Biochemical activities of the ParA partition protein of the P1 plasmid

The unit-copy P1 plasmid depends for stability on a plasmid-encoded partition region called par, consisting of the parA and parB genes and the parS site. ParA is absolutely required for partition, but its partition-critical role is not known. Purified ParA protein is shown to possess an ATPase activity in vitro which is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression of parA and parB, and purified ParA has an ATP-dependent, site-specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The role of the ATP-dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, is discussed.     

Fine-structure analysis of the P1 plasmid partition site.

P1 plasmid partition requires two plasmid-encoded Par proteins and a cis-acting site. The site, parS, lies in a region consisting of a 13-bp palindrome and an adjacent AT-rich sequence. A series of point mutations were analyzed for their effects on partition site activity. The results indicated that only the left arm of the palindrome and some adjacent bases were needed. The limits of the functional site were further refined to a maximum of 22 bp, which includes binding sites for the P1 ParB protein. Mutations in the 22-bp site cause concomitant defects in partition and the ability to exert partition-mediated incompatibility. Like the region immediately to the left of the 22-bp region, the right arm of the palindrome is not essential for partition but does contain information that affects the specificity of incompatibility.     

ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS

Localization of the P1 plasmid requires two proteins, ParA and ParB, which act on the plasmid partition site, parS. ParB is a site-specific DNA-binding protein and ParA is a Walker-type ATPase with non-specific DNA-binding activity. In vivo ParA binds the bacterial nucleoid and forms dynamic patterns that are governed by the ParB-parS partition complex on the plasmid. How these interactions drive plasmid movement and localization is not well understood. Here we have identified a large protein-DNA complex in vitro that requires ParA, ParB and ATP, and have characterized its assembly by sucrose gradient sedimentation and light scattering assays. ATP binding and hydrolysis mediated the assembly and disassembly of this complex, while ADP antagonized complex formation. The complex was not dependent on, but was stabilized by, parS. The properties indicate that ParA and ParB are binding and bridging multiple DNA molecules to create a large meshwork of protein-DNA molecules that involves both specific and non-specific DNA. We propose that this complex represents a dynamic adaptor complex between the plasmid and nucleoid, and further, that this interaction drives the redistribution of partition proteins and the plasmid over the nucleoid during partition.     

P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities

The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis-acting site parS. parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex. ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process. In this study we demonstrate a direct interaction between ParA and the P1 partition complex. The interaction was strictly dependent on ParB and ATP. The consequence of this interaction depended on the ParB concentration. At high ParB levels, ParA was recruited to the partition complex via a ParA-ParB interaction, but at low ParB levels, ParA removed or disassembled ParB from the partition complex. ADP could not support these interactions, but could promote the site-specific DNA binding activity of ParA to parOP, the operator of the par operon. Conversely, ATP could not support a stable interaction of ParA with parOP in this assay. Our data suggest that ParA-ADP is the repressor of the par operon, and ParA-ATP, by interacting with the partition complex, plays a direct role in partition. Therefore, one role of adenine nucleotide binding and hydrolysis by ParA is that of a molecular switch controlling entry into two separate pathways in which ParA plays different roles.     

Fine-structure analysis of the P7 plasmid partition site.

The par region of bacteriophage P7 is responsible for active partition of the P7 plasmid prophage into daughter cells. The cis-acting partition site was defined precisely as a 75-bp sequence that was necessary and sufficient to promote correct segregation of an unstable vector plasmid when the two P7 partition proteins, ParA and ParB, were supplied in trans. Roughly the same region was necessary to exert partition-mediated incompatibility. The minimal site contains an integration host factor (IHF) protein binding site bracketed by regions containing heptamer repeat sequences that individually bind ParB. An additional sequence forms the left boundary of the site. Site-directed mutations in the latter sequence, as well as the IHF motif and the rightmost ParB box, blocked site function. Although the P7 site shares 55% sequence identity with its counterpart in bacteriophage P1, functional interactions between the partition sites and the Par proteins of the two plasmids were entirely species specific in vivo. The P1 sequence has similar IHF and ParB binding motifs, but the left boundary sequence differs radically and may define a point of species-specific contact with the Par proteins. No evidence was found for the existence of a functional P7 analog of the P1 parS core, a small subregion of the P1 site that, in isolation, acts as an enfeebled partition site with modified incompatibility properties.     

