Mechanisms of Chromosome Maintenance in Bacteria

我们的大部分项目都集中在了解霍乱弧菌细胞周期中如何控制crisi的复制。染色体被认为起源于质粒，但它在细胞周期的特定时间开始复制，类似于其他真正的染色体，但与质粒不同，质粒的复制时间通常在细胞周期中不固定。我们正试图理解细胞周期中起始的随机时间是如何固定下来的。chrI复制似乎也依赖于chrI复制。两条染色体如何沟通以协调复制在很大程度上是未知的，这一知识是我们理解细菌（如霍乱弧菌）中多条染色体如何维持的基础。最后，chrII的复制是由分离蛋白控制的。分离对复制影响的发现是细菌染色体动力学领域的最新进展。我们在了解这些过程方面的进展报告如下。从质粒到染色体复制模式的转变：我们发现，chrII启动器特异性地结合两种位点：一种位点由iterons组成，相当于重复序列，这是质粒家族复制起源的特征。另一种称为39-mers的位点由不相关的序列组成。然而，与质粒内含子不同的是，chrII内含子必须在它们的腺嘌呤残基上被甲基化，才能与启动子结合。39-mers是复制的主要负调控因子。它们增强了使起源失活的相互作用。iteron反过来也可以抑制39-mers的抑制活性。这两种位点之间的相互作用是如何协调细胞周期的chrII复制的。该模型还考虑了这样一个事实，即在起源激活后半甲基化的起源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，除了质粒系统中存在的特征外，chrII控制系统还具有其他特征，这些特征很可能允许细胞周期特异性复制。chrI对chrI复制的控制：我们假设霍乱弧菌两条染色体的及时复制和分离需要它们之间的通信，以便两条染色体都能在细胞分裂之前完成这一过程。染色体间通讯的初步证据已经获得。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。crisi的复制也由分离蛋白控制：直到最近才知道分离可以影响复制。我们对细菌中染色体分离的认识主要来自于质粒的研究，在质粒中首次发现了专门用于质粒分离的基因（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。当两个par基因（parB）中的一个缺失时，该染色体的复制被特异性地促进。在枯草芽孢杆菌中也有类似的发现。在枯草芽孢杆菌和霍乱弧菌中，普遍的细菌引发剂dna被发现是Par蛋白的直接靶点。Par蛋白如何刺激枯草芽孢杆菌的dna活性已被研究，其机制可能与霍乱弧菌相似。因此，我们不再继续这个项目，而是询问分离蛋白是否也会影响chrII的复制。我们发现，chrII的着丝粒结合蛋白ParB2可以扩散到着丝粒之外，覆盖起源的一个强复制抑制位点（39-mer）。这可能会干扰39-mer与启动物RctB的相互作用，这种相互作用是抑制复制所必需的。出乎意料的是，ParB2也可以通过直接结合不同的39-mer来促进复制，而不需要着丝粒。ParB2和RctB竞争与该位点的结合，这可能会损害其抑制活性。因此，ParB2似乎通过与两个强复制抑制位点的结合竞争来促进复制，通过扩散到一个位点并特异性地结合到另一个位点。扩散代表了一种新的远程复制控制机制；非着丝体结合是由分离蛋白控制的一种新的机制。这些研究确立了分离蛋白影响复制的几种途径。我们认为，在霍乱弧菌中，分离蛋白直接参与复制控制是获得的质粒作为第二染色体永久居留的假设适应性的一个特征。弧菌特异性抗菌剂的产生：鉴于致病性弧菌的多药耐药性日益普遍，需要新的靶点和药物来对抗这些病原体。crisi特异性启动子RctB仅在弧菌科中保守。这种蛋白质似乎非常适合开发潜在的抗弧菌特异性药物。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，因为它们需要伴侣蛋白重塑和/或与特定DNA结合才能产生活性。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。这些较小的衍生品可能更适合于结构研究。细菌细胞大小和DNA复制的起始：细菌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，我们确定了这些小尺寸突变体细胞周期中复制起始的时间（起始年龄）。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义务。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需的，但控制其供应的机制似乎并不保守。了解这种差异的机制基础需要详细了解这两种细菌的复制控制系统，这对于我们的资源来说太过雄心勃勃了。我们不打算继续这个项目。

Most of our projects are centered on understanding how the replication of chrII is controlled in the cell cycle of V. cholerae. The chromosome is believed to have originated from a plasmid, yet it initiates replication at a particular time of the cell cycle, similar to other bona fide chromosomes but unlike plasmids whose timing of replication is generally not fixed in the cell cycle. We are trying to understand how the random timing of initiation has become fixed in the cell cycle. ChrII replication seems to also depend on chrI replication. How the two chromosomes communicate to coordinate replication is largely unknown and this knowledge is basic to our understanding of how multiple chromosomes are maintained in bacteria, such as V. cholerae. Finally, chrII replication is controlled by a segregation protein. The discovery of the influence of segregation on replication is a recent development in the field of chromosome dynamics in bacteria. Our progress in understanding these processes is reported below.Transition from plasmid to chromosomal mode of replication:We discovered that the chrII initiator binds specifically to two kinds of site: One kind of site consists of iterons, equivalent to repeats that characterize replication origin of a family of plasmids. The other kind of site, called 39-mers, consists of unrelated sequence. The chrII iterons, however, have to be methylated at their adenine residue before they can bind the initiator, unlike the situation in plasmid iterons. The 39-mers are the primary negative regulators of replication. They enhance interactions that inactivate the origins. The iterons in turn can also dampen the inhibitory activity of the 39-mers. How the interplay between the two kinds of site might coordinate chrII replication with the cell cycle has been modeled. The model also takes into