Mechanisms of Chromosome Maintenance in Bacteria

我们的兴趣在于控制染色体复制和分离的机制。这些基本机制在生物体中广泛共享，对它们的生长和发育至关重要。这些机制的破坏导致非整倍体，这是癌细胞的标志。因此，基因组稳定性的重要性怎么强调都不过分。更好地理解细菌模型中的作用机制应该具有普遍的意义，并指导疾病，特别是细菌感染的治疗方法的合理发展。在大肠杆菌和霍乱弧菌中，后者具有两条染色体（chrI和chrII），这为研究遗传上易处理的细菌中复制和分离的协调提供了机会。在过去的一年里，我们已经结束了一些关于低拷贝数E的复制控制的项目。coli质粒、P1. Nilangshu Das在数学家Johan Paulsson（剑桥大学，UK），已经表征了赋予质粒更高拷贝数的控制缺陷型起始突变体。突变体的性质最好的解释假设多种模式的控制，涉及转录自抑制的启动基因，启动失活的二聚体，和起源失活配对。当三种机制合作时，控制是最好的。近年来，E.大肠杆菌DNA复制是在复制起点发现起始DnaA的额外结合位点，称为I-sites（IHF蛋白依赖性DnaA结合位点）。这些位点只识别ATP结合的DNA。DNA复制中I位点的普遍性仍有待确定，因为没有一致序列可以从E. coli来源。Richard Fekete在质粒P1中鉴定出一个可能的I位点依赖性复制起点。这种起源对DnaA的ATP结合形式的依赖性正在测试中。由于ATP-DnaA水平在染色体复制启动后下降，这可能是质粒复制与染色体复制相关的一种方式，并为质粒复制控制增加了一个新的范例。该基因的一个强启动子被鉴定，并被发现是自阻遏的，以及受未流序列和全局调节因子IHF和Dam甲基化酶的调节。反式增加RctB增加了携带chrII起点的微型质粒的拷贝数，这意味着RctB可以限制chrII复制的速率。对RctB的多种控制模式预期减少引发剂浓度的波动，从而有助于维持染色体拷贝数稳态。为了研究chrII复制的调节，Tatiana Venkova-Canova定义了起点及其负控制元件。起源类似于那些从iteron型质粒，但负控制基因座更广泛和复杂。已经清楚的是，复制的调节已经明显不同于在iteron型质粒或E.与低拷贝数质粒和许多其他细菌不同，大肠杆菌的质粒拷贝数很低。大肠杆菌不含有与任何已知分离系统的同源性。寻找E.大肠杆菌着丝粒，Richard Fekete标记了同一个大肠杆菌上的不同位点。使用LacI-YFP和Lambda cI-CFP融合蛋白结合到其各自结合位点的阵列。使用荧光显微镜分析几对这样的位点的迁移表明了一个潜在的着丝粒位点，因为它先于其他位点（包括复制起点）向细胞极迁移。Preeti Srivastava正在使用荧光显微镜和流式细胞术研究霍乱弧菌的染色体动力学。

Our interest is in mechanisms that control chromosome replication and segregation. These basic mechanisms are widely shared among organisms and are fundamental to their growth and development. Subversion of the mechanisms causes aneuploidy, the hallmark of cancer cells. The importance of genome stability, therefore, can hardly be overstated. A greater understanding of the mechanisms operating in bacterial models should be of general interest and guide the rational development of therapeutics for diseases, particularly bacterial infections.We are studying the control mechanisms in E. coli and in V. cholerae, the latter having two chromosomes (chrI and chrII) provides an opportunity to study coordination of replication and segregation in a genetically tractable bacterium. In the last year, we have brought to closure a number of projects on replication control of a low copy number E. coli plasmid, P1. Nilangshu Das with help from a mathematician, Johan Paulsson (Cambridge U., UK), has characterized control-defective initiator mutants that confer higher copy number to the plasmid. The properties of the mutants are best explained assuming multiple modes of control involving transcriptional autorepression of the initiator gene, initiator inactivation by dimerization, and origin inactivation by pairing. The control is best when the three mechanisms cooperate. A new development in the field of E. coli DNA replication is the finding of additional binding sites for the initiator DnaA in the origin of replication, called I-sites (IHF protein-dependent DnaA binding sites). These sites recognize only ATP-bound DnaA. The generality of I-sites in DNA replication remains to be established as no consensus sequence could be derived from the three I-sites present in the E. coli origin. Richard Fekete has identified a possible I-site dependent replication origin in plasmid P1. The dependence of this origin on the ATP bound form of DnaA is being tested. Since ATP-DnaA level decreases after the initiation of chromosomal replication, this may be a way for the plasmid to correlate its replication with that of the chromosome and add a new paradigm in plasmid replication control.Knowing the importance for controlling initiator proteins in DNA replication control, Debasish Pal has studied the regulation of the RctB initiator for chrII replication. A strong promoter for the gene was identified and found to be autorepressed as well as regulated by unstream sequences and global regulators, IHF and Dam methylase. Increasing RctB in trans increased the copy number of a miniplasmid carrying the chrII origin, implying that RctB can be rate-limiting for chrII replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis. To study the regulation of replication of chrII, Tatiana Venkova-Canova has defined the origin and its negative control elements. The origin resembles those from iteron-type plasmids but the negative control locus is more extended and complex. It is already clear that the regulation of replication has diverged significantly from the ones operating in iteron-type plasmids or in E. coli.Unlike low copy number plasmids and many other bacteria, E. coli contains no homology to any known segregation system. To search for the E. coli centromere, Richard Fekete has labeled different loci on the same E. coli chromosome using LacI-YFP and Lambda cI-CFP fusion proteins bound to arrays of their respective binding sites. Analysis of migration of a few such pairs of loci using fluorescence microscopy suggested a potential centromere site since it migrated towards the cell pole ahead of the other loci, including the origin of replication.Preeti Srivastava is studying chromosome dynamics in V. cholera using fluorescence microscopy and flow cytometry.