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.     

Functioning of the TA cassette of streptococcal plasmid pSM19035 in various Gram-positive bacteria

Toxin-antitoxin (TA) systems are common in microorganisms and are frequently found in the chromosomes and low-copy number plasmids of bacterial pathogens. One such system is carried by the low copy number plasmid pSM19035 of the pathogenic bacterium Streptococcus pyogenes. This plasmid encodes an omega-epsilon-zeta cassette that ensures its stable maintenance by post-segregational killing of plasmid-free cells. In this study, the activity of the ω-ε-ζ cassette was examined in various Gram-positive bacteria with a low G/C content in their DNA. The broad host range of pSM19035 was confirmed and the copy number of a truncated derivative in transformed strains was determined by real-time qPCR.     

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.     

The P1 plasmid partition complex at parS. The influence of Escherichia coli integration host factor and of substrate topology

The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage. ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS. In this report the role of IHF in the formation of the partition complex has been explored. First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution. Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions). IHF induced a strong bend in the DNA at its binding site. Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo. Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold. Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence. These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.     

P1 partition complex assembly involves several modes of protein-DNA recognition

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.     

Site-specific recombination by the beta protein from the streptococcal plasmid pSM19035: minimal recombination sequences and crossing over site.

The beta recombinase from the broad host range Grampositive plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented recombination sites in the presence of a chromatin-associated protein (Hbsu). The recombination site had been localized to a 447 bp DNA segment from pSM19035. This segment includes a 90 bp region that contains two adjacent binding sites (I and II) for beta protein dimers. Using in vitro recombination assays, we show that this 90 bp region is necessary and sufficient for beta protein-mediated recombination; this defines the six site as the region required for beta protein binding. The point of crossing over has been localized to the center of site I. Hbsu has a strong binding affinity for an unknown site located within the 447 bp segment containing the six site. We discuss the possibility that Hbsu recognizes an altered DNA structure, rather than a specific sequence, generated in the synaptic complex.     

Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.

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.     

Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants.

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.     

Characterization of a novel partition system encoded by the delta and omega genes from the streptococcal plasmid pSM19035

High segregational stability of the streptococcal plasmid pSM19035 is achieved by the concerted action of systems involved in plasmid copy number control, multimer resolution, and postsegregational killing. In this study, we demonstrate the role of two genes, delta and omega, in plasmid stabilization by a partition mechanism. We show that these two genes can stabilize the native pSM19035 replicon as well as other theta- and sigma-type plasmids in Bacillus subtilis. In contrast to other known partition systems, in this case the two genes are transcribed separately; however, they are coregulated by the product of the parB-like gene omega. Analysis of mutants of the parA-like gene delta showed that the Walker A ATPase motif is necessary for plasmid stabilization. The ParB-like product of the omega gene binds to three regions containing repeated WATCACW heptamers, localized in the copS (regulation of plasmid copy number), delta, and omega promoter regions. We demonstrate that all three of these regions can cause partition-mediated incompatibility. Moreover, our data suggest that each of these could play the role of a centromere-like sequence. We conclude that delta and omega constitute a novel type of plasmid stabilization system.     

Mutational analysis of a site-specific recombinase: characterization of the catalytic and dimerization domains of the β recombinase of pSM19035

The β recombinase encoded by the streptococcal plasmid pSM19035, which shows 28 to 34% identity with DNA resolvases and DNA invertases, can catalyze formation of deletions or inversions between properly oriented target sites. We have constructed a number of site-directed mutations at residues that are conserved between the β protein and other DNA recombinases of the resolvase/invertase family. The analysis of the recombination and DNA-binding ability of each mutant protein shows that the mutations affect the catalytic activity and, in two cases, the dimerization of the protein. The results suggest that the β protein probably mediates recombination by a catalytic mechanism similar to that proposed for the resolvase/invertase family. Since the β recombinase differs from DNA resolvases and DNA invertases in its lack of bias towards either of these reactions, the results presented support the hypothesis that its unique properties might depend on details of the architecture or assembly of the recombination complex. In addition, two β protein mutants that can no longer form dimers in solution have provided new insights into the way the protein binds to DNA     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

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.     

