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

Abstract

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.