Characterization of Copy Number Control of Two Haloferax volcanii Replication Origins Using Deletion Mutants and Haloarchaeal Artificial Chromosomes
Characterization of Copy Number Control of Two Haloferax volcanii Replication Origins Using Deletion Mutants and Haloarchaeal Artificial Chromosomes
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DOI：
10.1128/jb.00517-17
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发表时间：
2018-01-01
影响因子：
3.2
通讯作者：
Soppa, Joerg
中科院分区：
文献类型：
--
作者：
Maurer, Sandy;Ludt, Katharina;Soppa, Joerg
沃氏哈夫拉菌是多倍体，在最佳条件下含有约20个基因组拷贝。然而，染色体拷贝数是高度调节的，范围从磷酸盐饥饿期间的2到磷酸盐过剩条件下的40多个。本研究的目的是表征两个复制起点对基因组拷贝数的影响。缺失了起始重复序列和编码起始识别复合物（ORC）蛋白的基因。核心起源oriC 1-orc 1（ori 1）缺失突变体具有比野生型更低的基因组拷贝数和更高的适应性水平，与oriC 2-orc 5（ori 2）缺失突变体形成鲜明对比。与ori 1相邻的基因不能被删除，因此，它们中的至少两个可能是必需的，而与ori 2相邻的基因的删除是可能的。将起源及其周围的各种片段克隆到自杀质粒中以产生盐古菌人工染色体（HAC）。oriC 1-orc 1 HAC的拷贝数远高于oriC 2-orc 5 HAC。相邻基因的添加影响HAC拷贝数和染色体拷贝数。结果表明，H. volcanii是不是独立的，但拷贝数是通过周围的origins.IMPORTANCE基因网络调控几个物种的古菌有一个以上的复制在其主要染色体上的起源，因此是唯一已知的原核生物物种，允许多起点复制的进化分析。被广泛研究的Holoferax火山H26菌株有一条主染色体，有四个复制起点。两个起源，ori 1和ori 2，被选择用于使用缺失突变体和盐古菌人工染色体进行深入分析。该分析不限于核心起源区域;还包括起源邻近基因。因为H. volconii是多倍体，对染色体拷贝数的影响特别重要。结果显示，这两种起源之间存在着极大的差异。
Hafoferax volcanii is polyploid and contains about 20 genome copies under optimal conditions. However, the chromosome copy number is highly regulated and ranges from two during phosphate starvation to more than 40 under conditions of phosphate surplus. The aim of this study was the characterization of the influence of two replication origins on the genome copy number. The origin repeats and the genes encoding origin recognition complex (ORC) proteins were deleted. The core origin oriC1-orc1 (ori1) deletion mutant had a lower genome copy number and a higher level of fitness than the wild type, in stark contrast to the oriC2-orc5 (ori2) deletion mutant. The genes adjacent to ori1 could not be deleted, and thus, at least two of them are probably essential, while deletion of the genes adjacent to ori2 was possible. Various fragments of and around the origins were cloned into a suicide plasmid to generate haloarchaeal artificial chromosomes (HACs). The copy number of the oriC1-orc1 HAC was much higher than that of the oriC2-orc5 HAC. The addition of adjacent genes influenced both the HAC copy number and the chromosome copy number. The results indicate that the origins of H. volcanii are not independent but that the copy number is regulated via a network of genes around the origins.IMPORTANCE Several species of archaea have more than one origin of replication on their major chromosome and are thus the only known prokaryotic species that allow the analysis of the evolution of multiorigin replication. The widely studied Holoferax volcano H26 strain has a major chromosome with four origins of replication. Two origins, ori1 and ori2, were chosen for an in-depth analysis using deletion mutants and haloarchaeal artificial chromosomes. The analysis was not restricted to the core origin regions; origin-adjacent genes were also included. Because H. volconii is polyploid, the effects on the chromosome copy number were of specific importance. The results revealed extreme differences between the two origins.