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Stoichiometry of P1 Plasmid Partition Complexes*

  • Jean-Yves Bouet
    Footnotes
    Affiliations
    Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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  • Jennifer A. Surtees
    Affiliations
    Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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  • Barbara E. Funnell
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular and Medical Genetics, Medical Sciences Bldg., University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1665; Fax: 416-978-6885
    Affiliations
    Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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  • Author Footnotes
    * This work was supported by a University of Toronto Open Fellowship (to J. A. S.) and a grant from the Medical Research Council of Canada (to B. E. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Present address: Laboratoire de Microbiologie et de Génétique Moléculaire du CNRS, 118 route de Narbonne, 31062 Toulouse cedex, France.
Open AccessPublished:March 17, 2000DOI:https://doi.org/10.1074/jbc.275.11.8213
      The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1. We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend inparS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.
      IHF
      integration host factor
      OP-Cu
      1,10-phenanthroline-copper
      PVDF
      polyvinylidene difluoride
      bp
      base pair(s)
      I complex
      IHF complex
      I + B1
      IHF plus ParB complex 1
      I + hB1
      IHF plus His-ParB complex 1
      I + B2
      IHF plus ParB complex 2
      I + hB2
      IHF plus His-ParB complex 2
      I + B2/hB2
      IHF plus ParB/His-ParB complex 2
      I + B3
      IHF plus ParB complex 3
      Low copy number plasmids in bacteria, such as the P1 plasmid/prophage, are faithfully maintained in growing cell populations. This stable inheritance is dependent on active partition systems that are responsible for proper intracellular localization of these plasmids (reviewed in Refs.
      • Williams D.R.
      • Thomas C.M.
      and
      • Hiraga S.
      ). Fluorescently tagged plasmids and fluorescence in situ hybridization have been used to show that P1 and F plasmids are specifically located at the ¼ and ¾ positions in the cell for most of the cell cycle (
      • Niki H.
      • Hiraga S.
      ,
      • Gordon G.S.
      • Sitnikov D.
      • Webb C.D.
      • Teleman A.
      • Straight A.
      • Losick R.
      • Murray A.W.
      • Wright A.
      ). Localization is directed via a partition complex, which consists of a plasmid protein bound specifically to acis-acting plasmid site.
      In P1, the par operon contains three elements that are essential for proper segregation: the parA andparB genes and a centromere-like site, parS (
      • Abeles A.L.
      • Friedman S.A.
      • Austin S.J.
      ). ParB and the Escherichia coli integration host factor (IHF)1 assemble specifically at parS to form the partition complex (
      • Funnell B.E.
      ,
      • Davis M.A.
      • Austin S.J.
      ). A highly organized nucleoprotein structure has also been reported for F plasmid (
      • Lynch A.S.
      • Wang J.C.
      ,
      • Biek D.P.
      • Strings J.
      ), and a similar complex may form on the Bacillus subtilis chromosome at the binding sites of the SpoOJ protein, a ParB homolog (
      • Ireton K.
      • Gunther N.W.
      • Grossman A.D.
      ,
      • Lin D.C.-H.
      • Grossman A.D.
      ). In addition to their role as the substrate for localization, it is speculated that partition complexes serve to promote pairing between newly replicated replicons, which are subsequently separated by an as yet unidentified mechanism. Pairing between partition complexes has recently been described for the R1 plasmid, which has a similar but nonhomologous partition system (
      • Jensen R.B.
      • Lurz R.
      • Gerdes K.
      ).
      The current picture of the P1 partition complex is derived from a variety of protein-DNA binding experiments in vitro and examination of mutant parS sites in vivo(
      • Funnell B.E.
      ,
      • Davis M.A.
      • Martin K.A.
      • Austin S.J.
      ,
      • Funnell B.E.
      • Gagnier L.
      ,
      • Funnell B.E.
      • Gagnier L.
      ,
      • Hayes F.
      • Austin S.
      ). ParB recognizes two distinct sets of repeated sequences, called box A and box B repeats, that flank an IHF binding site inparS (Fig. 1 A) (
      • Funnell B.E.
      ,
      • Funnell B.E.
      ). Binding of IHF toparS creates a large bend in the DNA, which greatly increases ParB's affinity for parS (
      • Funnell B.E.
      ). Proper phasing of the A and B boxes across this bend is functionally important for formation of the partition complex (
      • Funnell B.E.
      • Gagnier L.
      ,
      • Hayes F.
      • Austin S.
      ). ParB affinity is also greatly increased by superhelicity in the DNA substrate. All of these data suggest a partition complex structure in which the DNA is wrapped around a core composed of IHF and ParB. IHF binds to its specific sites as one IHFα/IHFβ heterodimer (
      • Yang C.-C.
      • Nash H.A.
      ,
      • Rice P.A.
      • Yang S.W.
      • Mizuuchi K.
      • Nash H.A.
      ), but the stoichiometry of ParB assembled on the partition complex at parS is not known.
      Figure thumbnail gr1
      Figure 1The P1 parS site. Gray and white boxes indicating the ParB box A and box B recognition motifs, respectively, are drawnbehind the DNA sequence. The left endis 25 bp from a TaqI restriction site in P1; the sequence between the TaqI and StyI restriction sites represents the P1 sequence in all of the DNA fragments used in this study (see “Experimental Procedures”). The IHF binding site, determined by DNase I footprinting studies (
      • Funnell B.E.
      ), is highlighted withdashed lines.
      Another characteristic of the centromeric parS site is the ability to silence neighboring genes when ParB is present (
      • Lobocka M.
      • Yarmolinsky M.
      ,
      • Rodionov O.
      • Lobocka M.
      • Yarmolinsky M.
      ). It has been proposed that silencing occurs when ParB molecules spread along and cover the DNA from a nucleation point, which isparS. The role of this larger structure in plasmid segregation is unknown, but this observation suggests that the partition complex can adopt different structures in vivo.
      One important question that remains is the stoichiometry of ParB in the P1 partition complex. ParB is a dimer in solution (
      • Funnell B.E.
      ). In this study, we have examined the nature and stoichiometry of the binding of ParB toparS by gel electrophoresis. We find that one dimer is sufficient to interact with the ParB recognition sequences that span the IHF-directed bend, resulting in the high affinity binding toparS that is observed for ParB and IHF. At higher concentrations of ParB, more dimers of ParB join this complex to create even higher order protein-DNA complexes.

