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

Driving apart and segregating genomes in Archaea

Genome segregation is a fundamental biological process in organisms from all domains of life. How this stage of the cell cycle unfolds in Eukarya has been clearly defined and considerable progress has been made to unravel chromosome partition in Bacteria. The picture is still elusive in Archaea. The lineages of this domain exhibit different cell-cycle lifestyles and wide-ranging chromosome copy numbers, fluctuating from 1 up to 55. This plurality of patterns suggests that a variety of mechanisms might underpin disentangling and delivery of DNA molecules to daughter cells. Here I describe recent developments in archaeal genome maintenance, including investigations of novel genome segregation machines that point to unforeseen bacterial and eukaryotic connections

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

ParB silencing test for wild type and mutated versions of <i>parS</i>.

<p><i>E</i>. <i>coli</i> DH5α transformants carrying pGB2 derivatives with individual wt <i>parS</i> sequences or their mutated versions were transformed with pKLB2 (<i>tacp-parB</i>_{<i>P</i>.<i>a</i>.}). Three independent transformation experiments were conducted and representative results are demonstrated. Undiluted transformation mixtures and their 10- and 100-fold dilutions were plated on three types of selection plates. Numbers of colonies growing on L-agar plates with Pn (blue bar), Pn and Sm (red bar), and double selection plates with 0.5 mM IPTG (green bar) are shown for the undiluted samples.</p

The <i>parS</i> sites and their localization in the <i>Pseudomonas aeruginosa</i> genome.

<p><b>(A)</b> Circular map of the <i>P</i>. <i>aeruginosa</i> genome with locations of putative ParB binding sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120867#pone.0120867.ref009" target="_blank">9</a>]. Position of the <i>parAparB</i> operon is shown as black rectangle, grey arrow marks <i>oriC</i>, black arrows indicate predicted <i>parS</i> sites. <b>(B)</b> Nucleotide sequences, genomic coordinates and gene locations of the <i>parS</i> sites. The sequences are presented in a clockwise configuration. The coordinates are given according to the genomic sequence of the PAO1-UW strain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120867#pone.0120867.ref069" target="_blank">69</a>]. <b>(C)</b> Sequence logo for all twenty 8-bp half-sites in the <i>P</i>. <i>aeruginosa</i> PAO1-UW genome <b>Error! Bookmark not defined.</b>). Nucleotides at positions 2 and 5 are invariant in all half-sites.</p

Phenotypes of selected PAO1161 <i>parS</i> mutants.

<p>Colony morphology, swarming motility, intracellular ParB localization with the use of FITC-conjugated anti-ParB antibodies, and proportion of anucleate cells after DAPI staining are shown for representative mutant strains. As the controls wt PAO1161 Rif^R and <i>parA</i>_null and <i>parB</i>_null mutants were tested in each set of experiments. The percentage of anucleate cells is the mean from at least three independent experiments, with approximately 1000 cells counted in each experiment.</p

Summary of phenotypes of various <i>parS</i> mutants.

<p>The collection comprises 10 mutants with each individual <i>parS</i> modified (single) and 21 multiple mutants with between two and ten (<i>parS</i>_null) <i>parS</i> sequences modified (red dots). Mutants in columns highlighted in green demonstrate wild-type phenotypes (wt-like), those highlighted in yellow show phenotypes similar to those observed for the <i>parA</i>_null and <i>parB</i>_null mutants (<i>parAB</i>-like). The PAO1161 Rif^R<i>parS</i>mut29 mutant has ectopic <i>parS2</i> (green dot) replacing <i>parS7*</i> in <i>parS</i>_null mutant (highlighted in blue).</p