Pseudomonas aeruginosa partitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of a parS-related motif

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

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

Abstract: 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

Rola regulatorów transkrypcji w kontroli ekspresji genów prokariotycznych na przykładzie modelowej bakterii Pseudomonas aeruginosa

Bakterie Pseudomonas aeruginosa spotykane są często w środowisku naturalnym jak woda czy gleba, ale zaliczane są także do oportunistycznych patogenów ludzi i zwierząt. Charakteryzuje je duża zdolność do przetrwania w bardzo różnych niszach ekologicznych. Adaptacja i umiejętność dostosowania do zmiennych warunków środowiska wynika z rozbudowanych sieci regulacyjnych tych bakterii i wykorzystania bogatego repertuaru kodowanych w genomie białek, ścieżek metabolicznych, mechanizmów obronnych i adaptacyjnych. Regulatory transkrypcji są kluczowymi elementami regulacji ekspresji genów, które odpowiadają na sygnały z otoczenia, włączając lub wyłączając określone ścieżki. Kompleksowe badania regulatorów transkrypcji z wykorzystaniem metod transkryptomicznych, genomicznych, regulacyjnych dostarczają wiedzy na temat mechanizmów ich działania, regulowanych genów i procesów, umożliwiając poznanie i zrozumienie często skomplikowanych sieci regulacyjnych zawiadujących przeżyciem komórki w danym środowisku i warunkach. Celem niniejszej pracy jest przedstawienie kluczowych zagadnień związanych z regulacją transkrypcji bakteryjnej na podstawie przeglądu danych literaturowych oraz zobrazowanych na przykładzie P. aeruginosa i charakterystyki mechanizmów regulacji ekspresji genów zaangażowanych między innymi w wirulencję tej bakterii

Increased ParB level affects expression of stress response, adaptation and virulence operons and potentiates repression of promoters adjacent to the high affinity binding sites parS3 and parS4 in Pseudomonas aeruginosa

Similarly to its homologs in other bacteria, Pseudomonas aeruginosa partitioning protein ParB facilitates segregation of newly replicated chromosomes. Lack of ParB is not lethal but results in increased frequency of anucleate cells production, longer division time, cell elongation, altered colony morphology and defective swarming and swimming motility. 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. ParB overproduction affects growth rate, cell division and motility in a similar way as ParB deficiency. To identify primary ParB targets, here we analysed the impact of a slight increase in ParB level on P. aeruginosa transcriptome. ParB excess, which does not cause changes in growth rate and chromosome segregation, significantly alters the expression of 176 loci. Most notably, the mRNA level of genes adjacent to high affinity ParB binding sites parS1-4 close to oriC is reduced. Conversely, in cells lacking either parB or functional parS sequences the orfs adjacent to parS3 and parS4 are upregulated, indicating that direct ParB- parS3/ parS4 interactions repress the transcription in this region. In addition, increased ParB level brings about repression or activation of numerous genes including several transcriptional regulators involved in SOS response, virulence and adaptation. Overall, our data support the role of partitioning protein ParB as a transcriptional regulator in Pseudomonas aeruginosa

Genome sequence of Pseudomonas aeruginosa PAO1161, a PAO1 derivative with the ICEPae1161 integrative and conjugative element

Background: Pseudomonas aeruginosa is a cause of nosocomial infections, especially in patients with cystic fibrosis and burn wounds. PAO1 strain and its derivatives are widely used to study the biology of this bacterium, however recent studies demonstrated differences in the genomes and phenotypes of derivatives from different laboratories. Results: Here we report the genome sequence of P. aeruginosa PAO1161 laboratory strain, a leu-, Rif R , restriction- modification defective PAO1 derivative, described as the host of IncP-8 plasmid FP2, conferring the resistance to mercury. Comparison of PAO1161 genome with PAO1-UW sequence revealed lack of an inversion of a large genome segment between rRNA operons and 100 nucleotide polymorphisms, short insertions and deletions. These included a change in leuA, resulting in E108K substitution, which caused leucine auxotrophy and a mutation in rpoB, likely responsible for the rifampicin resistance. Nonsense mutations were detected in PA2735 and PA1939 encoding a DNA methyltransferase and a putative OLD family endonuclease, respectively. Analysis of revertants in these two genes showed that PA2735 is a component of a restriction-modification system, independent of PA1939. Moreover, a 12 kb RPG42 prophage and a novel 108 kb PAPI-1 like integrative conjugative element (ICE) encompassing a mercury resistance operon were identified. The ICEPae1161 was transferred to Pseudomonas putida cells, where it integrated in the genome and conferred the mercury resistance. Conclusions: The high-quality P. aeruginosa PAO1161 genome sequence provides a reference for further research including e.g. investigation of horizontal gene transfer or comparative genomics. The strain was found to carry ICEPae1161, a functional PAPI-1 family integrative conjugative element, containing loci conferring mercury resistance, in the past attributed to the FP2 plasmid of IncP-8 incompatibility group. This indicates that the only known member of IncP-8 is in fact an ICE

The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins, mainly acting as repressors, which are involved in response to various environmental signals. In this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the PA3459–PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase, a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We showed that PA3458-mediated control of PA3459–PA3461 expression is required for the adaptation of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in osmoadaptation control in P. aeruginosa

Transcriptional profiling of ParA and ParB mutants in actively dividing cells of an opportunistic human pathogen Pseudomonas aeruginosa.