      DISCUSSION

      The formation of the P1 partition complex is an essential step in the segregation of the unit-copy number P1 plasmid at cell division. We have shown that the initial ParB + IHF complex visualized by gel mobility shift assays (I + B1) contains a single dimer of ParB, while the next complex formed (I + B2) contains two dimers of ParB. At higher concentrations of ParB, complexes with increasingly slower migration are observed (I + B3, I + B4, etc.), which presumably result from the loading of additional ParB dimers onto the nucleoprotein complex.
      The stoichiometry determined here has interesting implications for the architecture of this partition complex. In particular, the observation that one ParB dimer is responsible for the I + B1 complex is intriguing given the number of specific sequences in parS that are recognized by ParB. parS contains two copies of the box B repeat and four copies of the box A repeat (Fig. 1). We favor a model in which one dimer of ParB is precisely docked to occupy both box B repeats and the box A2 and A3 repeat. First, since both box A2 and A3 repeats are essential for high affinity ParB binding, both must be filled to form the I + B1 complex. Second, although the OP-Cu footprints indicate an additional protection in the region of box A1 (Fig. 6), previous deletion and mutational analyses and DMS interference experiments have shown that boxes A1 and A4 inparS are not essential for partition or for high affinity binding by ParB (
      • Davis M.A.
      • Martin K.A.
      • Austin S.J.
      ,
      • Funnell B.E.
      • Gagnier L.
      ,
      • Funnell B.E.
      • Gagnier L.
      ). Previous DNase I footprinting experiments showed that ParB and IHF protected the region that contains box A1 from DNase I cleavage even when box A1 was mutated (changed by a 4-bp substitution mutation (
      • Davis M.A.
      • Martin K.A.
      • Austin S.J.
      )). This result implies that ParB is in the same position in the complex with or without box A1. Therefore, we propose that this region (box A1) is protected from both OP-Cu and DNase I cleavage by alterations in the geometry of the DNA, such as a compression of the minor groove caused by tightening of the bend for example. The alternative explanation is that the I + B1 complex is a mixture of orientations of ParB binding across parS, in which case the domain of ParB that is responsible for recognition of box A must be quite flexible with respect to the domain responsible for recognition of box B. Given that box A1 cannot substitute for either A2 or A3 (
      • Davis M.A.
      • Martin K.A.
      • Austin S.J.
      ,
      • Funnell B.E.
      • Gagnier L.
      ), this scenario seems less likely.
      Our previous data indicate that the bend produced by IHF binding toparS allows ParB to simultaneously contact its DNA recognition sequences that flank this bend (
      • Funnell B.E.
      • Gagnier L.
      ). Our present results show that it is one dimer of ParB that interacts across this bend. Dimerization of ParB is mediated through a domain located at the C terminus of the protein (
      • Lobocka M.
      • Yarmolinsky M.
      ,
      • Surtees J.A.
      • Funnell B.E.
      ). This region has also been shown to be involved in box B recognition (
      • Radnedge L.
      • Davis M.A.
      • Austin S.J.
      ). These observations lead to a model in which the dimerized C termini of one ParB dimer interact with both box B sequences simultaneously, perhaps threading the DNA between the monomers. In other words, the extreme ends of the parS site are brought together near or at the dimerization interface. This model is consistent with the biochemical data that indicate that the DNA is wrapped around a core of protein (
      • Funnell B.E.
      ,
      • Funnell B.E.
      • Gagnier L.
      ). It has been suggested that a putative helix-turn-helix motif in the center of ParB binds the box A motif (
      • Radnedge L.
      • Davis M.A.
      • Austin S.J.
      ,
      • Dodd I.B.
      • Egan J.B.
