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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms. All known plasmid-encoded par loci specify three components: a cis-acting centromere-like site and two trans-acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon that is autoregulated by the par-encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host-encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e. the poles or quarter-cell positions). Two types of partitioning ATPases are known: the Walker-type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin-like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus). A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid-encoded ATPases clustered into distinct subgroups. Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib. A second surprise was that almost all chromosome-encoded ATPases, including members from both Gram-negative and Gram-positive Bacteria and Archaea, clustered into one distinct subgroup. The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea. Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria. These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

Recent methodological progress has resulted in rapid advances in the mechanistic understanding of the DNA segregation process in prokaryotes. The inference is that prokaryotic cells probably contain a mitotic-like apparatus that is responsible for the active segregation of plasmids and chromosomes prior to cell division. The components of this alleged apparatus are largely unknown. However, bacterial plasmids encode partitioning gene cassettes (termed par) that are required for important events in the replicon segregation process. The par genes were discovered on low-copy-number plasmids replicating in Escherichia coli (Austin and Abeles, 1983a; Ogura and Hiraga, 1983; Gerdes et al., 1985). More recently, orthologues of the plasmid-encoded par genes were identified on bacterial chromosomes (Ogasawara and Yoshikawa, 1992; Ireton et al., 1994; Mohl and Gober, 1997). The molecular mechanism by which par genes stabilize plasmids and chromosomes remained obscure for many years, and the evidence for an active segregation process was indirect and elusive (Nordstrom and Austin, 1989; Austin and Nordstrom, 1990; Hiraga, 1992; Rothfield, 1994; Wake and Errington, 1995). However, cytological techniques have been used recently to show that the components encoded by the par genes in conjunction with host-encoded factors support directional movement and positioning of newly replicated plasmids and chromosomes (reviewed in Harry, 1997; Wheeler and Shapiro, 1997; Gordon and Wright, 1998; Jensen and Shapiro, 1999; Sharpe and Errington, 1999; Møller-Jensen et al. 2000). In this MicroReview, we focus on recent advances in the understanding of the role of par genes in the process of DNA segregation. We also describe a thorough phylogenetic analysis of par genes. This has not been accomplished recently and therefore seems timely (Motallebi-Veshareh et al., 1990; Williams and Thomas, 1992). As describe below, several surprises emerged from the phylogenetic analyses.

Plasmid-encoded partitioning genes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

The analysis of low-copy-number plasmids such as F, P1 and R1 revealed partitioning loci with a strikingly similar genetic set-up. In general, par loci are organized as gene cassettes that can stabilize heterologous replicons – that is par genes act independently of the replication control units of their replicons. Almost all known plasmid-encoded par loci consist of three components: a cis-acting centromere-like site and two trans-acting proteins that form a nucleoprotein complex at the centromere (the partition complex). The two proteins, often termed ParA and ParB, are encoded by an operon whose transcription is autoregulated by the Par proteins themselves (see Fig. 1). In all cases known, the upstream gene encodes an ATPase that is essential to the DNA segregation process, whereas the downstream gene encodes a protein that binds to the centromere-like region. Two types of partitioning ATPases are known, one that contains the Walker-type ATPase motif (Koonin, 1993) and one that belongs to the actin/hsp70 superfamily of ATPases (Bork et al., 1992). Thus, all known par loci can be divided into two families based on the type of ATPase encoded. Partitioning proteins containing the Walker-type ATPase motifs have been found on many sequenced prokaryotic chromosomes. The molecular biology and phylogeny of the two known par gene families are described below.

image

Figure 1. Proposed genetic organization of type I and type II partitioning loci. Partitioning loci encode two trans-acting proteins and a cis-acting, centromere-like site. The upstream genes of type I loci encode Walker-type ATPases (shown as crosshatced boxes), whereas the upstream genes of type II loci encode actin-like ATPases. Type I loci can be divided into the Ia and Ib subgroups based on their genetic organization and the sizes of their genes (see the text). The ATPases of type Ia loci contain DNA-binding domains in their N-terminal ends (shown as a black bar; Hayes et al., 1994). Prototypes of the type Ia, Ib and II loci are shown to the right in the Figure. Arrows pointing rightwards indicate the par promoters and direction of transcription. Black bars outside the genes indicate centromeres (sopC of F, parS of P1 and parC of R1 respectively). Many type Ib loci have multiple direct repeats both upstream and downstream of the par genes. Thus, the regions downstream of the par genes may also contribute the the cis-acting centromere-like sites required for optimal function of type Ib par loci.