Stoichiometry of P1 plasmid partition complexes

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 in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.     

Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis

The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP-binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created 'super-repressors', in which repressor activity was much stronger than in wild-type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super-repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super-repressors bound site-specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParA's repressor activity and suggest that ATP hydrolysis is essential for plasmid localization.     

Effects of the P1 plasmid centromere on expression of P1 partition genes

The partition operon of P1 plasmid encodes two proteins, ParA and ParB, required for the faithful segregation of plasmid copies to daughter cells. The operon is followed by a centromere analog, parS, at which ParB binds. ParA, a weak ATPase, represses the par promoter most effectively in its ADP-bound form. ParB can recruit ParA to parS, stimulate its ATPase, and significantly stimulate the repression. We report here that parS also participates in the regulation of expression of the par genes. A single chromosomal parS was shown to augment repression of several copies of the par promoter by severalfold. The repression increase was sensitive to the levels of ParA and ParB and to their ratio. The increase may be attributable to a conformational change in ParA mediated by the parS-ParB complex, possibly acting catalytically. We also observed an in cis effect of parS which enhanced expression of parB, presumably due to a selective modulation of the mRNA level. Although ParB had been earlier found to spread into and silence genes flanking parS, silencing of the par operon by ParB spreading was not significant. Based upon analogies between partitioning and septum placement, we speculate that the regulatory switch controlled by the parS-ParB complex might be essential for partitioning itself.     

Pairing of P1 plasmid partition sites by ParB

The mechanisms by which bacterial plasmids and chromosomes are partitioned are largely obscure, but it has long been assumed that the molecules to be separated are initially paired, as are sister chromatids in mitosis. We offer in vivo evidence that the partition protein ParB encoded by the bacterial plasmid P1 can pair cis-acting partition sites of P1 inserted in a small, multicopy plasmid. ParB was shown previously to be capable of extensive spreading along DNA flanking the partition sites. Experiments in which ParB spreading was constrained by physical roadblocks suggest that extensive spreading is not required for the pairing process.     

P1 and P7 plasmid partition: ParB protein bound to its partition site makes a separate discriminator contact with the DNA that determines species specificity.

The cis-acting P1 and P7 parS sites are responsible for active partition of P1 and P7 plasmids to daughter cells. The two sites are similar but function only with ParB proteins from the correct species. Using hybrid ParB proteins and hybrid parS sites, we show that specificity is determined by contacts between bases that lie within two parS hexamer boxes and a region in the ParB C-terminus. We refer to these contacts as discriminator contacts. The P7 discriminator contacts were mapped to 3 and 2 bp respectively within the two parS hexamer boxes, and a 10 amino acid region of P7 ParB. Similarly placed residues of different sequence are responsible for the P1 discriminator contact. The discriminator contacts are distinct from previously identified DNA binding contacts which involve different ParB and parS regions. Deletion of the ParB C-terminus that makes the discriminator contact does not diminish in vitro binding but renders it species independent. The discriminator contact is therefore a negative function, interfering with binding of the wrong ParB, but not providing energy for the binding of the correct one. Similar discriminator contacts might be responsible for specificities seen among families of eukaryotic DNA binding proteins that share common binding motifs.     

