The Maintenance of Plasmids in Pathogenic Organisms

<p>Low copy number plasmids in bacteria are of interest for two principle reasons. First, they act in many ways like small, dispensible chromosomes within the cell, and are therefore tractable models for the study of chromosome replication and segregation. Second, they are of considerable medical importance. They are the transmissible elements that spread antibiotic resistance among pathogenic bacteria and in some cases, are the determinants of the virulence of bacterial infection in human infectious disease.</p>

<p>The spread of antibiotic resistance threatens to make antibiotic therapy virtually useless in the next few decades. In addition, pathogenic bacteria containing virulence plasmids are increasingly the ultimate cause of death of cancer patients whose immune systems are often compromised by disease progression or chemotherapy. It is therefore of importance to try to understand how these plasmids are stably maintained in the bacterial population as a first step toward developing strategies for infectious disease therapy and remediation of plasmid spread.</p>

<p>We are particularly interested in the mechanisms that plasmids use to ensure their proper segregation to daughter cells. We study a family of elements known as partition genes (the P1par family), that are responsible for the segregation of several types of plasmid including the virulence plasmids of Salmonella and Shigella species responsible for enteric disease, and of Yersinia pestis; the causative organism for bubonic plague. In each case, we have shown that segregation is achieved by recognition of a cis-acting site parS, analogous to a centromere, and two plasmid encoded proteins, ParA and ParB. ParB binds specifically to parS and ParA is an ATPase that may be a motor for moving the plasmid during segregation. Members of the P1par family show unique species specificities. This is important, because, otherwise, plasmids of different types would compete with each other, limiting their spread in nature.</p>

<p>We have discovered that these species specificities reside in a novel interaction between the ParB protein and the parS site. This is not the interaction that provides the energy for ParB binding to the site. Rather, it is a special contact between the ParB N-terminus and a short motif in parS termed the B box. By changing the B box sequence by as little as one base, we can change the specificity of the system from one species to another. This mechanism appears to be a novel type of DNA-protein recognition that may have broad implications for how proteins act at a specific site when other potential binding sites exist.</p>

<p>This year, we have further explored the hypothesis that the contact between the BoxB sequences in the cis-acting partition site and the ParB protein are responsible for the species specificity of members of the P1par family of partition elements. By changing the pMT1parS site BoxB sequences to their P1 or P7 equvalents, complete switches of species specificity were acheived. In addition, we were able to show that the specificity of partition mediated plasmid incompatibility, that govens whether two plasmids can be maintained in the same cell is also swiched in these mutants. thus, species psecificity for par protein recognition and partition-mediated incompatibility are co-determined. Thus, the BoxB-ParB recognition is a system for rapid speciation and survival of the plasmid during evolution.</p>