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The par family encoding Walker-type ATPases (type I loci)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

The genetic organization of the paradigm par/sop loci of plasmids F and P1 is shown in Fig. 1 (the par locus of F is called sop for stability of plasmid). The ParB/SopB proteins of c. 330 aa bind as dimers to the parS/sopC DNA regions that are located downstream of and adjacent to parB/sopB (Davis and Austin, 1988; Mori et al., 1989; Lobocka and Yarmolinsky, 1996; Surtees and Funnell, 1999). par loci with this typical genetic set-up are here referred to as type Ia loci.

The parS/sopC loci are centromere-like regions at which the par/sop proteins act (see below). The regions exert partitioning-related incompatibility (i.e. destabilization) towards plasmids stabilized by par/sop (Gardner et al., 1982; Austin and Abeles, 1983b; Austin and Nordstrom, 1990).

The ParA/SopA genes encode Walker-type ATPases that are specifically stimulated by the B proteins and by DNA (Watanabe et al., 1989; Davis et al., 1992; Davey and Funnell, 1994; 1997). ParA does not bind directly to parS, but instead contacts the preformed partition complex through protein–protein interaction (Davis and Austin, 1988; Bouet and Funnell, 1999). The ParA/SopA proteins control transcription of the par operons via binding to operator sequences in the promoter regions (Mori et al., 1989; Davis et al., 1992; Hayes et al., 1994). Elegant domain-swapping experiments showed that the DNA-binding domain of the P1 ParA protein resides in its N-terminus (Hayes et al., 1994; Radnedge et al., 1998). Autoregulation by ParA/SopA was enhanced by the ParB/SopA proteins which act as corepressors of transcription (Friedman and Austin, 1988; Mori et al., 1989). The autoregulation ensures the production of appropriate levels of the Par proteins (Funnell, 1988). In F, the sopC centromere is required for maximal repression of transcription by SopA and SopB, indicating cis- or trans-looping between the promoter region and the centromere (Yates et al., 1999). Trans-looping would be compatible with a pairing reaction between two centromere-containing plasmids (see below). Thus, the A proteins have dual functions: they interact with the promoter regions to repress transcription and with the B proteins in the repression and in the partition complexes. A recent study showed that, in vitro, the ADP-bound form of ParA specifically binds to the promoter region, whereas the ParA–ATP form interacts with ParB in a preformed partition complex at parS (Bouet and Funnell, 1999; Yates et al., 1999). This suggests that the two forms of ParA play different roles that may be controlled by other factors (e.g. ParB).

By the use of fluorescent probes, it is now possible to pinpoint the localization of plasmids, specific parts of chromosomes and proteins to specific subcellular compartments. These new methods have resulted in rapid advances in the mechanistic understanding of the DNA segregation process. Using fluorescence in situ hybridization (FISH) (Niki and Hiraga, 1997) or GFP-LacI (Gordon et al., 1997), P1 and F were found to localize in a non-random and dynamic fashion within the bacterial cell that depended on the par loci: in cells with one plasmid, the plasmid was located at mid-cell, whereas in cells with two plasmids, they were located at the one-quarter and three-quarter positions of cell length. The quarter-cell positions are destined to become mid-cell in newborn cells. These observations suggest that plasmids are located at mid-cell right after cell division. Because the replication machinery was proposed to be located at mid-cell (Lemon and Grossman, 1998), the plasmids may replicate at mid-cell and rapidly segregate to the observed quarter-cell positions.

