Mechanisms of Chromosome Maintenance in Bacteria

DNA甲基化对chrI和chrI复制的要求：霍乱弧菌有两条染色体（chrI和chrI），而大多数细菌只有一条染色体。ChrI是主要染色体，因为它有大部分的管家基因，大部分是与大肠杆菌染色体上的对应基因同源的，包括复制基因。然而，据报道，chrI和大肠杆菌染色体的复制在甲基化需求方面有所不同。在这两种细菌中，复制起源具有异常高密度的腺嘌呤甲基化GATC位点。在大肠杆菌中，甲基化对于限制每个细胞周期只复制一次很重要，但这一要求对生存能力不是必需的。我们已经证明，这是复制的chrI的情况下也，和最初的主张，甲基化是必不可少的chrI复制是一个人为的实验设置。然而，甲基化对于crisi复制是必不可少的。crisi的起源不同于crisi的起源，与一些研究得很好的质粒起源相似。并不是所有的质粒起始点在每个细胞周期内都激活一次，但像其他染色体起始点一样，chrII起始点也会激活一次。我们发现每个细胞周期一次的chrII复制也依赖于腺嘌呤甲基化。对于crisi，甲基化是引发剂与起始点结合的额外需要，这是引发过程中的一个重要功能。虽然甲基化被广泛用于控制许多DNA交易，但它在介导起始物相互作用中的作用是一个前所未有的发现。质粒向染色体复制模式的转变：在所有生物体中，DNA复制被限制在细胞周期的特定阶段。了解这在具有不同复制机制的crisi中是如何发生的，可能会为这一基本过程提供一个新的视角。基于这一动机，我们正在详细研究对crisi复制的控制。我们发现，crisi启动子特异性地与两种位点结合：一种相当于重复序列，在一些质粒中很常见，称为iteron。然而，与质粒内含子不同的是，chrII内含子在与引发子结合之前必须被甲基化。另一种是39-mers，它在序列上与迭代子无关。39-mers是复制的主要负调控因子。它们增强了iterons之间的相互作用，使起源失活。iteron反过来也可以抑制39-mers的抑制活性。两种位点之间的相互作用如何使crisi复制与细胞周期一致已经被建模。该模型还包含了这样一个事实，即在起源激活后变得半甲基化的起源iterons，由于半甲基化特异性蛋白SeqA的结合，在细胞周期的三分之二时间内保持在失活状态。这种情况不会发生在质粒内，因为它们没有甲基化。因此，chrII控制系统与最可能满足细胞周期特异性复制需求的质粒系统有很大的分歧。霍乱弧菌的染色体间通讯：我们假设霍乱弧菌的两条染色体的及时复制和分离需要它们之间的通讯，以便两条染色体都能完成细胞分裂前的过程。染色体间通讯的初步迹象已经获得。我们已经能够找到可以选择性地阻止其中一条染色体复制的条件。似乎阻止chrI复制可以阻止/延迟II号染色体的复制，但反之并非如此。目前正在研究chrI如何决定chrI复制的机制。复制和分离之间的交流：在真核生物中，分离的准备而不是分离本身是从复制开始的。在细菌中，分离本身遵循复制起始（共复制分离），甚至可能取决于复制的行为。然而，直到最近才知道分离影响复制的反向过程。在细菌中，我们对染色体分离的认识主要来自对质粒的研究，在质粒中首次发现了专门用于质粒分裂的基因（par基因）。质粒par基因的同源物现已在大多数细菌（包括霍乱弧菌）的复制起源附近被鉴定出来。弧菌的两条染色体都有自己的par基因。我们成功地删除了chrI的par基因，而没有造成太多的分离或生长缺陷。相反，两个par基因中的一个（parB）的缺失促进了复制。在枯草芽孢杆菌中也有类似的发现。枯草芽孢杆菌和霍乱弧菌这两种细菌在10亿多年前就已经分化，但它们都保留了par基因，并将它们用于相似的目的。利用细菌和酵母双杂交系统，我们已经确定了细菌复制启动子dna是Par蛋白的直接靶点。Par蛋白如何刺激dna活性仍有待研究。细菌中细胞的大小和DNA复制的开始：细胞分裂必须等待染色体复制完成和两个姐妹染色体分离到相反的细胞一半。在真核生物中，DNA复制通过周期蛋白依赖激酶和相关因子的作用与细胞生长和分裂相耦合。细菌中细胞分裂的时间控制在很大程度上是未知的。最近，在大肠杆菌和枯草芽孢杆菌中发现了一些参与感知生长培养基中葡萄糖浓度的基因来调节细胞大小。这些基因的突变会使细胞变小30%，但不会改变它们的生长速度。在一项合作研究中，我们正在确定这些小尺寸突变体细胞周期中复制起始的时间（起始年龄）。起始年龄似乎取决于所讨论的细菌。在枯草芽孢杆菌中，突变体的年龄保持不变，这表明特定细胞大小的实现不是初始化的必要条件。然而，在大肠杆菌中，起始被延迟到突变体达到野生型起始的大小。起始的延迟被复制延伸率的增加所补偿，允许复制周期按时完成。通过过量产生启动蛋白dna，可以避免启动延迟。这些结果与dna是大肠杆菌复制起始的限速成分一致，并且引发剂的积累到起始的临界水平取决于生长。dna在枯草芽孢杆菌中也有速率限制，但似乎不受生长依赖方式的控制。因此，尽管dna可能是所有细菌起始所必需的，但控制其供应的机制可能并不保守。crisi引发物RctB结构域分析：面向弧菌特异性抗菌药物的制备。鉴于致病性弧菌中多药耐药性日益普遍，需要寻找新的靶点和药物来对抗这些病原体。RctB是一种仅在弧菌科中保守的crisi特异性启动子。这种蛋白质似乎非常适合开发抗弧菌特异性药物。如果知道目标蛋白的三维结构信息，将极大地促进药物设计。为了实现这个目标，我们已经开始对RctB进行系统的域分析。一般来说，复制启动子已被证明难以进行结构研究，因为它们具有可塑性，需要通过伴侣蛋白和/或与特定DNA结合来重塑活性。最初试图形成RctB晶体的尝试失败了。我们正试图通过删除分析来分离功能域。这些较小的衍生物可能更适合于结构研究。

DNA methyaltion requirement of chrI and chrII replication: V. cholerae has two chromosomes (chrI and chrII) whereas most bacteria have one. ChrI is the primary chromosome, as it has most of the house-keeping genes, mostly homologous to their counterparts in the E. coli chromosome, including the replication genes. However, replication of chrI and the E. coli chromososme was reported to differ in terms of the requirement of methylation. In both bacteria, the origin of replication has unusually high density of GATC sites for adenine methylation. In E. coli, methylation is important for restricting replication to only once per cell cycle but this requirement is not essential for viability. We have shown this to be the case for replication of chrI also, and the original claim that methylation is essential for chrI replication was an artefact of the experimental set up. Methylation, however, is essential for chrII replication. The chrII origin is distinct from that of