A Single parS Sequence from the Cluster of Four Sites Closest to oriC Is Necessary and Sufficient for Proper Chromosome Segregation in Pseudomonas aeruginosa
A Single parS Sequence from the Cluster of Four Sites Closest to oriC Is Necessary and Sufficient for Proper Chromosome Segregation in Pseudomonas aeruginosa
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DOI：
10.1371/journal.pone.0120867
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发表时间：
2015-03-20
期刊：
影响因子：
3.7
通讯作者：
Jagura-Burdzy, Grazyna
中科院分区：
文献类型：
--
作者：
Jecz, Paulina;Bartosik, Aneta A.;Jagura-Burdzy, Grazyna
在控制细菌染色体分离的机制中，高度保守的分配系统由三个组分组成：PARB蛋白(一种异常的Walker型ATPase)，PARB蛋白(一种DNA结合元件)和多个顺式作用的回文着丝粒样序列，称为PARS。在铜绿假单胞菌PAO1基因组中已经确定了10个可能的PARS位点，其中4个位于ORIC附近，6个从一个完整的回文序列中分离出来，分布在染色体上。在这里，我们构建并分析了不含每个PARS序列及其不同组合的铜绿假单胞菌突变体。分析包括对一组表型特征、染色体分离和PARB在细胞中的定位进行评估。研究发现，PARB与所有10个PARS位点都有特异性结合，尽管亲和力不同。具有全部10个pars位点修饰的铜绿假单胞菌parS突变体(ParSnull)是可行的，但它表现出parAnull或parBull突变体的表型特征：生长速度略慢，无核细胞出现频率高，运动能力缺陷。PARs的基因组位置和序列决定了其在铜绿假单胞菌生物学中的作用。结果表明，与PARB亲和力最高的ORIC(parS1至parS4)附近的四个PARS位点中的任何一个都是ParABS在染色体分割中发挥作用的必要条件和充分条件。当所有这四个位点同时突变时，菌株表现为parSnull表型，这表明在这一功能中，其余六个PARS位点都不能替代这四个ORIC-近端位点。单个异位parS2(插入到parSnull突变体中与ORIC相反的位置)有助于将PARB组织成间隔规则的浓缩焦点，并逆转一些突变表型，但不足以进行准确的染色体分离。
Among the mechanisms that control chromosome segregation in bacteria are highly-conserved partitioning systems comprising three components: ParA protein (a deviant Walker-type ATPase), ParB protein (a DNA-binding element) and multiple cis-acting palindromic centromere-like sequences, designated parS. Ten putative parS sites have been identified in the P. aeruginosa PAO1 genome, four localized in close proximity of oriC and six, diverged by more than one nucleotide from a perfect palindromic sequence, dispersed along the chromosome. Here, we constructed and analyzed P. aeruginosa mutants deprived of each single parS sequence and their different combinations. The analysis included evaluation of a set of phenotypic features, chromosome segregation, and ParB localization in the cells. It was found that ParB binds specifically to all ten parS sites, although with different affinities. The P. aeruginosa parS mutant with all ten parS sites modified (parSnull) is viable however it demonstrates the phenotype characteristic for parAnull or parBnull mutants: slightly slower growth rate, high frequency of anucleate cells, and defects in motility. The genomic position and sequence of parS determine its role in P. aeruginosa biology. It transpired that any one of the four parS sites proximal to oriC (parS1 to parS4), which are bound by ParB with the highest affinity, is necessary and sufficient for the parABS role in chromosome partitioning. When all these four sites are mutated simultaneously, the strain shows the parSnull phenotype, which indicates that none of the remaining six parS sites can substitute for these four oriC-proximal sites in this function. A single ectopic parS2 (inserted opposite oriC in the parSnull mutant) facilitates ParB organization into regularly spaced condensed foci and reverses some of the mutant phenotypes but is not sufficient for accurate chromosome segregation.