The subcellular localization of SopB and ParB has also been investigated. In one study, SopB–GFP localized towards the cell poles independently of other sop-encoded components (Kim and Wang, 1998). However, using immunoflourescence microscopy, Hirano et al. (1998) showed that SopA and SopB formed foci only when sopC was present. Both studies used cells overproducing the Sop proteins, and further experiments are needed to resolve this apparent discrepancy. In P1, formation of ParB foci depended on the presence of parS (Erdmann et al., 1999). ParB foci were formed in the absence of ParA but their regular localization at quarter-cell positions was dependent on ParA. Without ParA, ParB foci were predominantly found at mid-cell or at quarter-cell positions. Because P1 DNA also localizes at quarter-cell positions, it is reasonable to suggest that ParA somehow mediates tethering of the ParB–parS nucleoprotein complex to specific cellular receptors at quarter-cell positions (Erdmann et al., 1999). Alternatively, ParA releases ParB from mid-cell and ParB relocalizes to quarter-site receptors. ParA itself did not seem to form foci, neither in the presence nor the absence of other components encoded by par.

The IncP plasmid RK2 encodes a partitioning locus that in several aspects seems similar to those of F and P1 (Williams and Thomas, 1992). Here IncC (ParA homologue) and KorB (ParB analogue) together constitute a partitioning locus that reduces the loss rates of test plasmids (Macartney et al., 1997; Williams et al., 1998). Using immunoflourescence microscopy, it was shown that the formation of KorB foci was dependent on the presence of KorB-binding sites and that the KorB protein itself did not form foci (Bignell et al., 1999). The symmetrical distribution of KorB foci as well as plasmid stabilization was dependent on IncC. Thus, the findings with the RK2 partitioning locus are consistent with those obtained with F and P1, and support the notion that the ParB proteins need ParA to localize correctly.

The ParB and SopB proteins can silence genes adjacent to their DNA-binding sites (Biek and Shi, 1994; Lynch and Wang, 1995; Rodionov et al., 1999; Yarmolinsky, 2000). Two theories have been proposed to explain the phenomenon. The most straightforward explanation invokes the DNA binding properties of the proteins and proposes that extended formation of nucleoprotein filaments prevents access of the RNA polymerase to the DNA coated with ParB or SopB (Rodionov et al., 1999). However, silencing by SopB was independent of the DNA-binding domain used but dependent on the localization domain of SopB (Hanai et al., 1996; Kim and Wang, 1999). Thus, in the alternative model, silencing was explained as a result of sequestration of the DNA to a subcellular localization inaccessible by RNA polymerase. The ParB foci detected by Erdmann et al., 1999 contained almost all ParB present in a cell, and therefore probably represent several thousand molecules assembled at or in the vicinity of parS. Thus, it is probable that the locally high subcellular concentration of the ParB and SopB proteins sequester genes adjacent to their binding sites. Further experiments are required to reveal the mechanism of gene silencing.

Phylogeny of Walker-type ATPases involved in plasmid partitioning

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

Partitioning loci encoding the Walker type of ATPases have been identified on a large number of plasmids, and constitute the par/sop gene family (see, e.g. Motallebi-Veshareh et al., 1990; William and Thomas, 1992; Erdmann et al., 1999; Kearney et al., 2000). In many cases, these loci are organized like the par/sop loci of F and P1 (denoted type Ia in Fig. 1). However, there are exceptions. For example, the linear prophage N15 of E. coli has an operon homologous to sopAB close to one end, but has four sopC sites dispersed within its genome (Ravin and Lane, 1999). Nevertheless, such an arrangement is unusual. Another locus, par from the Agrobacterium tumefaciens pTAR plasmid, has a genetic set-up that is clearly distinct from that of P1 and F (Gallie and Kado, 1987; Kalnin et al., 2000). Here, the ParA and ParB proteins are significantly smaller (222 and 94 aa respectively), and the parS site coincides with the promoter region upstream parAB. par loci with this typical genetic set-up are also common and referred to as type Ib loci (Fig. 1). In the case of pTAR, the ParB protein autoregulates the par operon via binding to the centromere region (Kalnin et al., 2000). Consistently, the ParA ATPases of type Ib loci probably lack DNA-binding domains. We argue later that the ParB proteins of type Ib loci autoregulate the par operons via binding to the centromere regions that contain the par promoters. Because of the striking differences between type Ia and Ib loci we thought it interesting to establish a detailed phylogeny of the Walker type of ATPases.