Mapping of RNA polymerase binding sites in R12 derived plasmids carrying the replication-incompatibility region and the insertion element IS1.

Interactions between Escherichia coli RNA polymerase holoenzyme and three small plasmid DNAs (pSM1, pSM2, and pSM15) derived from the drug resistant factor R12 have been studied. These plasmids carry the copy number and incompatibility determinants, the origin of DNA replication and the rep gene(s) necessary for plasmid replication. They also contain the insertion element IS1 and the putative finO cistron. Thirteen DNA segments within the largest of the three plasmids (pSM2) were able to form either a binary and/or ternary complex with RNA polymerase. A unique strong binding site was mapped within the left end of IS1. Five binding sites were found within the rep-cop-inc region. Four of these are weak binding sites whereas the fifth does not form a stable binary complex and was detected by ternary complex formation. A strong binding site was located in the putative finO region whereas the remaining six binding sites are located in regions with unidentified genetic functions.     

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 ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome

The mycelial prokaryote Streptomyces coelicolor A3(2) possesses a large linear chromosome (8.67 Mb) with a centrally located origin of replication (oriC). Recently, chromosome partitioning genes (parA and parB) and putative ParB binding sites (parS sequences) were identified in its genome. The S. coelicolor chromosome contains more parS sequences than any other bacterial chromosome characterized so far. Twenty of the 24 parS sequences are densely packed within a relatively short distance (approximately 200 kb) around oriC. A series of in vitro and in vivo experiments showed that S. coelicolor ParB protein interacts specifically with the parS sequences, albeit with a rather low affinity. Our results suggested that the binding of ParB is not only determined by the parS sequence, but also by the location of target DNA close to oriC. The unusually high number and close proximity to each other of the parS sites, together with in vivo and in vitro evidence that multiple ParB molecules may assemble along the DNA from an initial ParB-parS complex, suggest that a large DNA segment around the replication origin may form a massive nucleoprotein complex as part of the replication-partitioning cycle.     

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.     

Cloning and expression in Bacillus subtilis of the gene for neutral protease of Bacillus brevis

Plasmids pCB20 and pCB22 were used for cloning and expression of the Bac brevis 7882 neutral protease gene in Bac. subtilis cells. The protease-containing fragments of 13 and 14 kb were cloned in pCB20 plasmid based on replication region of Streptococci plasmid pSM19035. Expression of the gene was shown to take place in Bac. subtilis. Application of vegetative promoters of the previously identified expression unit EU19035 greatly increases the expression of the protease in Bac. subtilis. Bac. subtilis cells, expressing the gene of Bac. brevis neutral protease, do not sporulate, are considerably larger than the cells which do not contain the gene and form multicellular structures.     

A Type Ib ParB protein involved in plasmid partitioning in a gram-positive bacterium

Our current understanding of segregation of prokaryotic plasmids has been derived mainly from the study of the gram-negative bacterial plasmids. We previously reported a replicon of the cryptic plasmid from a gram-positive bacterium, Leifsonia xyli subsp. cynodontis. The replicon contains a putative plasmid partition cassette including a Walker-type ATPase followed by open reading frame 4 without sequence homologue. Here we reported that the orf4 gene was essential for maintaining the plasmid stability in L. xyli subsp. cynodontis. Furthermore, the purified orf4 protein specifically and cooperatively bound to direct repeat sequences located upstream of the parA gene in vitro, indicating that orf4 is a parB gene and that the direct repeat DNA sequences constitute a partition site, parS. The location of parS and the features of ParA and ParB proteins suggest that this plasmid partition cassette belongs to type Ib, representing the first type Ib cassette identified from a gram-positive bacterial plasmid.