Genome-wide identification of ParB binding sites in Pseudomonas aeruginosa. Genome-wide identification of ParB binding sites in Pseudomonas aeruginosa

ParA and ParB homologs are involved in accurate chromosome segregation in bacteria. ParBs participate in proper folding and initial separation of ori domains by binding to specific parS sites (palindromic centromere-like sequences), mainly localized close to oriC. Bioinformatic analyses identified 10 parS sequences in the Pseudomonas aeruginosa PAO1 genome. One parS from the parS1-parS4 cluster is required for ParB mediated chromosome segregation. To verify the binding of ParB to all known parSs in vivo as well as to identify additional ParB binding sites we performed chromation immunoprecipitation (ChIP) using polyclonal anti-ParB antibodies followed by high throughput sequencing. ChIP was performed with P. aeruginosa PAO1161 (WT) cells, PAO1161 pKB9 (ParB+++) cells with a slight, non-toxic ParB overproduction as well as with 3 strains containing parS modifications impairing ParB binding to these sites. The data confirmed ParB binding to all known parS sequences with the exception of parS5. Moreover, we identified more than a 1000 of new ParB-bound regions, majority of which contained a DNA motif corresponding to inner 7 nt from one arm of the parS palindrome. ParB interactions with these numerous sites could affect chromosome topology, compaction and gene expression classifying P. aeruginosa ParB as a Nucleoid Associated Protein (NAP). Overall design: ChIP was performed with P. aeruginosa PAO1161 (WT) cells, PAO1161 pKB9 (araBADp-parB) cells with a 5-fold ParB overproduction. Additionally, three parS mutants were included: parSnull with all ten parS sites mutated, parS1-4 mutant with the four high affinity ParB sites inactivated, and parS2+ strain with nine parS sequences modified and only parS2 left intact (Jecz et al., 2015). PAO1161 parBnull mutant, with parB gene disrupted was used as a negative control. Each strain was analysed in duplicate. Additionally, input DNA (DNA before incubation with antibodies) was sequenced for WT strain.

ParA与ParB同源蛋白参与细菌的精准染色体分离过程。ParB蛋白可通过结合特异性parS位点（palindromic centromere-like sequences，回文着丝粒样序列）参与ori结构域的正确折叠与初始分离，这类位点主要定位于oriC。生物信息学分析在铜绿假单胞菌（Pseudomonas aeruginosa）PAO1基因组中鉴定出10个parS位点。其中parS1-parS4簇中的一个parS位点是ParB介导染色体分离所必需的。为验证ParB在体内与所有已知parS位点的结合情况，并鉴定额外的ParB结合位点，我们使用多克隆抗ParB抗体开展染色质免疫共沉淀（Chromatin Immunoprecipitation，ChIP）实验，后续结合高通量测序（High Throughput Sequencing）进行分析。本次实验分别以铜绿假单胞菌PAO1161（野生型，WT）细胞、带有轻度非毒性ParB过表达的PAO1161 pKB9（ParB+++）细胞，以及3株携带会削弱ParB结合能力的parS位点突变菌株作为实验材料。实验数据证实，ParB可结合除parS5之外的所有已知parS序列。此外，我们还鉴定出超过1000个新的ParB结合区域，其中大多数区域包含一段与parS回文序列单臂内侧7个核苷酸对应的DNA基序。ParB与这些大量位点的相互作用可能影响染色体拓扑结构、压缩状态与基因表达，据此可将铜绿假单胞菌的ParB归类为类核关联蛋白（Nucleoid Associated Protein，NAP）。实验整体设计：本研究分别对铜绿假单胞菌PAO1161（野生型，WT）细胞、带有5倍ParB过表达的PAO1161 pKB9（araBADp-parB）细胞开展ChIP实验。此外还设置了3株parS突变菌株：parSnull（10个parS位点均发生突变）、parS1-4突变体（4个高亲和力ParB结合位点失活）以及parS2+菌株（9个parS序列被修饰，仅保留parS2位点完整，Jecz等，2015）。同时以parB基因被敲除的PAO1161 parBnull突变菌株作为阴性对照。所有菌株均设置2次生物学重复。此外，还对野生型菌株的Input DNA（与抗体孵育前的DNA样本）进行了测序。