Species and Incompatibility Determination within the P1par Family of Plasmid Partition Elements

Low-copy-number bacterial plasmids contain active plasmid partition systems that move plasmid copies into daughter cells to ensure that each cell receives at least one copy after cell division. In each case studied so far, the system consists of a single locus containing an operon for two proteins and a cis-acting partition site. The first protein is an ATPase, and the second is a specific DNA binding protein that recognizes the cis-acting site (29). Related systems can be found on the chromosomes of a variety of bacterial species, where they are implicated in chromosome segregation (13). Partitioning systems can be divided into two types: one encoding an ATPase of the Walker-type ATPase superfamily (18) and the other an actin/hsp70 type ATPase (3). The Walker-type systems are common and widely distributed among plasmid species. They can be classified into two groups, designated types Ia and Ib (13). Type Ia is the best characterized and includes the intensively studied partition systems of the P1 and F plasmids of Escherichia coli (P1par and Fsop). Although the F and P1 systems show some homology, they have cis-acting sites with very different organizations. The F site (incD) consists of 12 contiguous 48-bp repeat sequences just downstream of sopB, the second partition protein open reading frame (17). The P1 partition site, parS, is similarly placed downstream of the parB open reading frame but consists of a single copy of a sequence approximately 80 bp long. Within it is a central integration host factor (IHF) binding site flanked by two ParB binding regions (7, 11). This type of site is characteristic of a distinct family of partition systems, the P1par family, found in a variety of plasmids from various gram-negative bacteria (12). Three members of the P1par family have been studied extensively. The P1 and P7 systems are from closely related bacteriophages that have plasmid prophage forms (1, 22). The pMT1par system is from the large virulence plasmid pMT1 of Yersinia pestis, the facultative intracellular agent of bubonic plague (9, 21). Despite the fact that these three systems are very similar in terms of sequence and organization, each shows a unique species specificity. The parS site of one species is unable to function using the Par proteins of another species (27). This specificity is speculated to be an advantage in preventing competition from other family members (2, 24). In the case of the P1 and P7 par systems, the critical information for species specificity has been mapped. It resides in a pair of direct six-base repeats in parS (the BoxB sequences), and in a short motif within the C-terminal domain of the ParB protein termed the discriminator recognition sequence (DRS) (25). These sequences recognize each other in a species-specific interaction (15, 24, 25). The localized contact between parS BoxB and ParB is distinct and separable from the binding contact between the parS site and the protein that provides the bulk of the binding energy (28). The latter involves a different set of parS motifs, i.e., the BoxA sequences, which bind to a helix-turn-helix motif in the interior of the ParB sequence (25, 28). We have speculated that the BoxB-ParB contact might constitute a special mechanism for defining species specificity in the P1par family. This idea receives support from the properties of pWR100par from the recently characterized large virulence plasmid pWR100 of Shigella flexneri (27). There are many sequence and organizational differences between the parS sites and the ParB proteins of pWR100 and P1. However, the BoxB sequences and the ParB DRS motif with which the boxes are thought to interact are very similar. As predicted, the pWR100 and P1par systems show identical species specificities (27). Here, we show that the unique species specificity of the pMT1par system is determined by a unique variation in the BoxB repeats. Plasmids of similar types exhibit incompatibility: they cannot be maintained together in the same cell. Partition systems are a major factor in determining this (2). Two plasmids with identical parS sites compete with each other for partition, leading to the loss of one plasmid or the other (2). Most P1par family members have unique incompatibility specificities: they interfere only with their own types. Here, we examine the incompatibility specificities of parS sites with altered BoxB sequences and show that species and incompatibility specificities switch in concert.