Figure 2 shows a Cladogram visualizing the complicated phylogenetic relationship of 81 ParA homologues. ParA proteins which have been identified by experiments are indicated by their names in parentheses after the species designations. The other ParA homologues listed in Fig. 2 are putative partitioning ATPases. The Cladogram contains most of the known chromosome-encoded ParA homologues but many more plasmid-encoded ParA homologues are present in the databases. However, an exhaustive compilation is beyond the scope of this MicroReview, but we do include a sufficient number of proteins as to establish a stable evolutionary pattern. The sizes of the ParA proteins and their corresponding putative ParB partners are also given in Fig. 2. In general, the ParB proteins are much more diverse than the ParA proteins, and a meaningful alignment of the ParB sequences is possible only within subgroups of the par loci. All ParA proteins included in the phylogram in Fig. 2 contain the deviant Walker-type ATPase motif (Boxes A, A′ and B, see the Figure legend) as described by Koonin (1993).

image

Figure 2. Phylogram of the Walker type partitioning ATPases. Unrooted evolutionary tree (Cladogram) showing 81 ParA proteins from Gram-negative (blue) and Gram-positive (red) bacteria, and from Archaea (green). The lengths of the horizontal lines reflect relative evolutionary distances, whereas the lengths of the vertical bars are arbitrary. Types Ia and Ib refer to the two types of plasmid-encoded partitioning loci shown in Fig. 1 and described in the text. For comparison, MinD homologues from the three prokaryotic domains, and the Mrp homologue from E. coli were included in the phylogram. As in the cases of the 81 ParA homologues, these four proteins contain the deviant Walker-type ATPase motif with the following consensus motifs: A-box: kGGxxK[ST], A′-box: gx[rk]u4dxDp, and B-box: duuUuD (x denotes any aa, u denotes a bulky hydrophobic aa, small letters indicate at least 80% conservation and large letters 100% conservation) (Koonin, 1993). Sharp parentheses show two alternative amino acids at a given position (degeneracy). The global sequence alignment of the 85 proteins analysed in Fig. 2 revealed that they all contain these three motifs and they may therefore all have ATPase activity. The sizes of the putative ParA and ParB proteins are given in the two columns at right in the Figure. NA (not available) indicates that ParB homologues were not identifiable in corresponding DNA sequences. The genbank Accession Nos of the ParA ATPases and their corresponding ParB partners as well as a global sequence alignment of the ParA proteins are available as supplementary material published on the Internet. The phylogram was constructed using pileup in the Wisconsin GCG package version 8.1.0(a).

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The phylogram reveals several surprises. First, the plasmid-encoded ParA proteins are quite diverse, but can be divided into several subgroups (clades). Most unexpectedly perhaps, most of the plasmid-encoded ParA proteins cluster into three separate clades that coincides with the genetic organization of the par loci. Thus, two of these groups consist entirely of type Ib loci, whereas the third group consists solely of type Ia loci (Fig. 1). Type Ia loci encode large ParA (251–420 aa) and ParB (182–336 aa) homologues and, in those cases investigated, a parS site located downstream of parB. In contrast, type Ib loci encode small ParA (208–227 aa) and very small ParB (46–113 aa) proteins, and a centromere that coincides with the promoter region upstream of the parAB operon. The genetic organizations of type Ia and Ib loci are compared in Fig. 1.

Furthermore, the phylogram in Fig. 2 reveals that all type Ia loci are from plasmids of Gram-negative bacteria, whereas type Ib loci are present in plasmids from both Gram-negative and Gram-positive bacteria. However, par loci from plasmids of Gram-positive origin are exclusively of the Ib type. It should be noted, that the type Ib par locus of the Lactococcus lactis plasmid pCI2000 is the first plasmid-encoded locus from Grampositive bacteria known to be active in plasmid stabilization (Kearney et al., 2000). The rep63B-orf6 genes of plasmid pAW63 from Bacillus thuringiensis probably also constitute a type Ib locus (Wilcks et al., 1999). The Borrelia and the broad-host-range ParA proteins of RK2/RP4 and R751 form two separate clades. The F-like proteins (ParA from F, pO157, N15, QpDV, pCD1 and pYVe227) and the P1-like proteins (ParA from P1, P7 and pMT1) form two distinct subgroups within the type Ia clade. ParA proteins from type Ib loci form two different clades, one that contains ParA homologues from Grampositive bacteria only and one that contains homologues from both these Bacterial divisions.

