Regional Control of Chromosome Segregation in Pseudomonas aeruginosa

Abstract

Chromosome segregation in bacteria occurs concomitantly with DNA replication, and the duplicated regions containing the replication origin oriC are generally the first to separate and migrate to their final specific location inside the cell. In numerous bacterial species, a three-component partition machinery called the ParABS system is crucial for chromosome segregation. This is the case in the gammaproteobacterium Pseudomonas aeruginosa, where impairing the ParABS system is very detrimental for growth, as it increases the generation time and leads to the formation of anucleate cells and to oriC mispositioning inside the cell. In this study, we investigate in vivo the ParABS system in P. aeruginosa. Using chromatin immuno-precipitation coupled with high throughput sequencing, we show that ParB binds to four parS site located within 15 kb of oriC in vivo, and that this binding promotes the formation of a high order nucleoprotein complex. We show that one parS site is enough to prevent anucleate cell formation, therefore for correct chromosome segregation. By displacing the parS site from its native position on the chromosome, we demonstrate that parS is the first chromosomal locus to be separated upon DNA replication, which indicates that it is the site of force exertion of the segregation process. We identify a region of approximatively 650 kb surrounding oriC in which the parS site must be positioned for chromosome segregation to proceed correctly, and we called it “competence zone” of the parS site. Mutant strains that have undergone specific genetic rearrangements allow us to propose that the distance between oriC and parS defines this “competence zone”. Implications for the control of chromosome segregation in P. aeruginosa are discussed.

A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids

“…This serves as a nucleation point for assembling high molecular weight ParB-parS partition complexes, as initially seen by the silencing of genes present in the vicinity of parS (Lynch & Wang, 1995;Lobocka & Yarmolinsky, 1996). ParB binds over 10 Kbp away from parS sites for all ParABS systems studied to date (Rodionov et al, 1999;Murray et al, 2006;Sanchez et al, 2015;Donczew et al, 2016;Lagage et al, 2016). This phenomenon, termed spreading, refers to the binding of ParB to centromere-flanking DNA regions in a non-specific manner.…”

ParB spreading on DNA requires cytidine triphosphate in vitro

“…The SMC-ParAB-parS complex is widely employed for chromosome segregation in bacteria 1-13 . The centromere parS is the first DNA locus to be segregated following chromosome replication 8,9,14,15 . ParB specifically nucleates on parS before spreading outwards to the flanking DNA and bridges/cages DNA together to form a nucleoprotein network in vivo [16][17][18][19][20][21][22][23] .…”

“…ParB-parS also interacts with an ATPase ParA to power the segregation of replicated chromosomes [26][27][28][29][30] . Engineered strains harboring a nucleation-competent but spreading-defective mutant of parB are either unviable 10 or have elevated number of anucleate cells 4,7,8,15,[31][32][33][34] . Despite the importance of spreading for proper chromosome segregation, the mechanism by which a few parS-bound ParB can recruit hundreds more ParB molecules to the vicinity of parS to assemble a high molecular-weight nucleoprotein complex is not fully understood.Since the first report in 1995 35 , ParB spreading has been observed in vivo by chromatin immunoprecipitation in multiple bacterial species 12,[15][16][17]19,36 .…”

A CTP-dependent gating mechanism enables ParB spreading on DNA

“…This tripartite complex consists of a Walker-box ATPase ParA, a centromere-binding protein ParB, and a centromere-like DNA sequence parS. The parS site is the first DNA locus to be segregated after chromosome replication (2,5,11,12). ParB, a DNA-binding protein, nucleates on parS before binding to adjacent non-specific DNA to form a network of protein-DNA complexes.…”

“…In Caulobacter crescentus, ParA and ParB are essential for chromosome segregation and cell viability (3,13). In other bacterial species, engineered strains lacking ParB are still viable but have elevated numbers of anucleate cells due to defects in chromosome segregation (2,7,9,12,16,(20)(21)(22).The binding of multiple ParB molecules onto non-specific DNA after nucleation at parS (i.e. spreading) is a crucial event; bacterial cells harboring a nucleation-competent but spreadingdefective parB allele are impaired in plasmid/chromosome segregation (23-25).…”