Distinct architectural requirements for the parS centromeric sequence of the pSM19035 plasmid partition machinery

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    Evaluation Summary:

    The work by Volante et al. studied a new plasmid partition system, in which the authors discovered that four or more contiguous ParS sequence repeats are required to assemble a stable partitioning ParAB complex and to activate the ParA ATPase. The work reveals a new plasmid partitioning mechanism in which the mechanic property of DNA and its interaction with the partition complex may drive the directional movement of the plasmid.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Three-component ParABS partition systems ensure stable inheritance of many bacterial chromosomes and low-copy-number plasmids. ParA localizes to the nucleoid through its ATP-dependent nonspecific DNA-binding activity, whereas centromere-like parS -DNA and ParB form partition complexes that activate ParA-ATPase to drive the system dynamics. The essential parS sequence arrangements vary among ParABS systems, reflecting the architectural diversity of their partition complexes. Here, we focus on the pSM19035 plasmid partition system that uses a ParB pSM of the ribbon-helix-helix (RHH) family. We show that parS pSM with four or more contiguous ParB pSM -binding sequence repeats is required to assemble a stable ParA pSM -ParB pSM complex and efficiently activate the ParA pSM -ATPase, stimulating complex disassembly. Disruption of the contiguity of the parS pSM sequence array destabilizes the ParA pSM -ParB pSM complex and prevents efficient ATPase activation. Our findings reveal the unique architecture of the pSM19035 partition complex and how it interacts with nucleoid-bound ParA pSM -ATP.

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  1. Author Response

    Evaluation Summary:

    The work by Volante et al. studied a new plasmid partition system, in which the authors discovered that four or more contiguous ParS sequence repeats are required to assemble a stable partitioning ParAB complex and to activate the ParA ATPase. The work reveals a new plasmid partitioning mechanism in which the mechanic property of DNA and its interaction with the partition complex may drive the directional movement of the plasmid.

    Thank you for the kind evaluation. But we wonder about the description of the pSM19035 partition system we studied here as “a new plasmid partition system”. This system itself is quite old. The editor might have meant “new” as a subject of a research, but plasmid partition systems involving RHH-ParB proteins have been studied by number of groups for some time, including …

  2. Evaluation Summary:

    The work by Volante et al. studied a new plasmid partition system, in which the authors discovered that four or more contiguous ParS sequence repeats are required to assemble a stable partitioning ParAB complex and to activate the ParA ATPase. The work reveals a new plasmid partitioning mechanism in which the mechanic property of DNA and its interaction with the partition complex may drive the directional movement of the plasmid.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. Reviewer #1 (Public Review):

    This is a very thorough biochemical work that investigated the ParABS system in pSM19035 by Volante et al. Volante et al showed convincingly that a specific architecture of the centromere (parS) of pSM19035 is required to assemble a stable/functional partition complex. Minimally, four consecutive parS are required for the formation of partition complex, and to efficiently activate the ATPase activity of ParA. The work is very interesting, and the discovery will allow the community to compare and contrast to the more widespread/more investigated canonical chromosomal ParABS system (where ParB is a sliding CTPase protein clamp, and a single parS site is often sufficient to assemble a working partition complex). All the main conclusions in the abstract are justified and supported by biochemical data with …

  4. Reviewer #2 (Public Review):

    ParBs come in two variations, RHH and HTH. In this study, the authors examine the in vitro behavior of the RHH system, which is less studied. Two activities were carefully monitored; ATPase activation and ParA removal from DNA. The system is quite complex, but the authors have done a good job of examining parameter space. One question concerns the physiological relevance. Can this be assessed by uncoupling ParA/ParB expression (making it inducible with IPTG from the chromosome, for example) and testing plasmids with the various constructs?

    The authors appear to suggest that the requirement for at least 4 ParB binding sites is due to the inability of ParBs of this type to spread inferring that for the ParB-HTH multiple ParBs bound to ParS are required. Has this been tested in this system? In any event, …

  5. Reviewer #3 (Public Review):

    Drs. Volante, Alonso, and Mizzuchi presented a milestone experimental finding on how the distinct architecture of centromere (ParS) on bacterial plasmid drives the ParABS-mediated genome partition process. Rather than driven by cytoskeletal filament pushing or pulling as its eukaryotic counterpart, the genome partition in prokaryotes is demonstrated to operate as a burnt-bridge Brownian Ratchet, first put forward by the Mizuuchi group. To drive directed and persistent movement without linear motor proteins, this Brownian Ratchet requires two factors: 1) enough bonds (10s' to 100s') bridging the PC-bound ParB to the nucleoid-bound ParA to largely quench the diffusive motion of the PC, and 2) the PC-bound ParB 'kicks" off the nucleoid-bound ParA that can replenish the nucleoid only after a sufficient time …