P1 Plasmid Segregation: Accurate Redistribution by Dynamic Plasmid Pairing and Separation

Low-copy-number plasmids, such as P1 and F, encode a type Ia partition system (P1par or Fsop) for active segregation of copies to daughter cells. Typical descriptions show a single central plasmid focus dividing and the products moving to the cell quarter regions, ensuring segregation. However, using improved optical and analytical tools and large cell populations, we show that P1 plasmid foci are very broadly distributed. Moreover, under most growth conditions, more than two foci are frequently present. Each focus contains either one or two plasmid copies. Replication and focus splitting occur at almost any position in the cell. The products then move rapidly apart for approximately 40% of the cell length. They then tend to maintain their relative positions. The segregating foci often pass close to or come to rest close to other foci in the cell. Foci frequently appear to fuse during these encounters. Such events occur several times in each cell and cell generation on average. We argue that foci pair with their neighbors and then actively separate again. The net result is an approximately even distribution of foci along the long cell axis on average. We show mathematically that trans-pairing and active separation could greatly increase the accuracy of segregation and would produce the distributions of foci that we observe. Plasmid pairing and separation may constitute a novel fine-tuning mechanism that takes the basic pattern created when plasmids separate after replication and converts it to a roughly even pattern that greatly improves the fidelity of plasmid segregation.Low-copy-number plasmids encode partition systems for proper distribution of the plasmids during cell division. The par loci encode a cis-acting centromere-like sequence, parS, and two trans-acting proteins, ParA and ParB. ParB is a DNA binding protein that binds to parS, and ParA is an ATPase that is thought to power plasmid segregation. Par systems are classified as type I or type II according to whether they have Walker-type or actin-type ATPase domains in ParA. Type I partition system can be further divided into Ia and Ib by their different genetic organizations and general sequence dissimilarity (12). P1 is a bacteriophage that is maintained as a stable low-copy-number plasmid in its Escherichia coli host (28). It contains a typical type Ia partition system that is essential for proper plasmid segregation (13, 17, 27). The P1par locus is an operon producing ParA and ParB proteins followed directly by the centromere analog site, parS (9). The ParB protein binds specifically to the parS site (35). The ParA protein is an ATPase that interacts with the ParB-parS complex (3, 4, 11). P1 ParA has a nonspecific DNA binding activity (5). F partition depends on the interaction of its ParA protein (SopA) with nonspecific DNA (2). The P1parS site loads ParB in two steps, first binding a core dimer onto the site and then loading additional ParB copies that spread out on the surrounding sequences (31, 32). The latter activity causes a fluorescence-labeled ParB to form a bright focus that marks the position of the plasmid in the living cell (23).Observations of fluorescently labeled F and P1 plasmids show that, when a single plasmid focus is present in the newborn cell, it is located approximately at the cell center.The focus divides, and the products migrate to positions approximating the cell quarters (15, 23, 26). Cell division results in one plasmid focus in each daughter cell. This evidence has been used to suggest that specific sites exist at the cell center and quarters to tether the plasmids (15, 26). However, the limited data presented show that the distribution of focus positions is rather broad and, at least in the P1 case, that the positions to which the plasmid foci migrate are quite variable (23). This casts some doubt on the existence of fixed cell locations. The unrelated type Ib par system of pB171 distributes plasmid foci evenly along the cell length without reference to any particular cell location (7). This distribution has been linked to observations of an oscillation in the local concentration of ParA protein that appears to sweep slowly through the cell between the boundaries of the nucleoid (7). A similar oscillation has been reported for the F plasmid ParA protein, SopA (16).At most growth rates, more than one P1 plasmid copy is present at cell birth. How are the additional copies accommodated? For the F plasmid, measurements of foci and plasmid copy numbers suggest that the center-to-quarters paradigm holds at increased copy numbers and that each migrating focus can contain more than one plasmid copy (15). In our initial studies, this appeared to hold for P1 also (23). In these studies, the fluorescent marker protein, green fluorescent protein (GFP)-ParB, was transiently induced to the highest practicable level in order to visualize the foci. We now know that this treatment has adverse effects on the plasmid. The plasmids are subject to a reduction in copy number and delayed segregation through the period of observation (data not shown). The advent of improved optics has allowed us to avoid these problems and to study foci when the fluorescent protein is expressed continuously at a low level. We quickly realized that multiple foci were present under these conditions, that the positions and behaviors of foci were more dynamic, and that the center-to-quarters paradigm did not hold for P1 in the majority of cells.