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1.
A plasmid partition system of the P1-P7par family from the pMT1 virulence plasmid of Yersinia pestis 总被引:5,自引:0,他引:5
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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. 相似文献
2.
Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri
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P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein. 相似文献
3.
Species and incompatibility determination within the P1par family of plasmid partition elements
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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. 相似文献
4.
How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species. 相似文献
5.
Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor. 总被引:27,自引:4,他引:23
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The P1 plasmid partition system is responsible for segregation of daughter plasmids during division of the Escherichia coli host cell. The P1-encoded elements consist of two essential proteins, ParA and ParB, and the cis-acting incB region. The incB region determines partition-mediated incompatibility and contains the centromere-like site parS. We have isolated and purified the two proteins. ParB binds specifically to the incB region in vitro. DNase I footprinting assays place a strong binding site over the 35-bp parS sequence previously shown to be sufficient for partition when the Par proteins are supplied in trans. A weaker site lies within the incB region in sequences that are important for specifying incompatibility, but are not essential for partition. Gel band retardation assays show that a host factor binds specifically to the incB sequence. The factor strongly stimulates binding of ParB. Cutting the region at a site between the two ParB binding sites yields two fragments that can bind ParB but not host factor. Thus, information for host-factor binding lies in the region determining the specificity of plasmid incompatibility. The roles of parB and the host factor in partition and the specificity of plasmid incompatibility are discussed. 相似文献
6.
7.
Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants. 总被引:4,自引:1,他引:3
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The partition system of the low-copy-number plasmid/prophage of bacteriophage P1 encodes two proteins, ParA and ParB, and contains a DNA site called parS. ParB and the Escherichia coli protein IHF bind to parS to form the partition complex, in which parS is wrapped around ParB and IHF in a precise three-dimensional conformation. Partition can be thought of as a positioning reaction; the plasmid-encoded components ensure that at least one copy of the plasmid is positioned within each new daughter cell. We have used an E. coli chromosomal partition mutant to test whether this positioning is mediated by direct plasmid-chromosomal attachment, for example, by pairing of the partition complex that forms at parS with a bacterial attachment site. The E. coli MukB protein is required for proper chromosomal positioning, so that mukB mutants generate some cells without chromosomes (anucleate cells) at each cell division. We analyzed the plasmid distribution in nucleate and anucleate mukB cells. We found that P1 plasmids are stable in mukB mutants and that they partition into both nucleate and anucleate cells. This indicates that the P1 partition complex is not used to pair plasmids with the host chromosome and that P1 plasmids must be responsible for their own proper cellular localization, presumably through host-plasmid protein-protein interactions. 相似文献
8.
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. 相似文献
9.
Altered ParA partition proteins of plasmid P1 act via the partition site to block plasmid propagation 总被引:8,自引:3,他引:5
The partition system of the P1 plasmid, P1 par consists of the ParA and ParB proteins and a cis -acting site, parS . It is responsible for the orderly segregation of plasmid copies to daughter cells. Plasmids with null mutations in parA or parB replicate normally, but missegregate. ParB binds specifically to the parS site, but the role of ParA and its ATPase activity in partition is unclear. We describe a novel class of parA mutants that cannot be established or maintained as plasmids unless complemented by the wild-type gene. One, parAM314I , is conditional: it can be maintained in cells in minimal medium but cannot be established in cells growing in L broth. The lack of plasmid propagation in L broth-grown cells was shown to be caused by a ParB-dependent activity of the mutant ParA protein that blocks plasmid propagation by an interaction at the parS site. Thus, ParA acts to modify the ParB– parS complex, probably by binding to it. Partition is thought to involve selection of pairs of plasmids before segregation, either by physical pairing of copies or by binding of copies to paired host sites. We suggest that ParA is involved in this reaction and that the mutant ParA protein forms paired complexes that cannot unpair. 相似文献
10.
11.
Vibrio cholerae, the cause of cholera, has two circular chromosomes. The parAB genes on each V. cholerae chromosome act to control chromosome segregation in a replicon-specific fashion. The chromosome I (ChrI) parAB genes (parAB1) govern the localization of the origin region of ChrI, while the chromosome II (ChrII) parAB genes (parAB2) control the segregation of ChrII. In addition to ParA and ParB proteins, Par systems require ParB binding sites (parS). Here we identified the parS sites on both V. cholerae chromosomes. We found three clustered origin-proximal ParB1 binding parS1 sites on ChrI. Deletion of these three parS1 sites abrogated yellow fluorescent protein (YFP)-ParB1 focus formation in vivo and resulted in mislocalization of the ChrI origin region. However, as observed in a parA1 mutant, mislocalization of the ChrI origin region in the parS1 mutant did not compromise V. cholerae growth, suggesting that additional (non-Par-related) mechanisms may mediate the partitioning of ChrI. We also identified 10 ParB2 binding parS2 sites, which differed in sequence from parS1. Fluorescent derivatives of ParB1 and ParB2 formed foci only with the cognate parS sequence. parABS2 appears to form a functional partitioning system, as we found that parABS2 was sufficient to stabilize an ordinarily unstable plasmid in Escherichia coli. Most parS2 sites were located within 70 kb of the ChrII origin of replication, but one parS2 site was found in the terminus region of ChrI. In contrast, in other sequenced vibrio species, the distribution of parS1 and parS2 sites was entirely chromosome specific. 相似文献
12.
