Genome-wide identification of ParB binding sites in Pseudomonas aeruginosa
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ABSTRACT:
PROVIDER: PRJNA432963 | ENA |
REPOSITORIES: ENA
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Project description: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).
2018-03-27 | GSE110158 | GEO
Project description:To identify ParB binding sites in vivo, we performed ChIP experiments followed by high throughput sequencing using a 3xflag tagged version of ParB, in different genetic backgrounds. This allowed us to show that ParB binds in vivo mostly to 4 parS sites, located within 15 kb of oriC. We also identify 9 secondary ParB binding sites.
2016-10-18 | GSE87409 | GEO
Project description: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 transcriptome of P. aeruginosa with mutated 25 half-parSs forming the strongest ParB ChIP-seq peaks. Inactivation of ParB binding to even a small fraction of these sites modulated the gene expression, however this effect is most likely indirect. Overall this work suggests complex relation between ParB binding to genome and P. aeruginosa transcriptome.
2023-08-03 | GSE233235 | GEO
Project description: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.
2023-08-03 | GSE233378 | GEO
Project description:In Pseudomonas aeruginosa, partitioning protein ParB facilitates segregation of newly replicated chromosomes but is not essential for cell survival. Unlike in other bacteria, inactivation of parB leads to major changes of the transcriptome, suggesting that, directly or indirectly, ParB plays a role in regulation of gene expression in this organism. To identify primary targets of ParB, we analysed the impact of a slight increase in ParB amount on the transcriptome using microarrays. A several-fold increased ParB level does not cause recognizable phenotypic changes but leads to significant changes in the expression of 211 loci, including transcriptional regulators of operons involved in SOS response, virulence and adaptation. Most notably, the mRNA level of genes adjacent to high affinity ParB binding sites parS1-4 close to oriC is reduced. Our data support the role of partitioning protein ParB as a transcriptional regulator in Pseudomonas aeruginosa.
2017-07-25 | GSE95647 | GEO
Project description:In the majority of bacterial species, the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s), assists in the chromosome partitioning. ParB forms large nucleoprotein complexes at parS(s), located in the vicinity of oriC, which after replication are subsequently relocated by ParA to polar positions. It was shown that ParB-parS complexes are loading platforms for structural maintenance of chromosome (Smc) proteins, which juxtapose the two arms of the circular chromosome. In this work, we characterized the Pseudomonas aeruginosa ParB interactions with DNA in the absence of Smc and interaction with the cognate ParA using chromatin immunoprecipitation-sequencing (ChIPseq). We show that in strains lacking Smc or strains with disturbed ParB-ParA interactions (ParA L84K or ParB G11A mutations) ParB is able to bind and spread around parS1-4 cluster and still binds to half-parS sites. Comparison of the ratio between the number of ChIP-seq reads mapping to region around parS1-4 with those mapping to ChIPseq peaks containing half-parSs indicate no major effect of the twod factors on the extent of ParB spreading, thus suggesting that the mechanisms controlling ParB association with the DNA do not involve interaction with cognate ParA partner and Smc.
2022-12-17 | GSE213881 | GEO
Project description:Representatives of two families of bacterial Par proteins, ParA and ParB, are encoded by the majority of bacterial chromosomes in the close vicinity of oriC. ParA(Soj) and ParB(Spo0J) proteins of Pseudomonas aeruginosa are both important for optimal growth, nucleoids segregation, cell division and different types of motility. Comparative transcriptome analysis of parAnull, parBnull mutants versus parental PAO1161 strain of P. aeruginosa demonstrated global changes in genes expression pattern in logarithmic phase of planktonic cultures grown on rich medium. The set of genes that were similarly regulated in both mutant strains as compared to the wild-type strain as well as two sets of genes uniquely affected in the particular mutant were defined suggesting that ParA and ParB may act in common and independently. In general, many genes involved in cell division, DNA and RNA processing and metabolic processes were down-regulated in mutant cells, in contrast genes which products play a role in adaptation, protection, motility, cell-to-cell signaling as well as signal transduction increased their expression in par mutant cells. Besides their role in chromosome segregation, ParA and ParB seem to have the potential to regulate genes transcription. The altered expression of a large number of genes encoding known or predicted transcriptional regulators and genes coding for products involved in c-di-GMP signalling, suggests that the part of observed global changes in genes expression pattern in parAnull and parBnull mutants might be the effect of indirect regulation mediated by regulatory genes under ParA and ParB control. The extended regulatory network provides the mechanism to modulate genes expression in response to the stage of the chromosome segregation process and cell cycle. Pseudomonas aeruginosa PAO1161 (leu, r-, RifR), derivative of PAO1, as a control (reference) strain, Pseudomonas aeruginosa PAO1161 parA1-40::smh (parAnull) and Pseudomonas aeruginosa PAO1161 parB1-18::TcR (parBnull) disruption mutant strains were used in the experiments. Three independent biological replicates of total RNA were isolated for each strain from logarithmic (Log) phase of planktonic culture grown on rich medium (L broth) at 37oC. In total, nine samples of RNA were prepared.
2014-02-12 | E-GEOD-47031 | biostudies-arrayexpress
Project description:Representatives of two families of bacterial Par proteins, ParA and ParB, are encoded by the majority of bacterial chromosomes in the close vicinity of oriC. ParA(Soj) and ParB(Spo0J) proteins of Pseudomonas aeruginosa are both important for optimal growth, nucleoids segregation, cell division and different types of motility. Comparative transcriptome analysis of parAnull, parBnull mutants versus parental PAO1161 strain of P. aeruginosa demonstrated global changes in genes expression pattern in logarithmic phase of planktonic cultures grown on rich medium. The set of genes that were similarly regulated in both mutant strains as compared to the wild-type strain as well as two sets of genes uniquely affected in the particular mutant were defined suggesting that ParA and ParB may act in common and independently. In general, many genes involved in cell division, DNA and RNA processing and metabolic processes were down-regulated in mutant cells, in contrast genes which products play a role in adaptation, protection, motility, cell-to-cell signaling as well as signal transduction increased their expression in par mutant cells. Besides their role in chromosome segregation, ParA and ParB seem to have the potential to regulate genes transcription. The altered expression of a large number of genes encoding known or predicted transcriptional regulators and genes coding for products involved in c-di-GMP signalling, suggests that the part of observed global changes in genes expression pattern in parAnull and parBnull mutants might be the effect of indirect regulation mediated by regulatory genes under ParA and ParB control. The extended regulatory network provides the mechanism to modulate genes expression in response to the stage of the chromosome segregation process and cell cycle.
2014-02-12 | GSE47031 | GEO
Project description:Identification of ParB binding sites in vivo in Pseudomonas aeruginosa
| PRJNA344750 | ENA
Project description:We studied the binding of SMC protein to Streptomyces venezuelae chromosome during development of aerial hyphae (14 hour of growth) using SMC-FLAG protein. Additionally investigated the role of ParB protein in SMC DNA binding using parB deletion strain.
2021-05-28 | E-MTAB-9822 | biostudies-arrayexpress