account the fact that the origin iterons, which becomes hemimethylated after 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 features additional to those present in plasmid systems, which most likely allow cell cycle specific replication. Control of chrII replication by chrI:We hypothesize that the timely replication and segregation of the two chromosomes of V. cholerae requires communication between them, so that both of the chromosomes can complete the processes prior to cell division. Preliminary evidence for 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 currently being investigated. ChrII replication is also controlled by a segregation protein:That segregation can influence replication was not known until recently. Our knowledge of chromosome segregation in bacteria comes primarily from studies of plasmids, where genes dedicated to plasmid segregation (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 of the Vibrio chromosomes have their own par genes. Upon deletion of one of the two par genes (parB) of chrI replication of that chromosome was specifically promoted. A similar finding has also been made in B. subtilis. In both B. subtilis and V. cholerae, the universal bacterial initiator, DnaA, was found to be the direct target of Par proteins. How the Par proteins stimulate the activity of DnaA has been studied in B. subtilis, and the mechanism is likely to be similar in V. cholerae. For this reason, we are not continuing with this project, but rather asking whether segregation proteins could also influence chrII replication.We have found that the centromere-binding protein ParB2 of chrII can spread beyond the centromere and cover a strong replication inhibitory site (a 39-mer) of the origin. This likely interferes with the 39-mer interaction with the initiator RctB, an interaction required for replication inhibition. Unexpectedly, ParB2 can also promote replication without requiring the centromere by directly binding to a different 39-mer. ParB2 and RctB compete for binding to this site, which could compromise its inhibitory activity. ParB2 thus appears to promote replication by competing with the initiator for binding to two strong replication inhibitory sites, by spreading into one and binding specifically to the other. Spreading represents a novel replication control mechanism acting from a distance; non-centromeric binding is a novel mechanism for control by a segregation protein. These studies establish several ways by which segregation proteins could influence replication. We suggest that involving a segregation protein directly in replication control is a feature of the putative adaptation of an acquired plasmid to permanent residency as a second chromosome in V. cholerae.Generation of 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. The chrII-specific initiator RctB is conserved only in the family Vibrionaceae. The protein appears ideally suited for developing potential 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 require remodeling by chaperone proteins and/or binding to specific DNA for activity. 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 to structural studies. Cell size and the initiation of DNA replication in bacteria: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 have determined the timing of replication initiation (initiation age) in the cell cycle of these small size mutants. In B. subtilis, the age remains unchanged in the mutants, indicating that the 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 for by an increase in the 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 appear not to be conserved. Understanding the mechanistic basis of the difference will require detailed understanding of the replication control systems of the two bacteria, which would be too ambitious for our resources. We do not plan to proceed with this project.