Fig 4 - uploaded by Flavien Pillet
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Binding scores of ParB12 J for parSc3 non-parSc3 sequences and derivatives. Blue lines correspond to the BS of the palindromic sequence parSc3 (A) and non parSc3 (B). Legend is the same as in Fig 3.
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Bacterial centromeres–also called parS, are cis-acting DNA sequences which, together with the proteins ParA and ParB, are involved in the segregation of chromosomes and plasmids. The specific binding of ParB to parS nucleates the assembly of a large ParB/DNA complex from which ParA—the motor protein, segregates the sister replicons. Closely related...
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Citations
... The pathogenic B. cenocepacia strain J2315 maintains three chromosomes and a large, lowcopy-number plasmid (41). The type I (ParABS) partitioning systems of these replicons have coevolved to become distinct so that each replicon is partitioned independently (41)(42)(43)(44). Likewise, Rhizobium leguminosarum bv. ...
Toxins produced by the Gram-positive pathogen Clostridium perfringens are primarily encoded by genes found on different conjugative plasmids. These plasmids encode highly similar replication proteins and therefore should be incompatible, but they are often found to coexist within the same isolate.
... et seraient responsables de la régulation de l'expression génique(Kawalek et al., 2018). ParB est effectivement capable de se lier à des demi-sites parS synthétiques ou naturels à une plus faible affinité qu'au site complet (Figure 44B)(Pillet et al., 2011;Venkova-Canova et al., 2013;Pillet et al., 2017). La délétion des sites parS1-4 de P. aeruginosa n'abroge pas la liaison spécifique de ParB à ces sites secondaires (figure 44A)(Lagage et al., 2016). ...
La ségrégation des chromosomes et des plasmides à bas nombre de copies chez les bactéries est basée sur un mécanisme actif de positionnement. Il repose sur des systèmes de partition qui assurent la répartition intracellulaire des réplicons afin qu'ils soient transmis de façon fidèle à la descendance. Ils font intervenir trois partenaires encodés par la molécule à ségréger. Une protéine de liaison à l'ADN (ParB) s'assemble en un complexe de partition autour d'une séquence centromérique (parS). Une protéine NTPase, interagissant avec le complexe de partition, est responsable de l'activité de ségrégation et du positionnement du complexe de partition et ainsi du plasmide. Mon projet de thèse vise d'abord à approfondir le mécanisme d'assemblage du complexe de partition du système majoritaire de type I des plasmides F et pESBL et ensuite, à déchiffrer le mécanisme global de partition du système atypique de R388 récemment découvert, n'utilisant pas de NTPase codée par le plasmide pour assurer son positionnement. Ainsi, mon projet s'articule en trois parties. (i) Comprendre par une approche mutationnelle le mécanisme d'initiation de l'auto assemblage de l'ensemble des ParB du plasmide F en un complexe de haut poids moléculaire dynamique, autour de parS. (ii) Identifier les partenaires du système de partition du plasmide pESBL, caractériser in vitro l'interaction de ParB avec parS et déterminer par une étude in silico, le groupe auquel il appartient. (iii) Identifier le rôle des différents domaines de la protéine de liaison à l'ADN StbA du plasmide R388 dans ses activités et caractériser les modalités d'interaction de StbA avec son site spécifique sur le plasmide, par des approches de séquençage à haut débit et de biochimie, pour comprendre l'architecture du complexe de partition. Cette étude a permis d'approfondir nos connaissances sur la biologie des plasmides portant un système de partition de type I et a mis en lumière les spécificités d'interactions ADN/protéines d'un système atypique, ouvrant la voie à la compréhension de son mécanisme de stabilisation.