The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex

Stable maintenance of P1 plasmids in Escherichia coli is mediated by a high affinity nucleoprotein complex called the partition complex, which consists of ParB and the E. coli integration host factor (IHF) bound specifically to the P1 parS site. IHF strongly stimulates ParB binding to parS, and the minimal partition complex contains a single dimer of ParB. To examine the architecture of the partition complex, we have investigated the DNA binding activity of various ParB fragments. Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for the formation of the minimal, high affinity partition complex. A fragment missing only the last 16 amino acids of ParB bound specifically to parS, but binding was weak and was no longer stimulated by IHF. The ability of IHF to stimulate ParB binding to parS correlated with the ability of ParB to dimerize via its C terminus. Using full and partial parS sites, we show that two regions of ParB, one in the center and the other near the C terminus of the protein, interact with distinct sequences within parS. Based on these data, we have proposed a model of how the ParB dimer binds parS to form the minimal partition complex.     

Specificity switching of the P1 plasmid centromere-like site.

The P1 plasmid partition site acts like a centromere, promoting accurate segregation of copies to daughter cells. A 34 bp segment is essential for partition and binds the plasmid ParB protein. Additional sequences act as specificity elements that direct the choice of copies for partition. They include a second ParB binding site and a site for the host integration host factor protein. Sites lacking one or more of these additional elements are switched to a different specificity. Defined mutants were scored for partition specificity and protein binding. The results suggest that the wild-type site is folded in a specific DNA-protein complex. Disruption of the complex leads to an open configuration which, while still active in partition, has altered recognition specificity.     

A plasmid partition system of the P1-P7par family from the pMT1 virulence plasmid of Yersinia pestis

The complete sequence of the virulence plasmid pMT1 of Yersinia pestis KIM5 revealed a region homologous to the plasmid partition (par) region of the P7 plasmid prophage of Escherichia coli. The essential genes parA and parB and the downstream partition site gene, parS, are highly conserved in sequence and organization. The pMT1parS site and the parA-parB operon were separately inserted into vectors that could be maintained in E. coli. A mini-P1 vector containing pMT1parS was stably maintained when the pMT1 ParA and ParB proteins were supplied in trans, showing that the pMT1par system is fully functional for plasmid partition in E. coli. The pMT1par system exerted a plasmid silencing activity similar to, but weaker than those of P7par and P1par. In spite of the high degree of similarity, especially to P7par, it showed unique specificities with respect to the interactions of key components. Neither the P7 nor P1 Par proteins could support partition via the pMT1parS site, and the pMT1 Par proteins failed to support partition with P1parS or P7parS. Typical of other partition sites, supernumerary copies of pMT1parS exerted incompatibility toward plasmids supported by pMT1par. However, no interspecies incompatibility effect was observed between pMT1par, P7par, and P1par.     

The P1 plasmid in action: time-lapse photomicroscopy reveals some unexpected aspects of plasmid partition

The prophage of bacteriophage P1 is a low copy number plasmid in Escherichia coli and is segregated to daughter cells by an active partition system. The dynamics of the partition process have now been successfully followed by time-lapse photomicroscopy. The process appears to be fundamentally different from that previously inferred from statistical analysis of fixed cells. A focus containing several plasmid copies is captured at the cell center. Immediately before cell division, the copies eject bi-directionally along the long axis of the cell. Cell division traps one or more plasmid copies in each daughter cell. These copies are free to move, associate, and disassociate. Later, they are captured to the new cell center to re-start the cycle. Studies with mutants suggest that the ability to segregate accurately at a very late stage in the cell cycle is dependent on a novel ability of the plasmid to control cell division. Should segregation be delayed, cell division is also delayed until segregation is successfully completed.     

Species and incompatibility determination within the P1par family of plasmid partition elements