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. 相似文献
13.
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. 相似文献
14.
Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems. 总被引:10,自引:1,他引:9
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The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules. 相似文献
15.
Plasmid-partition functions of the P7 prophage 总被引:12,自引:0,他引:12
16.
Mini-P1 plasmid partitioning: excess ParB protein destabilizes plasmids containing the centromere parS. 总被引:17,自引:4,他引:13
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B E Funnell 《Journal of bacteriology》1988,170(2):954-960
The partition system of the unit-copy plasmid P1 consists of two proteins, the parA and parB gene products, and a cis-acting site, parS. Production of high levels of the P1 ParB protein, from an external promoter on a high-copy-number vector, inhibits the propagation of lambda-mini-P1 prophages and destabilizes other P1-derived plasmids. The interference by ParB protein depends on the parS site, or centromere, of the P1 partition region; plasmids lacking parS are unaffected. The defect is more severe than the defect due to mutations that simply eliminate par function. In the presence of excess ParB protein, plasmids carrying parS are more unstable than would be predicted from a random distribution at cell division. The destabilization is a segregation defect, as the copy number of parS-bearing plasmids is not decreased under these conditions. Thus, it appears that ParB protein binds to parS; if too much protein is present, it sequesters such plasmids so they cannot be properly, or even randomly, partitioned. This suggests that under normal conditions, ParB protein recognizes and binds to parS and may be the protein responsible for pairing plasmids during the process of partitioning at cell division. 相似文献
17.
P1 and P7 plasmid partition: ParB protein bound to its partition site makes a separate discriminator contact with the DNA that determines species specificity. 总被引:10,自引:1,他引:9
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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. 相似文献
18.
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. 相似文献
19.
Vecchiarelli AG Schumacher MA Funnell BE 《The Journal of biological chemistry》2007,282(15):10944-10952
Assembly of P1 plasmid partition complexes at the partition site, parS, is nucleated by a dimer of P1 ParB and Escherichia coli integration host factor (IHF), which promotes loading of more ParB dimers and the pairing of plasmids during the cell cycle. ParB binds several copies of two distinct recognition motifs, known as A- and B-boxes, which flank a bend in parS created by IHF binding. The recent crystal structure of ParB bound to a partial parS site revealed two relatively independent DNA-binding domains and raised the question of how a dimer of ParB recognizes its complicated arrangement of recognition motifs when it loads onto the full parS site in the presence of IHF. In this study, we addressed this question by examining ParB binding activities to parS mutants containing different combinations of the A- and B-box motifs in parS. Binding was measured to linear and supercoiled DNA in electrophoretic and filter binding assays, respectively. ParB showed preferences for certain motifs that are dependent on position and on plasmid topology. In the simplest arrangement, one motif on either side of the bend was sufficient to form a complex, although affinity differed depending on the motifs. Therefore, a ParB dimer can load onto parS in different ways, so that the initial ParB-IHF-parS complex consists of a mixture of different orientations of ParB. This arrangement supports a model in which parS motifs are available for interas well as intramolecular parS recognition. 相似文献
20.
Lee MJ Liu CH Wang SY Huang CT Huang H 《Biochemical and biophysical research communications》2006,342(3):744-750
The Soj and Spo0J proteins, together with one or more parS sequences, are crucial to chromosome segregation and the progression of cell cycle in many bacteria. In Helicobacter pylori, genes coding for Soj and a plasmid replication-partition-related protein containing a Spo0J or ParB conserved domain, together with two putative parS sites identified in this study, were found to be located within the origin-proximal 20-30% of the circular chromosome. Recombinant H. pylori Spo0J bound specifically to the two putative parS sequences and that of Bacillus subtilis. In addition, hydrolysis of ATP by H. pylori Soj was accelerated in the presence of parS and/or Spo0J. Protein-protein interactions, intracellular levels, and subcellular localization of Soj and Spo0J were analyzed through polyclonal antibodies directed against recombinant Soj and Spo0J. This study was the first implication of the existence of a functional parABS system in H. pylori. 相似文献