... Caulobacter crescentus 5/5/t/cGTTt/cCACGTGAAAca [80,81] essential Indispensable, severe chromosome segregation defects, ParB depletion results in defective Z-ring formation and cell division, formation of long polyploid cells [82] Hyphomonas neptunium 2/2/TGTTTCACGTGAAACA [83] essential anucleate buds [83] parB mutants could not be obtained, depletion of ParA blocks cell division [83] Betaproteobacteria Burkholderia cenocepacia chrI: 2/2/tGTTNCACGTGAAACa chrII: 6/6/gTTTATGCGCATAAAc [84][85][86][87] non-essential 1-14% (depending on mutated system) [85] Reduced growth rate, reduction in cell size, compromised viability, defects in ori positioning [85] Deltaproteobacteria ParB depletion: aberrant cell morphology, anomalies in DNA segregation and cell death [88] Gammaproteobacteria Pseudomonas aeruginosa 9/4/TGTTCCACGtGGAACa half-parSs GTTCCAC or GTTTCAC [89][90][91] non-essential 2-4% in LB medium, to 7% in an M9 medium (wt < 0.01%) [92] Reduced growth rate, 10-15% increase in cell size and 10% longer generation time, altered colony morphology, affected motility; decreased ParA stability [92] Pseudomonas putida ?*/3 TGTTCCACGTGGAACA [63] non-essential 5-10% in minimal medium during the transition from exponential to stationary phase [93] Defects in chromosome partitioning, abnormal cell morphologies during the deceleration phase of growth independent of the medium used [63,93] Vibrio cholerae chrI: 3/3/NGTTNCACGTGAAACN chrII: 10/9/NTTTACANTGTAAAN [94] non-essential chr1 essential chr2 no change in parB1 mutant [95] Increased frequency of replication initiation, disturbed ori positioning in cell poles [95], no segregation defect for V. cholerae chrI [94] Deinococci Deinococcus radiodurans chrI: 3/1/NGTTTcgcGtgaAACN [96] non-essential 8%-13% for ∆parB1, (wt >1%) [96] Reduced growth rate for ∆parB1 [96] Thermus thermophilus 1/1/TGTTTCCCGTGAAACA [97] non-essential 3% for ∆parAB (wt 1.2%) [97] No apparent growth defects for ∆parAB [97,98] Abbreviations: chrI/chrII-primary/secondary chromosome in the multipartite genome; wt-wild-type; ?* -only contig with P. putida oriC was analyzed for presence of parSs in the cited reference. ...
The segregation of newly replicated chromosomes in bacterial cells is a highly coordinated spatiotemporal process. In the majority of bacterial species, a tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target(s) parS sequence(s), facilitates the initial steps of chromosome partitioning. ParB nucleates around parS(s) located in the vicinity of newly replicated oriCs to form large nucleoprotein complexes, which are subsequently relocated by ParA to distal cellular compartments. In this review, we describe the role of ParB in various processes within bacterial cells, pointing out interspecies differences. We outline recent progress in understanding the ParB nucleoprotein complex formation and its role in DNA segregation, including ori positioning and anchoring, DNA condensation, and loading of the structural maintenance of chromosome (SMC) proteins. The auxiliary roles of ParBs in the control of chromosome replication initiation and cell division, as well as the regulation of gene expression, are discussed. Moreover, we catalog ParB interacting proteins. Overall, this work highlights how different bacterial species adapt the DNA partitioning ParAB-parS system to meet their specific requirements.
... is partitioned independently(46)(47)(48)(49). Likewise, Rhizobium leguminosarum bv. ...
Plasmids that encode the same replication machinery are generally unable to coexist in the same bacterial cell. However, Clostridium perfringens strains often carry multiple conjugative toxin or antibiotic resistance plasmids that are closely related and encode similar Rep proteins. In many bacteria, plasmid partitioning upon cell division involves a ParMRC system and there are ~10 different ParMRC families in C. perfringens, with differences in amino acid sequences between each ParM family (15% - 54% identity). Since plasmids encoding genes belonging to the same ParMRC family are not observed in the same strain, these families appear to represent the basis for plasmid compatibility in C. perfringens. To understand this process, we examined the key recognition steps between ParR DNA-binding proteins and their parC binding sites. The ParR proteins bound to sequences within a parC site from the same ParMRC family, but could not interact with a parC site from a different ParMRC family. These data provide evidence that compatibility of the conjugative toxin plasmids of C. perfringens is mediated by their parMRC-like partitioning systems. This process provides a selective advantage by enabling the host bacterium to maintain separate plasmids that encode toxins that are specific for different host targets.
