The Maintenance of Plasmids in Pathogenic Organisms

细菌中的低拷贝数质粒之所以令人感兴趣，主要有两个原因。首先，它们在许多方面都像细胞内的小染色体，因此是研究染色体复制和分离的易于处理的模型。其次，它们在医学上具有相当重要的意义。它们是在病原菌之间传播抗生素耐药性的可传播元件，在某些情况下，是细菌感染在人类传染病中毒力的决定因素。抗生素耐药性的传播可能会使抗生素治疗在未来几十年变得几乎毫无用处。此外，含有毒力质粒的病原菌越来越多地成为癌症患者死亡的最终原因，他们的免疫系统经常因疾病进展或化疗而受到损害。因此，重要的是要试图了解这些质粒是如何在细菌种群中稳定保持的，这是开发新的传染病治疗和补救质粒传播策略的第一步。我们特别感兴趣的是，质粒用来确保其正确分离到子代细胞的机制。我们研究了一组被称为分割基因的元素(P1par家族)，它们负责分离几种类型的质粒，包括引起肠道疾病的沙门氏菌和志贺氏菌的毒力质粒，以及引起腺鼠疫的鼠疫耶尔森氏菌。在每种情况下，我们都证明了分离是通过识别一个类似于着丝粒的顺式作用位点PARS和两个编码蛋白质的质粒ParA和PARB来实现的。PARB与PARS特异结合，而PARB是一种ATPase，它可能是分离过程中推动质粒移动的马达。P1par家族的成员表现出独特的物种特异性。这一点很重要，因为，否则，不同类型的质粒会相互竞争，限制它们在自然界的传播。我们发现，这些物种特异性存在于PARB蛋白和PARS位点之间的一种新的相互作用中。这不是为PARB与位点结合提供能量的相互作用。相反，它是PARB N-末端与PARS中一个称为B盒的短基序之间的一种特殊接触。只要将B盒序列改变一个碱基，我们就可以将系统的特异性从一个物种改变到另一个物种。这种机制似乎是一种新型的DNA-蛋白质识别，可能对蛋白质在存在其他潜在结合位点时在特定位置的作用具有广泛的影响。今年，我们进一步探索了这样的假设，即顺式作用分配位点上的boxB序列与PARB蛋白之间的接触是导致P1par分配元件家族成员的物种特异性的原因。以最近发表的P1 PARB蛋白的晶体结构为指导，我们已经能够准确地定位PARB蛋白表面的位置，该位置指定了单个质粒DNA PARS位点上的物种决定因素的识别。通过改变P1PAR蛋白中的单一氨基酸，我们已经能够将其识别特异性完全转换为P7的识别特异性。这加强了我们的信念，即这些相关质粒之间的PARB/PARS识别系统代表了一种新的机制，用于新的质粒类型的快速进化。

Low copy number plasmids in bacteria are of interest for two principle reasons. First, they act in many ways like small, dispensable 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. 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 novel strategies for infectious disease therapy and remediation of plasmid spread. 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. 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. 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. Using the recently published crystal structure of the P1 ParB protein as a guide, we have been able to pinpoint the position on the ParB protein surface that specifies recognition of species determinants on the individual plasmid DNA parS sites. By changing a single amino acid in the P1 Par protein we have been able to switch its recognition specificity completely to that of P7. This reinforces our belief that the ParB/parS recognition system among these related plasmids represents a novel mechanism for the rapid evolution of novel plasmid types.