Antagonizing Role of LeuO in Transcriptional Activation of vvpS gene by SmcR and CRP
Jeong-A Kim and Kyu-Ho Lee
“Background” Vibrio vulnificus produces multiple exoproteases including a quorum-sensing regulated elastase (VvpE) which is one of its major virulence factors. In addition to VvpE, serine protease (VvpS) has been identified from a zymographic analysis of V. vulnificus secretome. In this study, regulatory mechanisms for vvpS transcription were investigated to understand the expression characteristics of the potential virulence factor.
“Methods ” Expression of vvpS was monitored by using a luxAB -transcriptional fusion and the secreted VvpS was monitored by western blot. Transcription factors for vvpS expression were isolated by ligand-fishing experiments and identified by MALDI-TOF MS analyses. Their interactions with
DNA were examined using EMSA and DNase I footprint assays.
“Results ” LeuO, a LysR-type transcription factor, was isolated from a ligand-fishing experiment. To examine the effect of LeuO on vvpS expression, vvpS transcription was monitored in the leuO -deletion mutant. The mutant showed increased vvpS transcription compared to wildtype, suggesting that vvpS transcription was negatively regulated by LeuO. Interestingly, LeuO bound two independent sites in the upstream region of vvpS and its binding sites were localized at -189 to -145 relative to the transcription start site (LBS- I) and -142 to -115 (LBS-II), both of which are far from the typical sites for transcription repressors. Further screening of the transcription factors involving in vvpS transcription resulted in isolation of CRP and SmcR as activators for vvpS expression. Analyses of interactions of both activators with the vvpS upstream region showed that their binding sites were overlapped with two LeuO-binding sites: CRP bound the Site II and SmcR bound the Site I.
Additional DNase I footprinting experiments showed that each site occupied by LeuO was competitively exchanged by the other transcription factor.
“Conclusions ” vvpS was minimally expressed during exponential phase by a transcription repressor,
LeuO. As cells entered the stationary phase, the cellular content of LeuO was declined but the cellular levels of CRP and SmcR were increased. Then, the repression by LeuO was relieved by competitive binding of SmcR and cAMP-CRP complex to both LeuO-binding sites, which resulted in maximal induction of vvpS .