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.     

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.     

A type Ib plasmid segregation machinery of the Advenella kashmirensis plasmid pBTK445

pBTK445 is a newly described large (～60Kb), low-copy number, conjugative plasmid indigenous to the sulfur-chemolithoautotroph Advenella kashmirensis. Based on its minimal replication region, a shuttle vector, pBTKS was constructed which can be used for diverse Alcaligenaceae members. The construct was found to be stably maintained both in the native host as well as in Escherichia coli in the absence of selective pressure which indicated that pBTKS harbors the stabilizing system of pBTK445, that are commonly coded by low-copy-number plasmids. Deletion analyzes of pBTKS confirmed the essentiality of parA (encoding a Walker-type ATPase of 214 amino acids) and the downstream located small parB (encoding an 85 amino acid protein having no sequence homolog in the database) in the faithful partitioning of pBTK445. A 1075bp PCR product, containing parA, parB and an upstream sequence having nine 11bp direct repeats (parS site) was found to comprise the partition functions of pBTK445, stabilizing both low-copy or high-copy number homologous and heterologous replicons in diverse hosts. The incompatibility determinant and the par promoter, P(par) were both found to be present within a 191bp iterated sequence present upstream of parA. ParB was found to regulate the expression of the Par proteins from P(par). The presence of a typical Walker-type ATPase motif in ParA, a short phylogenetically unrelated ParB, that acts as a repressor of P(par), and location of the iterated parS site upstream of parA, confirm that the active partition system of pBTK445 belongs to the type Ib.     

Altered ParA partition proteins of plasmid P1 act via the partition site to block plasmid propagation

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.     

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.     

Mini-P1 plasmid partitioning: excess ParB protein destabilizes plasmids containing the centromere parS.

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.     

Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor.

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.     

Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri

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.     

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.     

Centromere pairing by a plasmid-encoded type I ParB protein

The par2 locus of Escherichia coli plasmid pB171 encodes two trans-acting proteins, ParA and ParB, and two cis-acting sites, parC1 and parC2, to which ParB binds cooperatively. ParA is related to MinD and oscillates in helical structures and thereby positions ParB/parC-carrying plasmids regularly over the nucleoid. ParB ribbon-helix-helix dimers bind cooperatively to direct repeats in parC1 and parC2. Using four different assays we obtain solid evidence that ParB can pair parC1- and parC2-encoding DNA fragments in vitro. Convincingly, electron microscopy revealed that ParB mediates binary pairing of parC fragments. In addition to binary complexes, ParB mediated the formation of higher order complexes consisting of several DNA fragments joined by ParB at centromere site parC. N-terminal truncated versions of ParB still possessing specific DNA binding activity were incompetent in pairing, hence identifying the N terminus of ParB as a requirement for ParB-mediated centromere pairing. These observations suggest that centromere pairing is an important intermediate step in plasmid partitioning mediated by the common type I loci.     

The bacterial ParA-ParB partitioning proteins

A pair of genes designated parA and parB are encoded by many low copy number plasmids and bacterial chromosomes. They work with one or more cis-acting sites termed centromere-like sequences to ensure better than random predivisional partitioning of the DNA molecule that encodes them. The centromere-like sequences nucleate binding of ParB and titrate sufficient protein to create foci, which are easily visible by immuno-fluorescence microscopy. These foci normally follow the plasmid or the chromosomal replication oriC complexes. ParA is a membrane-associated ATPase that is essential for this symmetric movement of the ParB foci. In Bacillus subtilis ParA oscillates from end to end of the cell as does MinD of E. coli, a relative of the ParA family. ParA may facilitate ParB movement along the inner surface of the cytoplasmic membrane to encounter and become tethered to the next replication zone. The ATP-bound form of ParA appears to adopt the conformation needed to drive partition. Hydrolysis to create ParA-ADP or free ParA appears to favour a form that is not located at the pole and binds to DNA rather than the partition complex. Definition of the protein domains needed for interaction with membranes and the conformational changes that occur on interaction with ATP/ADP will provide insights into the partitioning mechanism and possible targets for inhibitors of partitioning.     

Bacterial mitosis: partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells with a single plasmid focus, the focus located preferentially at mid-cell. In cells with two foci, these located at quarter-cell positions. In the absence of ParB and parC1/parC2, ParA-GFP formed stationary helices extending from one end of the nucleoid to the other. In the presence of ParB and parC1/parC2, ParA-GFP oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid and subsequently separate them into daughter cells.     

Distinct centromere-like parS sites on the two chromosomes of Vibrio spp

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.     

Characterization of the partitioning system of Myxococcus plasmid pMF1

pMF1 is the only autonomously replicating plasmid that has been recently identified in myxobacteria. This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMF1.21-pMF1.23 (parCAB). The pMF1.22 ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that the pMF1.23 (parB) product is a DNA-binding protein of iteron DNA sequences, while the product of pMF1.21 (parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ib par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1 par system has novel structural and functional characteristics.