Binding and Spreading of ParB on DNA Determine Its Biological Function in Pseudomonas aeruginosa
Binding and Spreading of ParB on DNA Determine Its Biological Function in Pseudomonas aeruginosa
复制标题
DOI：
10.1128/jb.00328-11
复制
发表时间：
2011-07-01
影响因子：
3.2
通讯作者：
Jagura-Burdzy, Grazyna
中科院分区：
文献类型：
--
作者：
Kusiak, Magdalena;Gapczynska, Anna;Jagura-Burdzy, Grazyna
铜绿假单胞菌的ParB蛋白属于广泛存在的ParB家族，该家族包括染色体编码和质粒编码的IA型分配蛋白。铜绿假单胞菌染色体上分散着10个假定的parS位点，其中8个位于oriC区域。ParB与parS结合后，会在DNA上扩散，导致附近基因的转录沉默（A. A. 巴尔托西克等人，《细菌学杂志》186:6983 - 6998，2004年）。我们研究了因DNA结合能力丧失或寡聚化受损而扩散能力有缺陷的ParB衍生物。我们确定了负责DNA结合的螺旋 - 转角 - 螺旋基序之外的特定决定因素。分析证实了ParB主要二聚化结构域位于C末端，但也在N末端结构域定位了另一个自我相互作用结构域。利用反向遗传学将5个扩散能力受损的parB等位基因引入铜绿假单胞菌染色体。ParB中的单个氨基酸替换导致寡聚化缺陷但不影响DNA结合，这会引起染色体分离缺陷、生长速率减慢以及运动能力受损，与parB缺失突变体的多效表型相似，表明扩散能力对ParB在细胞中的功能至关重要。假单胞菌属中ParB过量产生的毒性不是由于扩散，因为几种寡聚化有缺陷的ParB衍生物在过量提供时对铜绿假单胞菌仍然有毒性。
ParB protein of Pseudomonas aeruginosa belongs to a widely represented ParB family of chromosomally and plasmid-encoded partitioning type IA proteins. Ten putative parS sites are dispersed in the P. aeruginosa chromosome, with eight of them localizing in the oriC domain. After binding to parS, ParB spreads on the DNA, causing transcriptional silencing of nearby genes (A. A. Bartosik et al., J. Bacteriol. 186:6983-6998, 2004). We have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomerization. We defined specific determinants outside of the helix-turn-helix motif responsible for DNA binding. Analysis confirmed the localization of the main dimerization domain in the C terminus of ParB but also mapped another self-interactive domain in the N-terminal domain. Reverse genetics were used to introduce five parB alleles impaired in spreading into the P. aeruginosa chromosome. The single amino acid substitutions in ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect, slowed the growth rate, and impaired motilities, similarly to the pleiotropic phenotype of parB-null mutants, indicating that the ability to spread is vital for ParB function in the cell. The toxicity of ParB overproduction in Pseudomonas spp. is not due to the spreading since several ParB derivatives defective in oligomerization were still toxic for P. aeruginosa when provided in excess.