      ). In this case, these regions of each monomer would be directed toward box A2 and A3 (which make an inverted repeat sequence) but would be positioned on one side of the IHF bend rather than flanking it.
      An important question is whether the I + B1 complex is sufficient for partition in vivo. The affinity of ParB for the I + B2 complex is about 100-fold lower than its affinity for the I + B1 complex. ParB exists at relatively high concentrations in the cell (micromolar amounts) (
      • Funnell B.E.
      ,
      • Funnell B.E.
      • Gagnier L.
      ), and therefore progressive loading of ParB onto the DNA to form higher complexes probably also occursin vivo. This is supported by the observation that ParB binding can spread a great distance along the DNA on both sides ofparS under conditions where ParB can silence genes that are located close to parS (
      • Lobocka M.
      • Yarmolinsky M.
      ,
      • Rodionov O.
      • Lobocka M.
      • Yarmolinsky M.
      ). In addition, immunofluorescence analysis shows that ParB forms bright, discrete foci at the intracellular locations occupied by P1 plasmids, which suggests that much of the ParB in the cell converges on P1 at theparS site (
      • Erdmann N.
      • Petroff T.
      • Funnell B.E.
      ). However, the minimal amount of ParB that is necessary for partition in vivo has not been measured. Larger complexes (I + B2, I + B3, etc.) may be necessary to interact with ParA or with host factors. Alternatively, the I + B1 complex may be sufficient for partition, but not for competition (incompatibility). We have observed that weaker par sites (weakened by mutation of parS, for example (
      • Funnell B.E.
      • Gagnier L.
      )) are competent for partition but unable to compete with wild-type sites.
      Presumably binding of ParB to complex I + B1 to form complex I + B2 is mediated chiefly by protein-protein (dimer-dimer) interactions, which are weaker than the protein-DNA interactions that mediate complex I + B1. Such dimer-dimer interactions may occur via a self-association domain recently identified near the N terminus of ParB (
      • Surtees J.A.
      • Funnell B.E.
      ). In the OP-Cu footprints (Fig. 7), additional DNA on both edges ofparS (including box A4) becomes protected in the I + B2 complex, so it seems likely that both specific and nonspecific DNA contacts also contribute to the formation of complex I + B2.
      We reported recently that ParA interacts with ParB on the partition complex (
      • Bouet J.-Y.
      • Funnell B.E.
      ). The nature of this interaction is dependent on ParB concentration. At high ParB concentration, ParA is recruited to the partition complex, whereas at low ParB concentration, ParA interfered with ParB binding to parS. These interactions required magnesium in the gel and running buffer. Under these conditions, the two discrete complexes, corresponding to I + B1 and I + B2, are not observed, although the ParB complex migrates more slowly with increasing ParB concentrations (
      • Bouet J.-Y.
      • Funnell B.E.
      ). While the experimental conditions are not identical, it is possible, for example, that ParA is recruited only to a partition complex that contains two ParB dimers (complex I + B2 or higher), perhaps explaining a requirement for high concentrations of ParB.
      The architecture of ParB binding to parS is intriguing, given the organization of box A and box B motifs (Fig. 1) and the current result that one dimer interacts with these motifs to form the initial partition complex. Our results define the minimal protein requirements for parS binding. The high affinity core (complex I + B1) then recruits more ParB molecules by both protein-DNA and protein-protein interactions, and it will be important to determine how much ParB must bind the core structure in order for the complex to be competent for partition in vivo.

      Acknowledgments

      We thank Paul Sadowski and Marc Perry for critical reading of the manuscript.

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