Impact of culture conditions on ParB distribution in Pseudomonas aeruginosa and analysis of ParB distribution in spreading deficient strains as well as half-parS25 strain
Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1-parS4), which is positioned within the cell by ParA. Remarkably, ParB of P. aeruginosa binds to multiple heptanucleotides (half-parSs) scattered in the genome. In this work we analysed the influence of culturing conditions on ParB binding to DNA. Using chromatin immunoprecipitation-sequencing (ChIP-seq), we analysed patterns of genome occupancy by ParB in cells, with either coupling or uncoupling between replication and cell division. Our data indicated no altered preference of ParB to bind to individual half-parS sites under varying growth conditions, however a shift from parSs to half-parSs was evident in response to extended cell division time. The ChIP-seq analysis of strains expressing ParB variants unable to dislocate from parSs showed that ParB spreading ability is not required for ParB binding to half-parSs. Finally, a P. aeruginosa strain with mutated 27 half-parSs forming the strongest ParB ChIP-seq peaks was constructed and analysed showing changes in the ParB coverage of oriC region. Overall this work suggests the role of half-parSs in retaining ParB on the nucleoid within P. aeruginosa cells. Overall design: Pseudomonas aeruginosa PAO1161 (leu-, r-, RifR) derivative of PAO1, was used in the experiment (Kawalek et al., 2020; BMC Genomics, 21:14). ParB ChIP-seq experiments were performed using cells of PAO1161 (WT) strain grown on rich medium (LB) as well as minimal M9 medium with 0.5% glucose at two different temperatures (37°C and 30°C), harvested at exponential and stationary phases of culture growth.  ?parB strain was used as a background control. Additionally strains expressing spreading defficient variants (ParB R94C and ParB A97T) as well as C-terminally truncated ParB1-283 variant impaired in dimerization was analyzed (these strains were grown only in LB at 37°C , and harvested at log phase of the culture growth). The immunoprecipitation was performed with polyclonal anti ParB antibodies, raised in rabbit and affinity purified (as in Reznekov et al., 1996, Genes Cells 1:529-542) and two replicates were performed for each strain/conditions, with the exception of ?parB .