression of a tfoX VC ::lacZ translational fusion constructed in Escherichia coli. Using a tfoX VC ::lacZ reporter system in V. cholerae, we confirmed that tfoR positively regulated tfoX VC expression at the translational level. Deletion of tfoR abolished competence for exogenous DNA even when (GlcNAc) 2 was provided. The introduction of a plasmid clone carrying the tfoR ؉ gene into the tfoR deletion mutant complemented the competence deficiency. We also found that the tfoR gene encodes a 102-nucleotide small RNA (sRNA), which was transcriptionally activated in the presence of (GlcNAc) 2 . Finally, we showed that this sRNA activated translation from tfoX VC mRNA in a highly purified in vitro translation system. Taking these results together, we propose that in the presence of (GlcNAc) 2 , TfoR sRNA is expressed to activate the translation of tfoX VC , which leads to the induction of natural competence.

Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

“…With one exception, they were all encoded on chromosome I. Surprisingly, given that the tfox gene and the sRNA Tfor are both conserved in V. splendidus (Vsr152) (Supplemental Table S1) and that in V. cholerae Tfor targets the 59 UTR of the tfox message (Yamamoto et al 2011), the 59 UTR of the tfox message in V. splendidus appears to be specific to this species.…”

“…Only seven of these (including Qrr and one copy of CsrB) were encoded on chromosome II. These putative Vibrio core sRNAs include the previously characterized MicX (Davis and Waldor 2007), VrrA (Song et al 2008(Song et al , 2010Song and Wai 2009), and Tfor (Yamamoto et al 2011). Thirty-seven to 38 sRNAs are in common between V. splendidus and V. cholerae, depending on the strain (Supplemental Tables S1, S5A).…”

“…A prior search of the entire genome for known sRNAs using Rfam 10.1 had identified 18 cis-regulatory RNAs, including 11 riboswitches and three amino acid operon leaders, and 19 trans sRNAs, a set to which we added the recently described TfoR sRNA (Yamamoto et al 2011). TarB (Bradley et al 2011;Davies et al 2012), although present in several Vibrios, is absent in V. splendidus.…”

“…TarA is regulated by ToxT, a transcriptional activator of the cholera toxin gene, and targets the ptsG transcript, thus influencing glucose uptake (Richard et al 2010). Finally, the chitin-induced small RNA TfoR, which regulates the translation of tfoX, a positive regulator of natural competence, was recently described (Yamamoto et al 2011).…”

Genetics of Natural Competence in Vibrio cholerae and other Vibrios

“…A putative cognate response regulator for ChiS remains to be identified. Two recent studies have revealed an additional membrane-bound protein, transcription factor TfoS, which links chitin sensing to natural competence (66,67). Dalia et al demonstrated that E. coli expressing tfoS activate transcription of a noncoding small RNA (sRNA) TfoR in response to exogenous (GlcNAc) 2 (66).…”