Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism

Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2013; 110(15). DOI: 10.1073/pnas.1302745110
Source: PubMed


Increasingly diverse types of cargo are being found to be segregated and positioned by ParA-type ATPases. Several minimalistic systems described in bacteria are self-organizing and are known to affect the transport of plasmids, protein machineries, and chromosomal loci. One well-studied model is the F plasmid partition system, SopABC. In vivo, SopA ATPase forms dynamic patterns on the nucleoid in the presence of the ATPase stimulator, SopB, which binds to the sopC site on the plasmid, demarcating it as the cargo. To understand the relationship between nucleoid patterning and plasmid transport, we established a cell-free system to study plasmid partition reactions in a DNA-carpeted flowcell. We observed depletion zones of the partition ATPase on the DNA carpet surrounding partition complexes. The findings favor a diffusion-ratchet model for plasmid motion whereby partition complexes create an ATPase concentration gradient and then climb up this gradient toward higher concentrations of the ATPase. Here, we report on the dynamic properties of the Sop system on a DNA-carpet substrate, which further support the proposed diffusion-ratchet mechanism.

Download full-text


Available from: Ling Chin Hwang, Oct 06, 2015
1 Follower
107 Reads
  • Source
    • "Many studies on Par homologues (plasmid and chromosomally encoded) have demonstrated a role for a dynamic ParA scaffold and the importance of ParA–ParB interactions in bacterial DNA segregation (reviewed by Gerdes et al., 2010; Banigan et al., 2011; Hwang et al., 2013; Lim et al., 2014; Scholefield et al., 2011; Vecchiarelli et al., 2013). In the case of chromosomally encoded Par proteins, their roles in regulation of other cellular processes in a species-specific manner were also demonstrated (Kadoya et al., 2011; Murray & Errington, 2008; Scholefield et al., 2011; Thanbichler & Shapiro, 2006; Yamaichi et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: P. aeruginosa ParA belongs to a large subfamily of Walker-type ATPases acting as partitioning proteins in bacteria. ParA has the ability to both self-associate and interact with its partner ParB. Analysis of the deletion mutants defined the part of the protein involved in dimerization and interactions with ParB. Here, a set of the ParA alanine substitution mutants in the region between E67 and L85 was created and analyzed in vivo and in vitro. All mutants impaired in dimerization (substitutions at positions M74, H79, Y82, L84) were also defective in interactions with ParB suggesting that ParA-ParB interactions depend on the ability of ParA to dimerize. Mutants with alanine substitutions at positions E67, C68, L70, E72, F76, Q83 and L85 were not impaired in dimerization but defective in interactions with ParB. The dimerization interface partly overlaps the pseudo-hairpin, involved in interactions with ParB. ParA mutant derivatives tested in vitro showed no defects in ATPase activity. Two parA alleles, parA84, whose product can neither self-interact nor interact with ParB, and parA67, whose product is impaired in interactions with ParB but not in dimerization, were introduced into P. aeruginosa chromosome by homologous gene exchange. Both mutants showed defective separation of ParB foci but to different extents. Only PAO1161 parA84 was visibly impaired in chromosome segregation, growth rate and motilities similarly to a parAnull mutant.
    Microbiology 08/2014; 160(Pt 11). DOI:10.1099/mic.0.081216-0 · 2.56 Impact Factor
  • Source
    • " possibility that ParB / parS is simply diffusing and that its binding to the DNA - bound ParA - ATP dimers results in a biased diffusion along the ParA - ATP dimer gradient , as we previously suggested ( Schofield et al . , 2010 ) . This mechanism would be similar to the Brownian ratchet proposed for the P1 and F plasmids ( Hwang et al . , 2013 ; Vecchiarelli et al . , 2013 , 2014 ) ."
    [Show abstract] [Hide abstract]
    ABSTRACT: The widely conserved ParABS system plays a major role in bacterial chromosome segregation. How the components of this system work together to generate translocation force and directional motion remains uncertain. Here, we combine biochemical approaches, quantitative imaging and mathematical modeling to examine the mechanism by which ParA drives the translocation of the ParB/parS partition complex in Caulobacter crescentus. Our experiments, together with simulations grounded on experimentally-determined biochemical and cellular parameters, suggest a novel 'DNA-relay' mechanism in which the chromosome plays a mechanical function. In this model, DNA-bound ParA-ATP dimers serve as transient tethers that harness the elastic dynamics of the chromosome to relay the partition complex from one DNA region to another across a ParA-ATP dimer gradient. Since ParA-like proteins are implicated in the partitioning of various cytoplasmic cargos, the conservation of their DNA-binding activity suggests that the DNA-relay mechanism may be a general form of intracellular transport in bacteria. DOI:
    eLife Sciences 05/2014; 3(3):e02758. DOI:10.7554/eLife.02758 · 9.32 Impact Factor
  • Source
    • "Based on the phylogeny and type of NTPase involved, the bacterial par systems have been generally classified into three types (Gerdes et al., 2010). Type I systems contain ATPase (ParA) with a deviant Walker-box motif and actively segregate both bacterial plasmids and chromosomes by ParAs patterning on the nucleoid (Vecchiarelli et al., 2013). The most well-understood type II systems encode actin-like ATPase (ParM), which assembles into dynamic anti-parallel filaments and pushes the plasmids to opposite cell poles (Gayathri et al., 2012). "
    [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; 160(Pt_6). DOI:10.1099/mic.0.075465-0 · 2.56 Impact Factor
Show more