The division of par loci encoding the Walker-type ATPases into types Ia and Ib is novel and deserves scrutiny. From the global alignment of the ParA proteins (available as supplementary material on the Internet) it appears that type Ia proteins differ from almost all the other ParA proteins by having large N-terminal extensions of 108–130 aa. The type Ia proteins autoregulate their own transcription via binding to operators in their promoters. The DNA-binding domains reside in the N-terminal ends of the ParA proteins (Hayes et al., 1994; Radnedge et al., 1998). Thus, one possible difference between ParA proteins encoded by type Ia and Ib loci may be that the former autoregulate their own synthesis whereas the latter do not. This is consistent with the proposal that the ParB centromere-binding proteins from type Ib loci autoregulate the par operons via binding to the centromere regions upstream of the parAB operons (Kalnin et al., 2000). Thus, type Ib and type II loci may use the same theme for transcriptional autoregulation (see below).

For comparison, three MinD proteins from the three prokaryotic domains and the E. coli Mrp protein were included in the phylogenetic analysis in Fig. 2. The MinD and Mrp proteins, which also contain the deviant Walker-type ATPase motif, comprise a related subgroup within the plasmid-encoded ParA homologues, placed between two main plasmid-encoded clades (see below). The MinD proteins function in cell division to localize the septum at mid-cell, whereas the function of the E. coli Mrp protein is unknown.

The par family encoding actin-like ATPases (type II loci)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

The par locus (formerly parA) of plasmid R1 (identical to stb of plasmid R100) encodes two trans-acting proteins ParM and ParR and a cis-acting centromere-like region named parC (Dam and Gerdes, 1994; Gerdes and Molin, 1986). The genetic structure of par of R1 is shown in Fig. 1 as a type II locus. The centromere-like parC region is located upstream of parM and parR and has been mapped to a 160 bp region that contains the par promoter flanked by two sets of five 11 bp direct repeats (iterons; see Fig. 1). Resembling other centromere-like regions, the parC of R1 region exerts incompatibility towards plasmids stabilized by par (Dam and Gerdes, 1994; Breuner et al., 1996).

The par operon of R1 is transcribed from a single promoter located in the parC region (Min et al., 1991; Jensen et al., 1994). The promoter is repressed by ParR whereas ParM is not involved in the regulation (Tabuchi et al., 1992; Jensen et al., 1994). Consistently, the ParM protein does not effect the binding of ParR to parC in vitro (R.B.J., unpublished data). The autoregulation appears to be important for function because overproduction of ParR resulted in destabilization of a par-carrying plasmid (Dam and Gerdes, 1994). Thus, autoregulation by ParR appears to be to supply a suitable amount of both Par proteins (Jensen et al., 1994).

ParR was found to be a dimer in solution and to bind co-operatively to the 10 iterons in parC (M. Dam, unpublished results). The nucleo-protein complex formed at parC was examined using electron microscopy (Jensen et al., 1998). Unexpectedly, different complexes were formed when ParR was bound to linear and supercoiled DNA respectively. With supercoiled DNA, the complex was more condensed and the contour length of the protein-bound DNA was shortened by approximately 130 bp. This indicates that parC DNA wraps around a core of ParR protein. When ParR was bound to linear parC DNA or supercoiled DNA with truncated parC sites, no wrapping was observed (Jensen et al., 1998).

The ParM protein exhibits ATPase activity in vitro (Jensen and Gerdes, 1997). The ATPase activity was unaffected by addition of DNA, and the addition of ParR protein caused only a slight activation. However, ParR stimulated the ATPase activity of ParM in the presence of parC-containing DNA, indicating that ParM interacts with the ParR–parC complex. Mutant ParM proteins with severely reduced in vitro ATPase activity did not locate at cell poles and did not support plasmid partitioning, indicating that the ATPase activity of ParM is required for both processed.

Using electron microscopy and a ligation kinetics assay, very efficient pairing of two parC containing DNA molecules was observed (Jensen et al., 1998). Replicon pairing required the presence of ParR, but was more efficient in the presence of ParM and ATP. Pairing was much more efficient when supercoiled DNA was used, indicating that the presence of the complex in which parC is wrapped around a core of ParR was optimal for pairing. The ATPase activity of ParM was required for the ParM mediated increase in pairing efficiency (Jensen et al., 1998). As discussed below, post-replicational pairing of plasmid in a rational way explains the observed incompatibility exerted by parC.

