Mechanisms of Chromosome Maintenance in Bacteria

无论何种生物体，基因组的稳定性要求整个DNA在每个细胞周期内复制一次且仅一次。即使在大肠杆菌这样一个研究得很好的系统中，保证复制启动和防止过早再启动的复杂调控机制仍在解开。在细菌质粒中对复制控制有更好的了解。然而，质粒复制子虽然在细菌中普遍存在，但很少在细菌染色体中发现。在多染色体细菌（如霍乱弧菌）的次级染色体（具有较少管家基因的染色体）中发现了例外。在这种细菌中，染色体II的复制起源具有经过充分研究的携带iteron质粒的所有特征，表明其质粒来源，但它在控制复制的位点上表现出新的特征。我们对这些特征感兴趣是为了了解染色体II是如何像其他染色体一样以细胞周期特定的方式复制的，而不是像质粒那样在整个细胞周期中复制。我们发现，虽然质粒在其控制位点上只有iterons，但染色体II有第二种位点，它们是39-mers，与iterons的序列无关。39-mers与干扰素结合相同的启动蛋白，但它们是复制的主要负调节因子。它们增强了手铐作用，这是一种已知的利用内含子使质粒起源失活的机制。然而，39-mers的活性受到控制位点的干扰素的抑制。因此，控制迭代子促进复制，这一作用在质粒复制中尚不清楚。这表明，尽管在起源区域有相似性，染色体II控制系统与质粒系统有很大的差异。染色体II的新功能可能使其复制与细胞周期一致。霍乱弧菌的两条染色体起源是不同的，这就提出了一个问题，即它们是如何在每个细胞周期内激活一次的。我们已经确定，尽管两个起源具有不同的特征，但每个细胞周期一次的复制需要两个起源在其GATC位点的腺嘌呤残基上完全甲基化。对于染色体I，其起源与大肠杆菌起源相似，甲基化被发现对复制起始是不必要的，但需要控制复制起始频率，如大肠杆菌的情况。对于染色体II，甲基化是启动子与起始iteron结合的额外需要，这是启动过程本身的基本功能。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。对引发剂结合的要求也使得甲基化成为霍乱弧菌的基本功能。多染色体细菌（不像大肠杆菌和枯草芽孢杆菌这样的单染色体细菌）可能为研究不同染色体的协调复制和分离机制提供了机会。已获得霍乱弧菌染色体间协调的初步迹象。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。阻止染色体I的复制似乎可以阻止/延迟染色体II的复制，但反之则不然。2号染色体比1号染色体小，这种延迟可能使两条染色体同时完成复制，这可能有利于它们的分离与细胞分裂的协调。延迟的机制还有待研究。与我们对真核生物中染色体如何分离的理解相比，我们对细菌中染色体如何分离的了解要少得多。直到最近，分离研究主要是在质粒中进行的，在质粒中可以找到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1号染色体上的par基因，而没有造成太多的分离缺陷。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。par基因在复制起点附近的广泛存在表明，这些基因可能在连接复制和分离中起作用。par基因如何促进复制目前还在研究中。利用细菌和酵母的双杂交系统，我们正试图识别可能与Par蛋白相互作用的蛋白质，这可能为它们的作用机制提供线索。细胞分裂必须等待染色体复制完成和两个姐妹染色体运动到相反的细胞半。在真核生物中，DNA复制通过严格调控的细胞周期依赖机制与细胞生长和分裂耦合。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，我们正在确定这些较小细胞变异的复制起始时间（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，这意味着在起始时，突变体的细胞大小比野生型小。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。起始的延迟由复制延伸率的增加来补偿，从而使复制周期按时完成。通过过量产生启动蛋白dna，可以避免启动延迟。因此，在大肠杆菌中，限速成分似乎是dna，而在枯草芽孢杆菌中则不是。与真核生物一样，枯草芽孢杆菌的定时复制起始可能通过细胞周期依赖机制与分裂相耦合。

Irrespective of the organism, genome stability requires that the entire DNA be replicated once and only once per cell cycle. The intricacies of regulatory mechanisms that guarantee initiation of replication and prevent premature reinitiation are still being unraveled even in a well-studied system like E. coli. Replication control is better understood in bacterial plasmids. However, plasmid replicons, although prevalent in bacteria, are seldom found in bacterial chromosomes. Exceptions have been found in secondary chromosomes (one with fewer house-keeping genes) of multichromosome bacteria, such as Vibrio cholerae. In this bacterium, the replication origin of chromosome II bears all the hallmarks of well-studied iteron-carrying plasmids, indicating its plasmid provenance, but it exhibits novel features in the locus that controls replication. We are interested in those features to understand how chromosome II replicates in a cell-cycle specific fashion like other chromosomes, rather than throughout the cell cycle, as do plasmids. We show that while plasmids have only iterons in their control locus, chromosome II has a second kind of sites, which are 39-mers and unrelated in sequence to iterons. The 39-mers bind the same initiator protein as do the iterons but they are the primary negative regulators of replication. They enhance handcuffing, a mechanism known to inactivate plasmid origins with iterons. The activity of the 39-mers is, however, dampened by iterons of the control locus. The control iterons thus facilitate replication, a role not known to be operative in plasmid replication. It appears that, in spite of the similarity in the origin region, the chromosome II control system