Aneta A. Bartosik

Deletion of the parA (soj) Homologue in Pseudomonas aeruginosa Causes ParB Instability and Affects Growth Rate, Chromosome Segregation, and Motility

The parA and parB genes of Pseudomonas aeruginosa are located approximately 8 kb anticlockwise from oriC. ParA is a cytosolic protein present at a level of around 600 molecules per cell in exponential phase, but the level drops about fivefold in stationary phase. Overproduction of full-length ParA or the N-terminal 85 amino acids severely inhibits growth of P. aeruginosa and P. putida. Both inactivation of parA and overexpression of parA in trans in P. aeruginosa also lead to accumulation of anucleate cells and changes in motility. Inactivation of parA also increases the turnover rate (degradation) of ParB. This may provide a mechanism for controlling the level of ParB in response to the growth rate and expression of the parAB operon.

ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters

Deletions leading to complete or partial removal of ParB were introduced into the Pseudomonas aeruginosa chromosome. Fluorescence microscopy of fixed cells showed that ParB mutants lacking the C-terminal domain or HTH motif formed multiple, less intense foci scattered irregularly, in contrast to the one to four ParB foci per cell symmetrically distributed in wild-type P. aeruginosa. All parB mutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation of parA of P. aeruginosa. As complete loss of ParA destabilized its partner ParB it was unclear deficiency of which protein is responsible for the mutant phenotypes. Analysis of four parB mutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all four parB mutants demonstrate the same defects it can be concluded that either ParB, or ParA and ParB in combination, plays an important role in nucleoid distribution, growth and motility 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.

Novel broad-host-range vehicles for cloning and shuffling of gene cassettes

Novel vectors for cloning and shuffling of gene cassettes based on minireplicon of broad-host-range RA3 plasmid from IncU incompatibility group were constructed. A series of minireplicon variants were prepared with copy number ranging from low (1-2 copies per chromosome), medium (10-15 copies per chromosome) to high copy number (80-90 copies per chromosome). The new cloning vectors are relatively small in size (4.5-5.4kb) and carry various resistance determinants: kanamycin (Km(R)), tetracycline (Tc(R)) or chloramphenicol (Cm(R)). The vectors were engineered to facilitate cloning and shuffling of the functional modules with or without transcriptional terminators. Using the described strategy, a bank of functional modules, ready for exchange, has been initiated.

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 (parS null) is viable however it demonstrates the phenotype characteristic for parA null or parB null 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 parS null 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 parS null mutant) facilitates ParB organization into regularly spaced condensed foci and reverses some of the mutant phenotypes but is not sufficient for accurate chromosome segregation.

Dissection of the region of Pseudomonas aeruginosa ParA that is important for dimerization and interactions with its partner ParB

Pseudomonas aeruginosa ParA belongs to a large subfamily of Walker-type ATPases acting as partitioning proteins in bacteria. ParA has the ability to both self-associate and interact with its partner ParB. Analysis of the deletion mutants defined the part of the protein involved in dimerization and interactions with ParB. Here, a set of ParA alanine substitution mutants in the region between E67 and L85 was created and analysed in vivo and in vitro. All mutants impaired in dimerization (substitutions at positions M74, H79, Y82 and L84) were also defective in interactions with ParB, suggesting that ParA–ParB interactions depend on the ability of ParA to dimerize. Mutants with alanine substitutions at positions E67, C68, L70, E72, F76, Q83 and L85 were not impaired in dimerization, but were defective in interactions with ParB. The dimerization interface partly overlapped the pseudo-hairpin, involved in interactions with ParB. ParA mutant derivatives tested in vitro showed no defects in ATPase activity. Two parA alleles (parA84, whose product can neither self-interact nor interact with ParB, and parA67, whose product is impaired in interactions with ParB, but not in dimerization) were introduced into the P. aeruginosa chromosome by homologous gene exchange. Both mutants showed defective separation of ParB foci, but to different extents. Only PAO1161 parA84 was visibly impaired in terms of chromosome segregation, growth rate and motility, similar to a parA-null mutant.

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.

Concerted action of NIC relaxase and auxiliary protein MobC in RA3 plasmid conjugation.

Conjugative transfer of the broad‐host‐range RA3 plasmid, the archetype of the IncU group, relies on the relaxase NIC that belongs to the as yet uncharacterized MOBP4 subfamily. NIC contains the signature motifs of HUH relaxases involved in Tyr nucleophilic attack. However, it differs in the residue involved in His activation for cation coordination and was shown here to have altered divalent cation requirements. NIC is encoded in the mobC‐nic operon preceded directly by oriT, where mobC encodes an auxiliary transfer protein with a dual function: autorepressor and stimulator of conjugative transfer. Here an interplay between MobC and NIC was demonstrated. MobC is required for efficient NIC cleavage of oriT in supercoiled DNA whereas NIC assists MobC in repression of the mobC‐nic operon. A 7‐bp arm of IR3 (IR3a) was identified as the binding site for NIC and the crucial nucleotides in IR3a for NIC recognition were defined. Fully active oriTRA3 was delineated to a 47‐bp DNA segment encompassing a conserved cleavage site sequence, the NIC binding site IR3a and the MobC binding site OM. This highly efficient RA3 conjugative system with defined requirements for minimal oriT could find ample applications in biotechnology and computational biology where simple conjugative systems are needed.

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

BackgroundLow-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.ResultsTo 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.ConclusionThe range of BHR vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species.

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

Abstract 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.