A Single parS Sequence from the Cluster of Four Sites Closest to oriC Is Necessary and Sufficient for Proper Chromosome Segregation in Pseudomonas aeruginosa

Abstract

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.

Regional Control of Chromosome Segregation in Pseudomonas aeruginosa

“…No obvious growth defect could be observed when parS1 , parS2 and parS3 were inactivated (alone or in combination), in contrast to when the four parS sites were inactivated, which is consistent with results from Jecz and colleagues [27]. Therefore, we focused our study on the mutant presenting only one functional parS site (Δ parS123 ) and the mutant defective in all four parS sites (Δ parS1234) .…”

“…aeruginosa ParABS system plays a role in chromosome segregation [26,27]. However, there has been controversy about number of parS sites on the genome.…”

“…The resulting strain (Δ parS1234 parS2 +6.5) presents a generation time and an amount of anucleate cells only slightly higher than the wild type strain (Fig 2). We also introduced a sequence that is not efficiently bound by ParB in vivo (TCTTCCTCGTGGAACA, referred as parS9 in [21] and [27]) at the exact same location. The resulting strain (Δ parS1234 parS9 +6.5) presents a generation time and an amount of anucleate cells similar to the Δ parS1234 mutant (Fig 2).…”

“…aeruginosa [21–26]. Ten parS sites scattered along the chromosome have been proposed, based on sequence homology and in vitro binding experiments [21,27]; however, another bioinformatics study predicted only 4 parS close to oriC in P . aeruginosa [6].…”

Omega (ParB) binding sites together with the RNA polymerase-recognized sequence are essential for centromeric functions of the Pω region in the partition system of pSM19035

“…Moreover, a single intact origin-proximal parS site (when all other sites were mutated) was sufficient to preserve the wild-type phenotype of P. aeruginosa, with regard to the process of chromosome partitioning (Jecz et al, 2015).…”