... ParB2 of V. cholerae was shown to bind a noncentromeric site containing a half-parS sequence (82). Additionally, in the multipartite genome bacterium Burkholderia cenocepacia ParBc1, ParBc3 and ParBpBC displayed enhanced affinity for the corresponding half-parS relative to random-sequence DNA (84). This data show that the ability of ParB to bind to one arm of parS is not restricted to P. aeruginosa. ...
ParA and ParB homologs are involved in accurate chromosome segregation in bacteria. ParBs participate in the separation of ori domains by binding to parS palindromes, mainly localized close to oriC. In Pseudomonas aeruginosa neither ParB deficiency nor modification of all 10 parSs is lethal. However, such mutants show not only defects in chromosome segregation but also growth retardation and motility dysfunctions. Moreover, a lack of parB alters expression of over 1000 genes, suggesting that ParB could interact with the chromosome outside its canonical parS targets. Here, we show that indeed ParB binds specifically to hundreds of sites in the genome. ChIP-seq analysis revealed 420 ParB-associated regions in wild-type strain and around 1000 in a ParB-overproducing strain and in various parS mutants. The vast majority of the ParB-enriched loci contained a heptanucleotide motif corresponding to one arm of the parS palindrome. All previously postulated parSs, except parS5, interacted with ParB in vivo. Whereas the ParB binding to the four parS sites closest to oriC, parS1-4, is involved in chromosome segregation, its genome-wide interactions with hundreds of parS half-sites could affect chromosome topology, compaction and gene expression, thus allowing P. aeruginosa ParB to be classified as a nucleoid-associated protein.
... ParB did not significantly bind to the other six suggested parS sites (Lagage et al., 2016), which diverged by two basepairs from the consensus sequence described from B. subtilis (Bartosik et al., 2004;Livny et al., 2007). This parS sequence divergence may confer different affinities for ParB, as was shown for parSF and parS of Burkholderia cenocepacia (Pillet et al., , 2017. Interestingly, the phenotypes of strains deleted for either ParA or ParB proteins, resulted in similar pleiotropic phenotypes with defects including cell growth, chromosome segregation, cell division, swarming, and swimming (Bartosik et al., 2009;Lasocki et al., 2007). ...
The active partition of low copy number plasmids and most bacterial chromosomes relies on the assembly of a nucleoprotein superstructure nucleated at centromere-like sites near the origin of replication. In the case of type I partition systems, the most widespread on plasmids and the only ones present on chromosomes, centromere binding proteins, ParB, display the capability to propagate along DNA from their nucleation point at the centromere-like site, parS. This structure, termed the partition complex, contains over 90% of the available ParB in the cell and interacts with ParA, a ParB- and DNA-dependent ATPase, to segregate and position replicons within the cell. Two concurrent models exist to explain the assembly mechanism of the partition complex: the spreading and bridging (Graham et al., 2014), and the nucleation and caging (Sanchez et al., 2015) models. We used the partition system of the archetypal plasmid F and the naturally occurring partition system of Vibrio cholerae's chromosome 1. First, we explored the mechanisms of centromere-based incompatibilities to gain insight into partition complex assembly in low levels of ParB through parS titration of ParB. Next, we describe an optimized a high- resolution ChIP-sequencing protocol from the lab bench to data analysis. Then, through ChIP-sequencing, epifluorescence microscopy and physico-mathematical modeling, we revealed that the partition complex assembly mechanism is robust and consistent with the nucleation and caging model on both chromosomal and plasmid systems.
