The par genes of Pseudomonas aeruginosa have been studied to increase the understanding of their mechanism of action and role in the bacterial cell. Key properties of the ParB protein have been identified and are associated with different parts of the protein. The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role. The dimerization domain of P. aeruginosa ParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group). The C-terminal part of ParB is also involved in ParB-ParA interactions. Purified ParB binds specifically to DNA containing a putative parS sequence based on the consensus sequence found in the chromosomes of Bacillus subtilis, Pseudomonas putida, and Streptomyces coelicolor. The overproduction of ParB was shown to inhibit the function of genes placed near parS. This “silencing” was dependent on the parS sequence and its orientation. The overproduction of P. aeruginosa ParB or its N-terminal part also causes inhibition of the growth of P. aeruginosa and P. putida but not Escherichia coli cells. Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus of P. aeruginosa ParB in interactions with host cell components.

TheparAandparBgenes ofPseudomonas aeruginosaare located approximately 8 kb anticlockwise fromoriC. 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 ofP. aeruginosaandP. putida. Both inactivation ofparAand overexpression ofparAintransinP. aeruginosaalso lead to accumulation of anucleate cells and changes in motility. Inactivation ofparAalso 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 theparABoperon.

Deletions leading to complete or partial removal of ParB were introduced into thePseudomonas aeruginosachromosome. 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-typeP. aeruginosa. AllparBmutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation ofparAofP. 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 fourparBmutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all fourparBmutants 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 inP. aeruginosa.

ParB protein of Pseudomonas aeruginosa belongs to a widely represented ParB family of chromosomally and plasmid-encoded partitioning type IA proteins. Ten putative parS sites are dispersed in the P. aeruginosa chromosome, with eight of them localizing in the oriC domain. After binding to parS , ParB spreads on the DNA, causing transcriptional silencing of nearby genes (A. A. Bartosik et al., J. Bacteriol. 186:6983–6998, 2004). We have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomerization. We defined specific determinants outside of the helix-turn-helix motif responsible for DNA binding. Analysis confirmed the localization of the main dimerization domain in the C terminus of ParB but also mapped another self-interactive domain in the N-terminal domain. Reverse genetics were used to introduce five parB alleles impaired in spreading into the P. aeruginosa chromosome. The single amino acid substitutions in ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect, slowed the growth rate, and impaired motilities, similarly to the pleiotropic phenotype of parB -null mutants, indicating that the ability to spread is vital for ParB function in the cell. The toxicity of ParB overproduction in Pseudomonas spp. is not due to the spreading since several ParB derivatives defective in oligomerization were still toxic for P. aeruginosa when provided in excess.