Cytological methods were used to investigate the subcellular distribution of par-carrying plasmids. The localization pattern resembled that of plasmids carrying the sop and P1-par loci, albeit with important differences. In cells containing only one plasmid focus, it was located close to one pole or at mid-cell, whereas in the majority of cells with two foci, the plasmids were located towards opposite cell poles (Jensen and Gerdes, 1999). In contrast, plasmids without par were asymmetrically positioned. Using the GFP fusions, we showed that ParM forms discrete foci which exhibited a simple, yet dynamic pattern. Newborn cells contained two ParM foci that were present close to opposite cell poles (Jensen and Gerdes, 1999). Concomitant with cell growth, a new focus formed at mid-cell. Prior to cell division, this ParM focus divided into two new foci that became polar after cell division. Using double labelling of single cells, we observed that ParM and par-carrying plasmids colocalized. These results show that par of R1 is responsible for a non-random intracellular positioning of its replicon and suggest that ParM tethers parC carrying plasmids to positions close to the cell poles. Recruitment of ParM to the cell poles was strictly dependent on its ATPase activity and occurred in the absence of other par-encoded components, indicating that localization is an intrinsic property of ParM. Similarly, ParA of P1 was required for the regular localization pattern of ParB foci. Because ParB binds to parS, this indicates that the ParA ATPase is responsible for the correct subcellular localization of P1 at quarter-cell positions (Erdmann et al., 1999). Thus, the ATPases of the two par families may have analogous functions.

Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

The ParM protein shares similarity with a superfamily of ATPases that includes actin, sugar kinases, hsp70/DnaK and the prokaryotic cell-cycle proteins FtsA and MreB (Bork et al., 1992). The members of the superfamily share extensive structural similarity in the core region, which contains the active site (Kabsch and Holmes, 1995; Hurley, 1996). Database searches revealed several new ParM homologues from enteric bacteria. One homologue was identified on the chromosome of Enterobacter aerogenes, but the sequence of this putative par locus was only partial (Okazaki et al., 1997). Curiously, the as-yet-annotated sequence of the Klebsiella pneumoniae chromosome contains two ParM homologues. Figure 3A shows the sequence alignment of five plasmid-encoded ParM homologues. As expected, the best-conserved regions coincide with the core regions of the actin type of ATPases (Bork et al., 1992). Cognate ParR homologues were identified downstream of the five ParM homologues. Their sequences are aligned in Fig. 3B. These proteins are extremely divergent with a few conserved residues only. Furthermore, two of the proteins (from R478 and R27) are almost twice as large as the other ParR homologues. In conclusion, our phylogenetic studies show that all known plasmid-encoded partitioning loci can be divided into two types on the basis of the ATPases encoded by them. By far, the most prevalent type of par loci encode the Walker type of ATPases.

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Figure 3. Sequence alignment of plasmid-encoded homologues of ParM and ParR from plasmid R1.

A. Sequence alignment of five ParM homologues belonging to the actin family of ATPases.

B. Sequence alignment of the corresponding ParR homologues. Plasmids R1 and R27 are from Salmonella typhimurium, whereas ColIb-P9, R478 and pB171 are E. coli plasmids.

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The genetic organization of type Ib and II loci are strikingly similar (Fig. 1). However, the two families of ATPases encoded by them do not exhibit sequence similarity. Thus, the two types of par loci may have evolved independently and is then an example of astonishing convergent evolution. Alternatively, the par loci may have exchanged components during their evolution. In the latter case, the ATPases probably play similar roles in partitioning. This question can now be addressed experimentally.

Chromosome-encoded partitioning loci

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

Chromosome segregation is an efficient process because chromosome-free cells are rarely detected (Hiraga et al., 1989; Bork et al., 1992; Hiraga, 1992; Lobner-Olesen and Kuempel, 1992; Ireton et al., 1994). Using GFP-LacI fusion and FISH techniques, the subcellular positions of the origin and terminus regions of Bacillus subtilis and E. coli have been visualized. In these cells, the oriC regions were located at quarter-cell positions or near the cell poles, whereas the terC regions were found predominantly at mid-cell (Gordon et al., 1997; Niki and Hiraga, 1997; Webb et al., 1997; 1998). In E. coli, duplication of oriC seemed to occur close to the cell pole, and one origin migrated to a position in the predivisional cell becoming the new pole (Gordon and Wright, 1998; Niki and Hiraga, 1998). In B. subtilis, a similar pattern was observed. In this case, rapid movement of the origin region was independent of cell growth (Webb et al., 1998) (see later). These results strongly suggest the presence of a mitotic-like apparatus that actively moves the origin regions apart and tethers them to specific cellular sites.