The P1par family of active plasmid partition systems consists of at least six members, broadly distributed in a variety of plasmid types and bacterial genera. Each encodes two Par proteins and contains a cis-acting parS site. Individual par systems can show distinct species specificities; the proteins from one type cannot function with the parS site of another. P1par-versus-P7par specificity resides within two hexamer BoxB repeats encoded by parS that contact the ParB protein near the carboxy terminus. Here, we examine the species specificity differences between Yersinia pestis pMT1parS and Escherichia coli P1 and P7parS. pMT1parS site specificity could be altered to that of either P1 or P7 by point mutation changes in the BoxB repeats. Just one base change in a single BoxB repeat sometimes sufficed. The BoxB sequence appears to be able to adopt a number of forms that define exclusive interactions with different ParB species. The looped parS structure may facilitate this repertoire of interaction specificities. Different P1par family members have different partition-mediated incompatibility specificities. This property defines whether two related plasmids can coexist in the same cell and is important in promoting the evolution of new plasmid species. BoxB sequence changes that switch species specificity between P1, P7, and pMT1 species switched partition-mediated plasmid incompatibility in concert. Thus, there is a direct mechanistic link between species specificity and partition-mediated incompatibility, and the BoxB-ParB interaction can be regarded as a special mechanism for facilitating plasmid evolution.     

The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells

The P1 ParA protein is an ATPase that recognizes the parA promoter region where it acts to autoregulate the P1 parA–parB operon. The ParB protein is essential for plasmid partition and recognizes the cis -acting partition site parS . The regulatory role of ParA is also essential because a controlled level of ParB protein is critical for partition. However, we show that this regulatory activity is not the only role for ParA in partition. Efficient partition can be achieved without autoregulation as long as Par protein levels are kept within a range of low values. The properties of ParA mutants in these conditions showed that ParA is essential for some critical step in the partition process that is independent of par operon regulation. The putative nucleotide-binding site for the ParA ATPase was identified and disrupted by mutation. The resulting mutant was substantially defective for autoregulation and completely inactive for partition in a system in which the need for autoregulation is abolished. Thus, the ParA nucleotide-binding site appears to be necessary both for the repressor activity of ParA and for some essential step in the partition process itself. We propose that the nucleotide-bound form of the enzyme adopts a configuration that favours binding to the operator, but that the ATPase activity of ParA is required for some energetic step in partition of the plasmid copies to daughter cells.     

Partitioning of P1 plasmids by gradual distribution of the ATPase ParA

Recently, it has been reported that prokaryotes also have a mitotic-like apparatus in which polymerized fibres govern the bipolar movement of chromosomes and plasmids. Here, we show evidence that a non-mitotic-like apparatus that does not form polymerized filaments carries out plasmid partitioning. P1 ParA, which is a DNA-binding ATPase protein, was found to be distributed through the whole nucleoid and formed a dense spot at the centre of the nucleoid. The fluorescent intensity of the ParA spot blinked, and then the spot gradually migrated from the midcell to a cell quarter position. Such distribution was not observed in anucleate cells, suggesting that the nucleoid could be a matrix for gradual distribution of ParA. Plasmid DNA constantly colocalized at the spot of ParA and migrated according to spot migration and separation. Thus, the gradient distribution of ParA determines the destination of partitioning plasmids and may direct plasmids to the cell quarters.     

Covalent cross-linking of tRNAGly1 to the ribosomal P site via the dihydrouridine loop

The dihydrouracil residue at position 20 of Escherichia coli tRNAGly1 has been replaced by the photoaffinity reagent, N-(4-azido-2-nitrophenyl)glycyl hydrazide (AGH). The location of the substituent was confirmed by the susceptibility of the modified tRNA to cleavage with aniline. When N-acetylglycyl-tRNAGly1 derivatized with AGH was bound noncovalently to the P site of E. coli 70 S ribosomes, 5-6% on average was photochemically cross-linked to the ribosomal particles in a reaction requiring poly(G,U), irradiation and the presence of the AGH label in the tRNA. Approximately two-thirds of the covalently attached tRNA was associated with 16 S RNA in the 30 S subunit. This material was judged to be in the P site by the criterion of puromycin reactivity. As partial RNAase digestion of the tRNA-16 S RNA complex produced labeled fragments from both 5' and 3' segments of the rRNA, there appeared to be more than one site of cross-linking in the 30 S subunit. The small amount of N-acetylglycyl-tRNAGly1 associated with the 50 S subunit was also linked mainly to rRNA, but it was not puromycin-reactive.