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.