Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative of<i>Bacillus subtilis</i>(<i>natto</i>) Plasmid pLS32

Takano, Teruo

doi:10.1128/jb.01312-09

Cited by 21 publications

(19 citation statements)

References 52 publications

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“…In contrast to type I and II systems, which could stabilize an unstable plasmid (27,28), the TubZ-encoding type III systems are required but insufficient for plasmid partition, and the pBsph and pBtoxis minireplicons carrying this system are segregationally highly unstable (8,21). Our previous studies demonstrated that pBsph encodes the polymerizing protein TubZ-Bs and the centromere-binding protein TubR-Bs and that their proper expression is essential for pBsph partitioning and stability (21).…”

Section: Discussionmentioning

confidence: 99%

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

Zhao

et al. 2014

J Bacteriol

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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.

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Section: Discussionmentioning

confidence: 99%

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

Zhao

et al. 2014

J Bacteriol

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“…Interestingly, we find that increasing the spacing between AlfBbinding repeats in parN increases the efficiency of nucleation. Either (i) there is no selective advantage to faster nucleation or (ii) other functions of AlfB, such as transcriptional repression (19), dictate its spacing along the DNA.…”

Section: Discussionmentioning

confidence: 99%

“…Two have been shown to encode proteins important for plasmid segregation: AlfA and AlfB. Loss of the third, AlfC, has little effect on plasmid stability (19). The AlfB protein is thought to bind three DNA repeats in the parN locus and form a kinetochore-like segrosome structure (19) that interacts with AlfA filaments.…”

Section: Regulation Of Alfa Polymer Dynamics By Elements Of the Alf Omentioning

confidence: 99%

Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling

Polka

Kollman

Mullins

2014

Proc. Natl. Acad. Sci. U.S.A.

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In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNAsegregating spindles.DNA segregation | bacterial cytoskeleton | Bacillus subtilis | reconstitution T he first filament-forming actin-like protein (ALP) was identified in bacteria in 2001 (1), and subsequent work identified more than 30 additional classes of ALPs in eubacteria and archaea (2). These proteins are involved in a variety of cellular processes, including assembly of the cell wall (1, 3-5), positioning of organelles (6), anchoring of cytokinesis machinery (7), and segregation of DNA (8). Most DNA-segregating ALPs participate in type II plasmid partitioning systems, which consist of three components: (i) a centromeric DNA sequence; (ii) a DNAbinding protein that interacts with the centromeric sequence to form a segrosome complex; and (iii) a polymer-forming ALP, whose self-assembly moves the segrosome through the cytoplasm (SI Appendix, Fig. S1). In the best understood type II segregation system, bidirectional polymerization of the ALP, ParM, pushes pairs of plasmids to opposite poles of rod-shaped cells. Extensive studies, both in vivo and in vitro, have produced detailed models of ParM-mediated DNA segregation (9, 10), but it is unclear to what extent these models can be generalized to describe other ALP-dependent DNA segregation systems.To better understand the diversity of molecular mechanisms underlying plasmid segregation, we studied a type II plasmid partitioning system encoded by the alf operon from the Bacillus subtilis plasmid pLS32 (11). The alf operon was first identified based on its ability to maintain plasmids through the process of sporulation, presumably by actively pushing a plasmid into the forespore at one end of the cell. In addition, the alf operon confers approximately eightfold greater stability to plasmids in rapidly dividing cells. The alf operon itself contains a centromeric sequence, parN, with three short, repeated sequences, and it encodes both an ALP, AlfA, and a DNA-binding protein, AlfB. The AlfA protein shares 15% sequence identity with ParM (2), whereas AlfB shares only 8% identity with ParR and has no significant BLAST hits (expect value < 1). In addition to sequence differences, AlfA...

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“…subtilis (27), but a highly homologous plasmid, pLS32, which is found in a more distant strain of B. subtilis subsp. natto, has been analyzed and sequenced (28). Notably, pBS32 has been shown to carry an additional rap-phr cassette, termed rapP-phrP, and RapP was specifically shown to contribute to the difference in biofilm-forming capabilities between strains 3610 and 168 (25) (Fig.…”

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confidence: 99%

The RapP-PhrP Quorum-Sensing System of Bacillus subtilis Strain NCIB3610 Affects Biofilm Formation through Multiple Targets, Due to an Atypical Signal-Insensitive Allele of RapP

et al. 2014

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The genome of Bacillus subtilis 168 encodes eight rap-phr quorum-sensing pairs. Rap proteins of all characterized Rap-Phr pairs inhibit the function of one or several important response regulators: ComA, Spo0F, or DegU. This inhibition is relieved upon binding of the peptide encoded by the cognate phr gene. Bacillus subtilis strain NCIB3610, the biofilm-proficient ancestor of strain 168, encodes, in addition, the rapP-phrP pair on the plasmid pBS32. RapP was shown to dephosphorylate Spo0F and to regulate biofilm formation, but unlike other Rap-Phr pairs, RapP does not interact with PhrP. In this work we extend the analysis of the RapP pathway by reexamining its transcriptional regulation, its effect on downstream targets, and its interaction with PhrP. At the transcriptional level, we show that rapP and phrP regulation is similar to that of other rap-phr pairs. We further find that RapP has an Spo0F-independent negative effect on biofilm-related genes, which is mediated by the response regulator ComA. Finally, we find that the insensitivity of RapP to PhrP is due to a substitution of a highly conserved residue in the peptide binding domain of the rapP allele of strain NCIB3610. Reversing this substitution to the consensus amino acid restores the PhrP dependence of RapP activity and eliminates the effects of the rapP-phrP locus on ComA activity and biofilm formation. Taken together, our results suggest that RapP strongly represses biofilm formation through multiple targets and that PhrP does not counteract RapP due to a rare mutation in rapP.T he behavior of bacterial communities is often regulated by quorum-sensing (QS) signaling pathways. In these pathways, a secreted molecular signal accumulates in the environment and activates a cognate receptor at sufficiently high concentrations. Gram-positive bacteria frequently utilize peptides as QS signals (1-3), thereby eliciting a variety of behaviors, including the secretion of various molecules (e.g., enzymes, antibiotics, surfactants, and exopolysaccharides [4]), the initiation of developmental processes (such as sporulation and biofilm formation), and horizontal gene transfer through transformation or conjugation (5-7).Members of the Rap-Phr family of QS systems in the Grampositive model Bacillus subtilis are involved in the regulation of competence, sporulation, and biofilm formation. The chromosome of strain B. subtilis 168 encodes eight full receptor signal systems and three orphan receptors from this family (6,8). In each of the full systems, the phr gene encodes a prepeptide, which is secreted through the major secretory system and then further processed outside the cell, resulting in the formation of a mature penta-or hexapeptide signal (9). The mature peptide is transported into the cell by the oligopeptide-permease (Opp) complex, where it interacts with its Rap targets within the cytoplasm (10). All characterized Rap proteins contain two domains (10-12), a tricopeptide repeat (TPR) domain that interacts with the signaling peptide and a second domain that f...

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Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative ofBacillus subtilis(natto) Plasmid pLS32

Cited by 21 publications

References 52 publications

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

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

Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling

The RapP-PhrP Quorum-Sensing System of Bacillus subtilis Strain NCIB3610 Affects Biofilm Formation through Multiple Targets, Due to an Atypical Signal-Insensitive Allele of RapP

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