A Novel Transcriptional Activator, tubX , Is Required for the Stability of Bacillus sphaericus Mosquitocidal Plasmid pBsph

Abstract

bStable 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 centromerelike DNA site, tubC, composed of three blocks (I, II, and III) of 12-bp degenerate repeats. Previous studies have shown that minipBsph 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.

The C-terminal region of the plasmid partitioning protein TubY is a tetramer that can bind membranes and DNA

“…Previously, tubY and tubRZ were shown to form a regulon whose gene products work cooperatively (15,16). The EMSA analyses in this study showed that BcY associates with the TubR-DNA complex to form a supramolecular structure, implying that BcY is a component of the segrosome.…”

Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

“…More complex mechanisms might involve that the initial growing end is captured to a cellular structure, which then contributes to pulling of DNA when disassembling. Additional components similar to organizing centers, spindle poles that anchor filaments as suggested for the PhuZ-based phage centering system (28), or other regulators may be required (29). One possibility for an anchor could be the nucleoid, and it has been noted that another gene, downstream of TubZRC, tubY (pBt158 on pBtoxis), might have DNA-binding activity and hence might be a candidate for this requirement (20).…”

Commensal Propionibacterium strain UF1 mitigates intestinal inflammation via Th17 cell regulation

“…Deletion of dlaT in P. UF1 resulted in significant transcriptic changes involved in the glycolytic and multiple metabolic pathways, including the metabolism of carbohydrates, nitrogen, folate, and cysteine biosynthesis (Supplemental Figure 4, A-C Figure 4D). To investigate the mechanistic paradigm associated with DlaT-specific Th17 cell differentiation, the dlaT gene was deleted from the bacterial chromosome by homologous recombination with a single crossover event (44), resulting in the ΔdlaT P. UF1 ( Figure 5, A-D). Importantly, the bacterial growth kinetics and detection of ΔdlaT P. UF1 in conventional C57BL/6 mouse fecal and cecal contents were comparable to P. UF1 ( Figure 5, E and F).…”