Probing plasmid partition with centromere‐based incompatibility

Bouet, Jean‐Yves; Rech, Jérôme; Egloff, Sylvain; Biek, Donald; Lane, David

doi:10.1111/j.1365-2958.2004.04396.x

Cited by 56 publications

(75 citation statements)

References 61 publications

(100 reference statements)

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“…SopA uniformly binds the DNA carpet away from SopB-bound cargo (sopC-bead). We have previously shown that, under these conditions, SopA binding density on the DNA carpet is less than 1% of saturation (6), which is comparable to the in vivo estimate of 0.2% binding density per bacterial chromosome (19). SopB stimulates the release of SopA on and around the cargo, forming a local SopA gradient on the carpet by a reaction-diffusion process.…”

Section: Discussionmentioning

confidence: 83%

A propagating ATPase gradient drives transport of surface-confined cellular cargo

Vecchiarelli

Neuman

Mizuuchi

2014

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

171

227

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Significance The process of DNA segregation is of central importance for all organisms. Although the basic mechanism of eukaryotic mitosis is relatively well established, the most common mechanism used for bacterial DNA segregation has been unclear. ParA ATPases form dynamic patterns on the bacterial nucleoid to spatially organize plasmids, chromosomes and other large cellular cargo, but the force-generating mechanism has been a source of controversy and debate. A dominant view proposes that ParA-mediated transport and cargo positioning occurs via a filament-based mechanism that resembles eukaryotic mitosis. Here, we present direct evidence against such models. Our cell-free reconstitution supports a non–filament-based mode of transport that may be as widely found in nature as filament-based mechanisms.

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Section: Discussionmentioning

confidence: 83%

A propagating ATPase gradient drives transport of surface-confined cellular cargo

Vecchiarelli

Neuman

Mizuuchi

2014

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

171

227

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“…pJYB57 was made by inserting the sopC sequence, amplified by PCR from pDAG114 (29), into pBSKS(ϩ) using EcoRV and EcoRI restriction sites. pYAS6 is a pTYBI derivative (NEBioLabs) into which, the sopB-G324 gene, PCR amplified from pDAG170 (30), was inserted using NdeI and SapI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein

Ah-Seng

Lopez

Pasta

et al. 2009

Journal of Biological Chemistry

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In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis.

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“…If one assumes that 1,000 SopA dimers exist inside an Escherichia coli cell with one chromosome (22), ∼0.2% of the chromosomal DNA would be bound by SopA. Also, nucleoid-associated proteins and transcription factors dynamically occupy a significant fraction of the bacterial chromosome and likely prevent SopA from forming stable higherorder complexes on the nucleoid surface.…”

mentioning

confidence: 99%

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

Vecchiarelli

Hwang

Mizuuchi

2013

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

138

185

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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.bacterial chromosome segregation | ParA ATPase | plasmid segregation | spatial pattern organization | chromosome dynamics P roper DNA segregation ensures the faithful inheritance of genomic information for all life forms. In bacteria, this fundamental process is poorly understood. Low-copy bacterial genomes, including plasmids and chromosomes, encode active partition (Par) systems to ensure stability. Par systems are minimalistic in that only three dedicated components are required: a partition site on the DNA, a partition site-binding protein, and a nucleoside triphosphatase (NTPase). Par systems have been classified according to the type of NTPase involved: Walker-type (generically called "ParA"), actin-like, or tubulin-like (reviewed in ref. 1). Reconstitution of purified Par components of R1 plasmid in a cell-free system unveiled the mechanism involving an actin-like ATPase, ParM, in which elongating filaments of the ATPase push plasmids to opposite cell poles (2). Tubulin-like GTPases also appear to function as a filament (3). However, all chromosome-based and most plasmid-based systems use ParAs, and mounting evidence shows that ParA-like ATPases also are responsible for transporting large protein machineries (reviewed in ref. 4). However, the underlying mechanism for reactions of this category remains unresolved.The Sop system (stability of plasmid) of F plasmid is one of the first Par systems to be identified (5, 6) and is considered a paradigm for the study of ParA-mediated DNA segregation. The three plasmid-encoded system components are SopA (the ParAtype ATPase), SopB (or ParB in other systems; i.e., the partition site-binding protein), and sopC (or parS in other systems; i.e., the cis-acting partition site on the plasmid). The first task of a partition system is to identify its DNA cargo. SopB accomplishes this task by loading onto sopC and forming a partition c...

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Probing plasmid partition with centromere‐based incompatibility

Cited by 56 publications

References 61 publications

A propagating ATPase gradient drives transport of surface-confined cellular cargo

A propagating ATPase gradient drives transport of surface-confined cellular cargo

Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein

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

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