Accurate chromosome segregation to progeny cells is a fundamental process ensuring proper inheritance of genetic material. In bacteria with simple cell cycle, chromosome segregation follows replication initiation since duplicated oriC domains start segregating to opposite halves of the cell soon after they are made. ParA and ParB proteins together with specific DNA sequences are parts of the segregation machinery. ParA and ParB proteins in Pseudomonas aeruginosa are important for optimal growth, nucleoid segregation, cell division and motility. Comparative transcriptome analysis of parA null and parB null mutants versus parental P. aeruginosa PAO1161 strain demonstrated global changes in gene expression pattern in logarithmically growing planktonic cultures. The set of genes similarly affected in both mutant strains is designated Par regulon and comprises 536 genes. The Par regulon includes genes controlled by two sigma factors (RpoN and PvdS) as well as known and putative transcriptional regulators. In the absence of Par proteins, a large number of genes from RpoS regulon is induced, reflecting the need for slowing down the cell growth rate and decelerating the metabolic processes. Changes in the expression profiles of genes involved in c-di-GMP turnover point out the role of this effector in such signal transmission. Microarray data for chosen genes were confirmed by RT-qPCR analysis. The promoter regions of selected genes were cloned upstream of the promoter-less lacZ gene and analyzed in the heterologous host E. coliΔlac. Regulation by ParA and ParB of P. aeruginosa was confirmed for some of the tested promoters. Our data demonstrate that ParA and ParB besides their role in accurate chromosome segregation may act as modulators of genes expression. Directly or indirectly, Par proteins are part of the wider regulatory network in P. aeruginosa linking the process of chromosome segregation with the cell growth, division and motility

Convenient broad-host-range unstable vectors for studying stabilization cassettes in diverse bacteria

Background: Low-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. The efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use. Results: To facilitate such analysis highly unstable, mobilizable, broad-host-range (BHR) vectors based on RK2 replicon were constructed. The vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding ParB homologues capable of spreading on DNA. The xylE or lacZ reporter systems facilitate easy monitoring of plasmid segregation. Conclusion: The range of BHR vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species

Functional Characterization of TetR-like Transcriptional Regulator PA3973 from Pseudomonas aeruginosa

Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa PA3973 encodes an uncharacterized TetR family transcriptional regulator. In this study, we applied a transcriptome profiling (RNA-seq), genome-wide identification of binding sites using ChIP-seq, as well as the phenotype analyses to unravel the biological role of PA3973. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes in the mRNA level of 648 genes. Concomitantly, ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome. A 13 bp sequence motif was indicated as the binding site of PA3973. The PA3973 regulon encompasses the PA3972-PA3971 genes encoding a probable acyl-CoA dehydrogenase and a thioesterase. In vitro analysis showed PA3973 binding to PA3973p. Accordingly, the lack of PA3973 triggered increased expression of PA3972 and PA3971. The DPA3972-71 PAO1161 strain demonstrated impaired growth in the presence of stress-inducing agents hydroxylamine or hydroxyurea, thus suggesting the role of PA3972-71 in pathogen survival upon stress. Overall our results showed that TetR-type transcriptional regulator PA3973 has multiple binding sites in the P. aeruginosa genome and influences the expression of diverse genes, including PA3972-PA3971, encoding proteins with a proposed role in stress response

A single parS sequence from the cluster of four sites closest to oriC is necessary and sufficient for proper chromosome segregation in Pseudomonas aeruginosa

Among the mechanisms that control chromosome segregation in bacteria are highly-conserved partitioning systems comprising three components: ParA protein (a deviant Walker-type ATPase), ParB protein (a DNA-binding element) and multiple cis-acting palindromic centromere-like sequences, designated parS. Ten putative parS sites have been identified in the P. aeruginosa PAO1 genome, four localized in close proximity of oriC and six, diverged by more than one nucleotide from a perfect palindromic sequence, dispersed along the chromosome. Here, we constructed and analyzed P. aeruginosa mutants deprived of each single parS sequence and their different combinations. The analysis included evaluation of a set of phenotypic features, chromosome segregation, and ParB localization in the cells. It was found that ParB binds specifically to all ten parS sites, although with different affinities. The P. aeruginosa parS mutant with all ten parS sites modified (parSnull) is viable however it demonstrates the phenotype characteristic for parAnull or parBnull mutants: slightly slower growth rate, high frequency of anucleate cells, and defects in motility. The genomic position and sequence of parS determine its role in P. aeruginosa biology. It transpired that any one of the four parS sites proximal to oriC (parS1 to parS4), which are bound by ParB with the highest affinity, is necessary and sufficient for the parABS role in chromosome partitioning. When all these four sites are mutated simultaneously, the strain shows the parSnull phenotype, which indicates that none of the remaining six parS sites can substitute for these four oriC-proximal sites in this function. A single ectopic parS2 (inserted opposite oriC in the parSnull mutant) facilitates ParB organization into regularly spaced condensed foci and reverses some of the mutant phenotypes but is not sufficient for accurate chromosome segregation