In B. subtilis, the replication machinery (i.e. DNA polymerase III) is located at mid-cell (in slowly growing cells) or quarter-cell positions (in rapidly growing cells) (Lemon and Grossman, 1998; Niki and Hiraga, 2000). The origin-localization data described above are consistent with a model in which the origin moves rapidly from the pole to mid-cell where it is duplicated. After pairing and directional separation, the origins rapidly move to the new and old cell poles respectively. However, it cannot be excluded that origin duplication occurs at the cell pole followed by rapid movement to the opposite pole.

The chromosome of B. subtilis encodes parA and parB homologues (called soj and spo0J). Deletion of soj and spo0J, which are located near oriC, resulted in a large increase in the frequency of anucleate cells (Ireton et al., 1994). Curiously, deletion of soj alone had no apparent effect on the chromosome segregation pattern. The ParB homologue Spo0J binds to eight parS-like sequences in the origin proximal part of the chromosome (Lin and Grossman, 1998). One Spo0J-binding site was sufficient to stabilize a plasmid in B. subtilis. In this case, stabilization was dependent on both soj and spo0J, providing indirect evidence that the parAB homologous locus is involved in the active segregation of the B. subtilis chromosome. Further support of this view came from cytological studies of Spo0J and Soj. The subcellular localization of Spo0J coincides with that of the origin region (Lewis and Errington, 1997; Lin and Grossman, 1998; Teleman et al., 1998), whereas Soj is located at the cell poles or at nucleoid ends (Marston and Errington, 1999; Quisel et al., 1999). Interestingly, the latter two studies showed that Soj oscillates from nucleoid to nucleoid (or from pole to pole) on a timescale of minutes. The oscillation depended on Spo0J. Furthermore, Marston and Errington (1999) obtained evidence that Soj plays a role in compacting the DNA in the origin region, probably via interaction with Spo0J bound to DNA. Soj also represses expression of sporulation genes by direct binding to their promoter regions. Because the repression of these genes by Soj was enhanced in the absence of Spo0J, it is reasonable to suggest that the soj spo0J locus functions in a cell cycle checkpoint that couples chromosome segregation to cell differentiation. The majority of soj spo0J minus cells exhibited a normal pattern of origin localization and movement, indicating that components other than those encoded by the par locus are involved in chromosome segregation (Webb et al., 1998).

The chromosome of Caulobacter crescentus also encodes parA and parB orthologues near oriC (Mohl and Gober, 1997). The par genes are essential for cell viability, and their overexpression leads to chromosome segregation defects. The ParB protein binds to an AT-rich region downstream of parB (parS), and the ParA and ParB proteins localize to both cell poles in predivisional cells. Thus, in two out of two cases investigated, B. subtilis and C. crescentus, the par genes seem to be involved in the active segregation of their chromosomes. By inference, other chromosome-encoded par loci might have similar, if not identical, functions. Therefore, we found it interesting to establish the phylogeny of chromosomal par loci.

