Mechanisms of Chromosome Maintenance in Bacteria

在过去的一年里，我们已经完成了对低拷贝数E. coli质粒、P1. Nilangshu Das在数学家Johan Paulsson（剑桥大学，UK），已经表征了控制缺陷型起始突变体，其赋予质粒相对不受阻碍的复制能力。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DNA A的弱结合位点，称为I位点。这些位点仅被ATP结合的DnaA识别。I位点在DNA复制中的普遍性仍有待确定。Richard Fekete在质粒P1的复制起点中发现了一个可能的I位点。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，为质粒复制控制增加了一种新的范式。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。其起源类似于P1型质粒，但阴性对照位点更广泛和复杂。已经清楚的是，复制的调控已经明显不同于质粒或大肠杆菌中的调控。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。到目前为止，她已经跟踪了两条染色体的起点和终点的运动。迁移模式似乎不同的两个染色体之间，以及从什么已经在其他系统中报道。新发现之一是两条染色体末端区域的凝聚力，这表明在这些区域可能存在分离信息。

In the past year, we have brought to closure 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 relatively unhindered replication profiency 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 weak (incognito) binding sites for the initiator DnaA in the origin of replication, called I-sites. These sites recognize only by ATP-bound DnaA. The generality of I-sites in DNA replication remained to be established. Richard Fekete has identified a possible I-site in the replication origin in plasmid P1. 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 chromosome II replication of V. cholerae. 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 of P1 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 plasmids or in E. coli.Preeti Srivastava is studying chromosome dynamics in V. cholera using fluorescence microscopy and flow cytometry. So far she has tracked the movement of the origins and terminii of both the chromosomes. The migration patterns seem to differ between the two chromosomes as well as from what has been reported in other systems. One of the novel findings is the cohesion of the terminus region of the two chromosomes, which suggests the possibility that there could be segregational information in those regions.