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 ParB(P.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.

Fig. 1.

Model of a dimer of C termini of ParB_P.a. (amino acids 242–290). The mutagenized residues are shown as sticks in the red subunit (labelled according to their position in the ParB_P.a. sequence). The indicated residues L282 and V285 in the grey subunit are possibly involved in a leucine zipper formation. The distance between R290 of one monomer and Q266 of another facilitates the electrostatic interactions (magnification at the left).

Fig. 2.

ParB_P.a. self-association and DNA binding in vitro. (a) Cross-linking with glutaraldehyde (GA) of ParB variants. Purified His₆-tagged proteins (0.1 mg ml⁻¹) were incubated at room temperature for 20 min without (0) or with increasing concentrations (1×, 2×, 5× and 10×10⁻³ %) of glutaraldehyde. The samples were separated by SDS-PAGE on 12 % gels and analysed by Western blotting with anti-ParB antibodies. Monomeric, dimeric and higher forms are indicated by m, d and h, respectively. (b) DNA binding affinity of ParB derivatives (EMSA). Purified His₆-tagged proteins (10, 20, 30 and 50 pmol) were incubated with 5.6 pmol double-stranded parS_P.a. oligonucleotide at 37 °C for 15 min. As a control, double-stranded oligonucleotides with an unrelated palindromic motif were used under the same conditions.

Fig. 3.

Heterodimer formation of ParB variants with wild-type ParB and ParA in vivo (BACTH). Double transformants of E. coli BTH101(pLKB4 derivatives)(pLKB2 derivatives) were streaked on MacConkey indicator medium supplemented with penicillin, kanamycin and 0.5 mM IPTG to visualize protein interactions. Dark streaks are indicative of interaction between the two proteins, whereas light streaks correspond to a lack of interaction.

Fig. 4.

Motility assays for P. aeruginosa PAO1161 Rif^R, parB mutants and merodiploid strains. Volume-standardized plates for swarming and swimming were inoculated with a sterile toothpick using material from a single colony and incubated for 24 h at 30 °C. The zones of growth/spreading are indicated in mm below the images; the boundaries of the swimming zones are marked by arrows.

Fig. 5.

Effect of ParB modifications on its turnover and cellular localization. (a) Intracellular levels of ParB in P. aeruginosa PAO1161 Rif^R pBBR1-MCS, parB mutants with pBBR1-MCS and merodiploids of parB mutants with the appropriate mutant allele on the pBBR1-MCS under control of tacp. Total cellular extracts from 1×10⁹ cells were separated by SDS-PAGE and analysed by Western blotting with anti-ParB antibodies. His-tagged purified ParB was run on the gel as a control. Two cultures of all merodiploid strains were analysed. The intensities of signals were estimated using ImageQuant and shown underneath as relative (%) to the values obtained for the wild-type (wt) strain. (b) Immunofluorescence/phase-contrast overlaid images showing ParB localization in cells of P. aeruginosa PAO1161 Rif^RparB mutants. Cells from the exponential growth phase (OD₆₀₀ 0.5), grown on Luria broth at 37 °C, were fixed. The dark background is a phase-contrast image; blue colour shows DAPI-stained chromosome; green colour indicates FITC-stained ParB_P.a..