A Novel Transcriptional Activator, tubX , Is Required for the Stability of Bacillus sphaericus Mosquitocidal Plasmid pBsph
| Autor: | Xiaomin Hu, Yong Ge, Ni Zhao, Tingyu Shi, Quanxin Cai, Zhiming Yuan |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
DNA
Bacterial Transcriptional Activation Transcription Genetic Operon Bacterial Toxins DNA Footprinting DNA footprinting Bacillus Electrophoretic Mobility Shift Assay Biology Microbiology Bacillus sphaericus Genomic Instability Plasmid Electrophoretic mobility shift assay Molecular Biology Regulation of gene expression Genetic Complementation Test Promoter Articles Gene Expression Regulation Bacterial biology.organism_classification Molecular biology Genes Bacterial Trans-Activators Ectopic expression Gene Deletion Plasmids Protein Binding |
| Zdroj: | Journal of Bacteriology. 196:4304-4314 |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.01855-14 |
| Popis: | Stable maintenance of the low-copy-number plasmid pBsph in Bacillus sphaericus requires a partitioning ( par ) system that consists of a filament-forming protein, B. sphaericus TubZ (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 the 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 orf187 (encoding TubX), a gene downstream 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 the level of tubRZ -Bs expression, and the TubX-null phenotype was suppressed by ectopic expression of a wild-type copy of tubX and additional tubRZ -Bs. An electrophoresis mobility shift assay (EMSA) and a DNase I footprinting assay revealed that the TubX protein bound directly to five 8-bp degenerate repeats located in the par promoter region and that TubX competed with TubR-Bs for binding to the par promoter. Further studies demonstrated that TubX significantly stimulated the transcription of the par operon in the absence of tubR -Bs, and a higher level of gene activation was observed when tubR -Bs was present. These results suggested that TubX positively regulates tubRZ -Bs transcription by interfering with TubR-Bs-mediated repression and binding directly to the tubRZ -Bs promoter region. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|