has diverged considerably from the plasmid system. The novel functions of chromosome II might align its replication to the cell cycle. The two chromosomal origins of V. cholerae are distinct, which raises the question how they fire once per cell cycle. We have determined that in spite of the distinct features of the two origins, once-per-cell-cycle replication requires that both the origins be fully methylated at the adenine residues of their GATC sites. For chromosome I, whose origin is similar to the E. coli origin, methylation was found to be dispensable for replication initiation but required to control replication initiation frequency, as is the case in E. coli. For chromosome II, methylation was additionally required for initiator binding to the origin iterons, an essential function in the initiation process per se. Although methylation is widely used to control many DNA transactions, its role in mediating initiator-origin interactions is an unprecedented finding. The requirement of initiator binding also makes methylation an essential function in V. cholerae. Multichromosomal bacteria (unlike such well-studied mono-chromosome bacteria as E. coli and B. subtilis) may offer opportunities to investigate mechanisms for coordinating replication and segregation of the different chromosomes. An initial indication of inter-chromosomal coordination in V. cholerae 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 chromosome I replication can prevent/delay chromosome II replication but the reverse is not true. Chromosome II is smaller than chromosome I and the delay might allow the two chromosomes to complete replication at the same time, which might facilitate the coordination of their segregation with the cell division. The mechanism of delay remains to be investigated. Compared to our understanding of how chromosomes segregate in eukaryotes, much less is known about how chromosomes segregate in bacteria. Until recently, segregation studies were done primarily in plasmids, where genes dedicated to plasmid partition (par genes) could be 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 chromosome I without causing much segregation 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. The widespread occurrence of par genes near the replication origins suggests that the genes might have a role in connecting replication and segregation. How might the par genes promote replication is under current investigation. Using both the bacterial and yeast two-hybrid systems we are trying to identify proteins that might interact with Par proteins, which might provide a clue as to their mechanism of action. Cell division must await completion of chromosome replication and movement of the two sister chromosomes to opposite cell halves. In eukaryotes, DNA replication is coupled to cell growth and division via tightly regulated cell cycle-dependent mechanisms. 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 these smaller cell variants. The initiation age seems to depend on the bacterium in question. In B. subtilis, the age remains unchanged in the mutants, meaning that at the time of initiation, the cell size is smaller in the case of mutants compared to the wild type. 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 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. The rate-limiting component thus appears to be DnaA in E. coli but not in B. subtilis. The timing replication initiation in B. subtilis might be coupled to division through a cell cycle dependent mechanism, as in eukaryotes.