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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.The ubiquitous superfamily of Walker-type ATPases is involved in many bacterial processes (7,34,45). One subfamily of these proteins, the ParA proteins, normally functions with a second protein, ParB, and in plasmid systems the combination of ParA and ParB has been associated with better-than-random segregation of low-copy-number plasmids (6,19,23). Although the plasmid partitioning systems fall into two groups on the basis of the ATPase type of component A (type I with Walker-type ATPase and type II with actin-like ATPase), the two groups seem to be unified in the general mechanism of plasmid pairing and directional separation of plasmid molecules prior to cell division. The ParB-like protein binds a centromere-like sequence and is proposed to form pairs of the plasmid molecules (12,17,30,54). The separation of replicons and their transfer towards opposite poles of the dividing cell correlate with the ability of the ParA-like protein to form a dynamic scaffold in the cells (1,3,11,41), its ATPase activity, and its ability to interact with the ParB-parS complex.Duplication of bacterial chromosomes is part of the cell cycle events leading to cell division. Data gathered during the last decade have revealed the role of many different proteins involved in replication and segregation, as well as the spatial and temporal sequence of these processes (4, 14). However, there are still many questions concerning the directional separation of chromosomes to the progeny cells and the regulation of this process. The discovery of highly conserved homologues of ParA (Walker-type ATPase) and ParB encoded by bacterial chromosomes in close vicinity to oriC (5, 18, 36, 44, 47) suggested a role for these proteins in chromosome segregation. To date, the potential roles of both the ParA and ParB proteins in cell biology have been related to regulatory cell cycle check points (8,16,37,44,48,49), positioning of oriC domains (38, 50), separation of replicated origins (37), and the translocation of the proteins to fixed cell locations (55, 61). Studies on chromosomal ParA and ParB also revealed a lack of uniformity in their action in different organisms despite the high degree of conservation at the levels of sequence and genetic organization (5,20,21,26,32,40,44,55,56,60).The aim of the work described in th...

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.

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.

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.