Phylogeny of Walker-type ATPases involved in chromosome partitioning

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

Figure 2 shows the phylogenetic relationship of 26 ParA homologues from the chromosomes of Gram-negative and Gram-positive Bacteria, and from Archaea. As in the case of the plasmid-encoded homologues, the chromosomal ParA proteins all contain the A, A′ and B boxes typical of the deviant type of the Walker-type ATPases (Koonin, 1993). A striking and surprising finding is, that all the chromosome-encoded ParA homologues (except for the ones from Aquifex aeolicus and Synechosystis) cluster in one distinct subgroup which contain only one plasmid-encoded ParA protein (from Methanococcus jannaschii pURB800). This was unexpected, because the chromosomal subgroup includes members from all three prokaryotic domains. This finding suggests that the chromosome-encoded ParA homologues have a conserved function not present in the plasmid-encoded proteins. Even although the chromosome-encoded ParA homologues form a separate subgroup, they are highly diverse and the phylogenetic relationship does not appear to follow a simple vertical pattern. For example, three Archaeal orthologues group together, but the fourth Archaeal ParA protein (from Methanococcus thermoautotrophicum) is more closely related to the Bacterial orthologues, perhaps indicating horizontal gene transfer. Furthermore, the ParB homologues from all four Archaeal species (97–110 aa) are all smaller than the bacterial counterparts (185–368 aa). A number of chromosomes were found to contain two or more parAB paralogues (e.g. S. coelicolor). In addition, some chromosomes have parA homologues without a typical parB homologue (e.g. in Helicobacter pylori) in addition to a complete parAB locus. Such homologues are not easily distinguished from minD homologues and not included in Fig. 2.

It is evident from this analysis that most prokaryotic chromosomes contain parAB loci near their origins of replication. However, some chromosomes seem to lack these genes. Thus, despite considerable effort, we have not been able to identify parAB homologous loci in E. coli and Haemophilus influenzae. The identity of the genes that accomplish chromosome segregation in these organisms remains obscure.

Models for plasmid segregation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

Our results with the par locus of plasmid R1 indicate that par-carrying plasmids are tethered close to the cell poles during most of the cell cycle. The colocalization of ParM with the plasmid suggests that ParM is the connection between a polar host-cell structure or receptor and the ParR–parC complex. One attractive model is that the plasmids move to mid-cell where they are replicated (Lemon and Grossman, 1998). After replication, the plasmids probably pair via ParR–parC interactions, separate, and move towards opposite cell poles, and the cycle is completed. The movement from mid-cell to near-polar or quarter-cell positions presumably requires interaction with a host-encoded mitotic-like apparatus. The data from F and P1 support a similar model. The major difference is that the tethering of F and P1 seems to occur at quarter-cell position, i.e. clearly distinct from that of R1. However, this makes the plasmid life cycle even simpler because quarter-cell positions become mid-cell in newborn cells. Thus, in the cases of F and P1, it is not necessary to postulate a step involving movement from pole to mid-cell.

The replicon pairing model is attractive because the host-encoded receptor that secure ordered segregation does not have to be unique and several compatible partitioning loci would be able to use the same host structure because the pairing-specificity-determinants are encoded by the plasmids themselves. Furthermore, the replicon-pairing model is strongly supported by the in vitro pairing observed for R1 and by the symmetric behaviour of the plasmid R1 segregation pattern (Jensen et al., 1998; Jensen and Gerdes, 1999). In the pairing model, incompatibility is best explained by the formation of mixed pairs of plasmids. The lack of incompatibility of functional but truncated P1 parS and R1 parC sites towards the cognate full-length sites (Breuner et al., 1996; Martin et al., 1987) can be explained if they can pair with themselves but not with the wild-type sites (i.e. the truncation of the centromeres results in a specificity switch). The replicon-pairing model in plasmid partitioning is currently favoured by most groups. Models that explain partitioning of bacterial chromosomes are more speculative (Harry, 1997; Wheeler and Shapiro, 1997; Jensen and Shapiro, 1999; Sharpe and Errington, 1999; Møller-Jensen et al., 2000).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information
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Footnotes
  1. †Present address: Department of Developmental Biology, Stanford University School of Medicine, Beckmann Center B300, Stanford, CA 94305–5329, USA.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Plasmid-encoded partitioning genes
  5. The par family encoding Walker-type ATPases (type I loci)
  6. Phylogeny of Walker-type ATPases involved in plasmid partitioning
  7. The par family encoding actin-like ATPases (type II loci)
  8. Phylogeny of plasmid-encoded par loci encoding the actin type of ATPases
  9. Chromosome-encoded partitioning loci
  10. Phylogeny of Walker-type ATPases involved in chromosome partitioning
  11. Models for plasmid segregation
  12. References
  13. Supporting Information

Global alignment of protein sequences Accession numbers

FilenameFormatSizeDescription
MMI_1975_sm_1.doc111KSupporting info item
MMI_1975_sm_2.doc30KSupporting info item

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