Identification of C-terminal hydrophobic residues important for dimerization and all known functions of ParB of Pseudomonas aeruginosa

Abstract

The ParB protein of Pseudomonas aeruginosa is important for growth, cell division, nucleoid segregation and different types of motility. To further understand its function we have demonstrated a vital role of the hydrophobic residues in the C terminus of ParBP.a.. By in silico modelling of the C-terminal domain (amino acids 242–290) the hydrophobic residues L282, V285 and I289 (but not L286) are engaged in leucine-zipper-like structure formation, whereas the charged residues R290 and Q266 are implicated in forming a salt bridge involved in protein stabilization. Five parB mutant alleles were constructed and their functionality was defined in vivo and in vitro. In agreement with model predictions, the substitution L286A had no effect on mutant protein activities. Two ParBs with single substitutions L282A or V285A and deletions of two or seven C-terminal amino acids were impaired in both dimerization and DNA binding and were not able to silence genes adjacent to parS, suggesting that dimerization through the C terminus is a prerequisite for spreading on DNA. The defect in dimerization also correlated with loss of ability to interact with partner protein ParA. Reverse genetics demonstrated that a parB mutant producing ParB lacking the two C-terminal amino acids as well as mutants producing ParB with single substitution L282A or V285A had defects similar to those of a parB null mutant. Thus so far all the properties of ParB seem to depend on dimerization.

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

“…aeruginosa cells ParB forms large, compact nucleoprotein complexes co-localizing with ori domains, visualized by use of immunofluorescence as 1 to 4 foci, depending on the stage of growth [ 55 ]. In parA null mutant ParB foci are much weaker and irregularly distributed whereas in various parB point mutants, defective in dimerization/ polymerization and interactions with ParA, multiple small foci are dispersed [ 54 , 55 , 67 , 68 ]. A transcriptomic analysis of P .…”

“…aeruginosa par mutants [ 56 ] has revealed changes in the expression of multiple operons indicating an important role of Par proteins (especially ParB) in coordinating different cell processes either directly through interactions with DNA or indirectly through interactions with putative partner proteins (Glabski K., unpublished). Molecular analysis of ParB derivatives has led to the identification of a dimerization domain [ 9 , 55 , 67 ], domains interacting with parS [ 55 , 68 ], and a polymerization domain responsible for spreading around parS sequence to form the nucleoprotein complex [ 68 ]. Also for ParA of P .…”

Pseudomonas aeruginosapartitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of aparS-related motif

“…To verify the ParB binding to these parS sequences in vivo , chromatin immunoprecipitation (ChIP) with anti-ParB antibodies followed by quantitative PCR analysis was applied. Since a lack of ParB in P. aeruginosa PAO1161 parB null mutant produced clear-cut effects under conditions of exponential growth in rich medium at 37°C, but not as severe as during growth in minimal medium (24, 76), such milder conditions were used for these experiments. The ChIP procedure was performed with PAO1161 (WT) strain, PAO1161 parB null mutant (negative control), and PAO1161/pKGB9 ( araC-araBAD p- parB ) strain (ParB+++) with 5-fold increased ParB level which does not retard bacterial growth (63).…”

Strain-Specific Identification ofBifidobacterium bifidumOLB6378 by PCR

“…Although the function of the product remains unknown for B. bifidum, that of Psudomonas aeruginosa has been reported to play a role in growth and cell division. [17][18][19] Thus, the parB gene may be common and widely distributed in other bacteria. On the other hand, the 1,074-bp PCR product did not show similarity to those of other species of Bifidobacterium, except for B. bifidum.…”