A novel transcriptional activator tubX is required for the stability of Bacillus sphaericus mosquitocidal plasmid pBsph. J Bacteriol. 2014.

　　Ge Y, Zhao N, Hu X, Shi T, Cai Q, Yuan Z.

      J Bacteriol. 2014 Sep 29. pii: JB.01855-14.

　　Abstract

　　Stable maintenance of the low-copy-number plasmid pBsph in Bacillus sphaericus requires partitioning (par) system that consists of a filament-forming protein TubZ-Bs, a centromere-binding protein TubR-Bs, and a centromere-like DNA site tubC, composed of three blocks (I, II and III) of 12-bp degenerate repeats. Previous studies have shown that mini-pBsph replicons encoding TubZ system are segregationally highly unstable, whereas the native pBsph is stably maintained. However, the mechanism underlying the stability discrepancy between pBsph and its minireplicon is poorly understood. Here, a downstream gene orf187 (encoding TubX) of tubZ-Bs was found to play a role in plasmid stabilization. Null mutation or overexpression of tubX resulted in a defect in pBsph stability and a significant decrease in tubRZ-Bs expression, and the TubX-minus phenotype was suppressed by ectopic expression of a wild-type copy of tubX and additional tubRZ-Bs. Electrophoresis mobility shift assay (EMSA) and DNase I footprinting revealed a direct binding of TubX protein to five 8-bp degenerate repeats located in the par promoter region, and that TubX competed for binding to the par promoter with TubR-Bs. Further studies demonstrated that TubX significantly stimulated transcription of the par operon in the absence of tubR-Bs, and a greater magnitude of gene activation was observed when tubR-Bs was present. These results suggested that TubX positively regulates the tubRZ-Bs transcription by interfering with TubR-Bs-mediated repression and binding directly to the tubRZ-Bs promoter region.

　　http://www.ncbi.nlm.nih.gov/pubmed/25266379