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.     

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.     

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.     

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.     

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.     

Structure and function of the F plasmid genes essential for partitioning

The F plasmid in Escherichia coli has its own partition mechanism controlled by the sopA and sopB genes, and by the cis-acting sopC region. The DNA sequence of the entire partition region and its flanking regions is described here. Two large open reading frames coding for 43,700 Mr and 35,400 Mr proteins correspond to sopA and sopB, respectively. The sopB reading frame is located immediately downstream from the sopA reading frame. Twelve 43 base-pair direct repeats exist in the sopC region without any spacer regions, and one pair of seven base-pair inverted repeats exists in each of the direct repeats. Analysis of deletions in the sopC region showed that the direct repeats play an important role in plasmid partition and IncD incompatibility. IncG incompatibility is exhibited by pBR322 derivatives carrying the sopB gene alone. When compared with the partition genes parA and parB of plasmid P1, homology in amino acid sequence was found between the SopA protein of F and the ParA protein of P1, and also between SopB protein of F and ParB protein of P1. In addition, homology was found between Rep proteins of F and P1.     

Deletion of the parA (soj) homologue in Pseudomonas aeruginosa causes ParB instability and affects growth rate, chromosome segregation, and motility

The parA and parB genes of Pseudomonas aeruginosa are located approximately 8 kb anticlockwise from oriC. ParA is a cytosolic protein present at a level of around 600 molecules per cell in exponential phase, but the level drops about fivefold in stationary phase. Overproduction of full-length ParA or the N-terminal 85 amino acids severely inhibits growth of P. aeruginosa and P. putida. Both inactivation of parA and overexpression of parA in trans in P. aeruginosa also lead to accumulation of anucleate cells and changes in motility. Inactivation of parA also increases the turnover rate (degradation) of ParB. This may provide a mechanism for controlling the level of ParB in response to the growth rate and expression of the parAB operon.     

Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci

Regulation of DNA replication and segregation is essential for all cells. Orthologs of the plasmid partitioning genes parA, parB, and parS are present in bacterial genomes throughout the prokaryotic evolutionary tree and are required for accurate chromosome segregation. However, the mechanism(s) by which parABS genes ensure proper DNA segregation have remained unclear. Here we report that the ParA ortholog in B. subtilis (Soj) controls the activity of the DNA replication initiator protein DnaA. Subcellular localization of several Soj mutants indicates that Soj acts as a spatially regulated molecular switch, capable of either inhibiting or activating DnaA. We show that the classical effect of Soj inhibiting sporulation is an indirect consequence of its action on DnaA through activation of the Sda DNA replication checkpoint. These results suggest that the pleiotropy manifested by chromosomal parABS mutations could be the indirect effects of a primary activity regulating DNA replication initiation.     

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.     

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.     

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.     

ParA‐mediated plasmid partition driven by protein pattern self‐organization

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low‐copy plasmids, such as the plasmids P1 and F, employ a three‐component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker‐type ATPase, typically called ParA, which also binds non‐specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP‐driven patterning is involved in partition is unknown. We reconstituted and visualized ParA‐mediated plasmid partition inside a DNA‐carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB‐stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion‐ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.