GTP-dependent polymerization of the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis.

Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
Molecular Microbiology (Impact Factor: 5.03). 03/2008; 67(4):881-90. DOI: 10.1111/j.1365-2958.2007.06100.x
Source: PubMed

ABSTRACT RepX protein encoded by the pXO1 plasmid of Bacillus anthracis is required for plasmid replication. RepX harbours the tubulin signature motif and contains limited amino acid sequence homology to the bacterial cell division protein FtsZ. Although replication proteins are not known to polymerize, here we show by electron microscopy that RepX undergoes GTP-dependent polymerization into long filaments. RepX filaments assembled in the presence of GTPgammaS were more stable than those assembled in the presence of GTP, suggesting a role for GTP hydrolysis in the depolymerization of the filaments. Light scattering studies showed that RepX underwent rapid polymerization, and substitution of GTP with GTPgammaS stabilized the filaments. RepX exhibited GTPase activity and a mutation in the tubulin signature motif severely impaired its GTPase activity and its polymerization in vitro. Unlike FtsZ homologues, RepX harbours a highly basic carboxyl-terminal region and exhibits GTP-dependent, non-specific DNA binding activity. We speculate that RepX may be involved in both the replication and segregation of the pXO1 plasmid.

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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 contains 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 TubZ-Bs are involved in the replication and probably in the partitioning of pBsph plasmid, and the information facilitates our understanding of the genetic particularity of TubZ systems.
    Microbiology 04/2014; DOI:10.1099/mic.0.075465-0 · 3.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA segregation in bacteria is mediated most frequently by proteins of the ParA superfamily that transport DNA molecules attached via the segrosome nucleoprotein complex. Segregation is governed by a cycle of ATP-induced polymerization and subsequent depolymerization of the ParA factor. Here, we establish that hyperactive ATPase variants of the ParA homolog, ParF, display altered segrosome dynamics that block accurate DNA segregation. An arginine finger-like motif in the ParG centromere binding factor augments ParF ATPase activity, but is ineffective in stimulating nucleotide hydrolysis by the hyperactive proteins. Moreover, whereas polymerization of wild-type ParF is accelerated by ATP and inhibited by ADP, filamentation of the mutated proteins is blocked indiscriminately by nucleotides. The mutations affect a triplet of conserved residues that are situated neither in canonical nucleotide binding and hydrolysis motifs in the ParF tertiary structure nor at interfaces implicated in ParF polymerization. Instead the residues are involved in shaping the contours of the binding pocket so that nucleotide binding locks the mutant proteins into a configuration that is refractory to polymerization. Thus, the architecture of the pocket is crucial not only for optimal ATPase kinetics, but also plays a key role in the polymerization dynamics of ParA proteins that drive DNA segregation ubiquitously in procaryotes.
    Journal of Biological Chemistry 10/2012; DOI:10.1074/jbc.M112.410324 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Far from being simple 'bags' of enzymes, bacteria are richly endowed with ultrastructures that challenge and expand standard definitions of the cytoskeleton. Here we review rods, rings, twisted pairs, tubes, sheets, spirals, moving patches, meshes and composites, and suggest defining the term 'bacterial cytoskeleton' as all cytoplasmic protein filaments and their superstructures that move or scaffold (stabilize/position/recruit) other cellular materials. The evolution of each superstructure has been driven by specific functional requirements. As a result, while homologous proteins with different functions have evolved to form surprisingly divergent superstructures, those of unrelated proteins with similar functions have converged.
    Current opinion in cell biology 11/2012; DOI:10.1016/ · 14.15 Impact Factor