chrI and is similar to some of the well-studied plasmid origins. Not all plasmid origins fire once per cell cycle but the chrII origin does, like other chromosomal origins. We found that the once-per-cell-cycle replication of chrII also depends on adenine methyaltion. For chrII, methylation was additionally required for initiator binding to the origin, an essential function in the initiation process. Although methylation is widely used to control many DNA transactions, its role in mediating initiator-origin interactions is an unprecedented finding. Transition of plasmid to chromosomal mode of replication: In all organisms, DNA replication is restricted to a specific stage of the cell cycle. Understanding of how this happens in chrII with distinct replication machinery might provide a fresh perspective on this fundamental process. With this motivation, we are studying the control of chrII replication in detail. We discovered that the chrII initiator binds site-specifically to two kinds of site: One is equivalent to repeats, common in some plasmids, called iterons. However, unlike the plasmid iterons, the chrII iterons have to be methylated before they can bind the initiator. The other kind is the 39-mers, which are unrelated in sequence to iterons. The 39-mers are the primary negative regulators of replication. They enhance interactions between iterons that inactivates the origins. The iterons in turn can also dampen the inhibitory activity of the 39-mers. How the interplay between two kinds of site might align chrII replication to the cell cycle has been modeled. The model also incorporates the fact that the origin iterons, which becomes hemimethylated after the origin firing, are kept in an inactivated state due to the binding of a hemimethylation-specific protein, SeqA, for two-thirds of the cell cycle. This does not happen to plasmid iterons because they are not methylated. The chrII control system thus has diverged considerably from the plasmid system most likely to meet the demand of cell cycle specific replication. Inter-chromosome communication in V. cholerae: We hypothesize that timely replication and segregation of the two chromosomes of V. cholerae will require communication between them, so that both the chromosomes can complete the processes before cell division. An initial indication of inter-chromosomal communication has been obtained. We have been able to find conditions where replication of one of the chromosomes could be selectively prevented. It appears that preventing chrI replication can prevent/delay chromosome II replication but the reverse is not true. The mechanism of how chrI dictates chrII replication is being investigated. Communication between replication and segregation: In eukaryotes, the preparation of segregation but not the segregation itself starts with replication. In bacteria, segregation itself follows replication initiation (coreplicational segregation) and may even depend upon the act of replication. However, the reverse process of segregation influencing replication was not known until recently. In bacteria, our knowledge of chromosome segregation primarily comes from studies in plasmids, where genes dedicated to plasmid partition (par genes) were first found. Homologues of plasmid par genes have now been identified near the origin of replication in most bacteria, including