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

<div><p>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 <i>parS</i>. Ten putative <i>parS</i> sites have been identified in the <i>P</i>. <i>aeruginosa</i> PAO1 genome, four localized in close proximity of <i>oriC</i> and six, diverged by more than one nucleotide from a perfect palindromic sequence, dispersed along the chromosome. Here, we constructed and analyzed <i>P</i>. <i>aeruginosa</i> mutants deprived of each single <i>parS</i> 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 <i>parS</i> sites, although with different affinities. The <i>P</i>. <i>aeruginosa parS</i> mutant with all ten <i>parS</i> sites modified (<i>parS</i>_null) is viable however it demonstrates the phenotype characteristic for <i>parA</i>_null or <i>parB</i>_null mutants: slightly slower growth rate, high frequency of anucleate cells, and defects in motility. The genomic position and sequence of <i>parS</i> determine its role in <i>P</i>. <i>aeruginosa</i> biology. It transpired that any one of the four <i>parS</i> sites proximal to <i>oriC</i> (<i>parS1</i> to <i>parS4</i>), which are bound by ParB with the highest affinity, is necessary and sufficient for the <i>parABS</i> role in chromosome partitioning. When all these four sites are mutated simultaneously, the strain shows the <i>parS</i>_null phenotype, which indicates that none of the remaining six <i>parS</i> sites can substitute for these four <i>oriC</i>-proximal sites in this function. A single ectopic <i>parS2</i> (inserted opposite <i>oriC</i> in the <i>parS</i>_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.</p>