A new tubRZ operon involved in the maintenance of the Bacillus sphaericus mosquitocidal plasmid pBsph

Abstract

A new tubRZ operon involved in the maintenance of the Bacillus sphaericus mosquitocidal plasmid pBsph pBsph is a mosquitocidal plasmid first identified from Bacillus sphaericus, encoding binary toxins (Bin toxins) that are highly toxic to mosquito larvae. This plasmid plays an important role in the maintenance and evolution of the bin genes in B. sphaericus. However, little is known about its replication and partitioning. Here, we identified a 2.4 kb minimal replicon of pBsph plasmid that contained an operon encoding TubR-Bs and TubZ-Bs, each of which was shown to be required for plasmid replication. TubR-Bs was shown to be a transcriptional repressor of tubRZ-Bs genes and could bind cooperatively to the replication origin of eleven 12 bp degenerate repeats in three blocks, and this binding was essential for plasmid replication. TubZ-Bs exhibited GTPase activities and interacted with TubR-Bs : DNA complex to form a ternary nucleoprotein apparatus. Electron and fluorescence microscopy revealed that TubZ-Bs assembled filaments both in vitro and in vivo, and two point mutations in TubZ-Bs (T114A and Y260A) that severely impaired the GTPase and polymerization activities were found to be defective for plasmid maintenance. Further investigation demonstrated that overproduction of TubZ-Bs-GFP or its mutant forms significantly reduced the stability of pBsph. Taken together, these results suggested that TubR-Bs and TubZBs are involved in the replication and probably in the partitioning of pBsph plasmid, increasing our understanding of the genetic particularity of TubZ systems.

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

“…The TubX protein with a six-histidine (His 6 ) tag at the C terminus was purified from E. coli, and the TubR-Bs protein was prepared as described previously (21). The pET28b-tubX expression plasmid was constructed by PCR amplification of the tubX sequence from the B. sphaericus C3-41 genome using primer pair 187-F/187-R.…”

“…The relative RNA abundance of each gene compared to that of rpoB was calculated using the 2 Ϫ⌬⌬CT comparative method (25,26), and the results were obtained from the analyses of at least four independent RNA samples. The copy number of the pBsph plasmid in B. sphaericus was measured by qPCR by the method described previously, using the chromosome-specific reference gene adk (21).…”

“…TubZ-Bs interacts with the TubRC complex to form a ternary nucleoprotein complex, which is demonstrated to be important for plasmid segregation. Additionally, miniplasmids involving the TubZ system are segregationally highly unstable, whereas the native plasmids are stably maintained in Bacillus species (8,21), indicating the presence of other plasmid-encoded stability determinants. However, remarkably little is known regarding the factors and mechanisms that contribute to this stability discrepancy between the native plasmid and its minireplicon.…”

“…The Bacillus sphaericus mosquitocidal plasmid pBsph is a large (178-kb) low-copy-number (1 to 2 copies per chromosome) plasmid (19,20) with its segregation/replication machinery encoded by the par operon, composed of tubC, tubR-Bs, and tubZ-Bs (21). The pBsph par centromere-like site tubC is composed of three blocks (I, II, and III) of 12-bp degenerate repeats (iterons) separated by a 54-bp promoter region and a 57-bp AT-rich spacer.…”

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

“…However, TubZRC alone maintains synthetic plasmids only under artificial selective pressure (7,11), and replication of pBtoxis occurs without the dnaAbox but not without TubZRC, prompting others to propose a role for the TubZRC system in plasmid replication (12). In vitro, TubZ assembles into two-and four-stranded polymers (13)(14)(15)(16), and structural studies suggested that the centromeric TubRC complex forms a ring-like structure (17), interacting with the long C-terminal TubZ extensions (18).…”