V. cholerae. Both the Vibrio chromosomes have their own par genes. We have succeeded in deleting the par genes of chrI without causing much segregation or growth defect. Rather, deletion of one the two par genes (parB) promoted replication. A similar finding has also been made in B. subtilis. The two bacteria, B. subtilis and V. cholerae, have diverged more than a billion years ago but both have retained the par genes and use them for similar purposes. Using both the bacterial and yeast two-hybrid systems we have identified the bacterial replication initiator DnaA to be the direct target of Par proteins. How the Par proteins stimulate the activity of DnaA remains to be studied. Cell size and the initiation of DNA replication in bacteria: Cell division must await completion of chromosome replication and segregation of the two sister chromosomes to opposite cell halves. In eukaryotes, DNA replication is coupled to cell growth and division through the actions of cyclin-dependent kinases and associated factors. The temporal control of cell division is largely unknown in bacteria. Recently, some of the genes involved in sensing glucose concentration in the growth media have been found to regulate cell size in E. coli and in B. subtilis. Mutations in these genes make cells smaller by about 30% but do not change their growth rates. In a collaborative study, we are determining the timing of replication initiation (initiation age) in the cell cycle of these small size mutants. The initiation age seems to depend on the bacterium in question. In B. subtilis, the age remains unchanged in the mutants, indicating that achievement of a particular cell size is not obligatory for initiation. In E. coli, however, initiation is delayed until the mutants reach the size at which initiation occurs in the wild type. The delay in initiation is compensated by an increase in replication elongation rate, allowing the replication cycle to complete on time. The initiation delay could be avoided by overproducing the initiator protein, DnaA. These results are consistent with DnaA being the rate-limiting component of replication initiation in E. coli, and that the accumulation of the initiator to a level critical for initiation depends upon growth. DnaA is also rate-limiting in B. subtilis but appears not to be controlled in a growth dependent manner. Thus, although DnaA is likely to be required for initiation in all bacteria, the mechanisms governing its supply might not to be conserved. Domain analysis of chrII initiator RctB: Towards generating Vibrio specific antimicrobial agents. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. RctB is a chrII-specific initiator and is conserved only in the family Vibrionaceae. The protein appears ideally suited for developing anti-vibrio specific drugs. Drug design is greatly facilitated if the 3-D structural information of the target protein is known. Towards this goal we have started a systematic domain analysis of RctB. Replication initiators in general have proven refractory to structural studies because they are plastic and require for activity remodeling by chaperone proteins and/or binding to specific DNA. Initial attempts to form crystals of RctB have failed. We are attempting deletion analysis to isolate functional domains. These smaller derivatives might be more amenable for structural studies.