Binding and spreading of ParB on DNA determine its biological function in Pseudomonas aeruginosa

Abstract

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.

Fig. 1.

Summary of “silencing” parB mutants of P. aeruginosa used in the present study. A schematic map of ParB (290 amino acids) with highly conserved motifs (5) marked in black and the approximate positions of amino acid substitutions in ParB derivatives defective in silencing ability (mutant alleles selected in pKLB2 tacp-parB) is shown. The deletion mutants are presented underneath the scheme. The ParB derivative with two regions modified A122V and Δ253-256 was originally encoded by allele parB40 and was also included in the analysis.

Fig. 2.

Circular dichroism spectra of ParB derivatives. Purified 6×His-tagged versions of mutated ParB were analyzed by CD as described in Materials and Methods. Since all spectra were similar, only four are shown as representative ParB derivatives for clarity.

Fig. 3.

Structural analysis of ParB derivatives. (A) Analytical ultracentrifugation of ParB derivatives. Purified His₆-tagged versions of mutated ParB at concentrations 0.1 mg ml⁻¹ were centrifuged in the Beckman XL-A ultracentrifuge in sets of eight tubes, with WT ParB and ParB 1-249 included in each set. “S” represents the sedimentation coefficient. Comparison of the S values for four ParB mutants are shown, where an S value close to 2 corresponds to dimeric form of the protein and an S value close to 1 corresponds to the monomeric form of the protein. (B) Cross-linking of ParB derivatives with glutaraldehyde (GA). Purified His₆-tagged proteins at 0.3 mg ml⁻¹ were incubated at room temperature for 20 min (18) without or with different concentrations of glutaraldehyde: 0.001, 0.002, 0.005, and 0.01%. Proteins were separated by SDS-PAGE on 12% gels and visualized by Western blotting with anti-ParB antibodies. Monomers are indicated as “M,” and higher-order are indicated as complexes as “P.” (C) Graphic summary of the ability of ParB mutants to form higher-order complexes. Localization of analyzed mutations is indicated by arrows. Black solid arrows indicate mutants with the ability to form higher-order complexes comparable to WT ParB. Black V-type arrows indicate mutants impaired in higher-order complex formation. The gray arrow indicates mutant ParB 1-249, which remains in the monomeric state.

Fig. 4.

DNA-binding activity of ParB derivatives. (A) EMSA for representative ParB derivatives. Different amounts (10, 20, 30, and 50 pmol) of purified ParB proteins were incubated with 5.6 pmol of parS double-stranded oligonucleotide at 37°C for 15 min. The samples were analyzed on 10% (wt/vol) nondenaturing polyacrylamide gels run in TBE. After electrophoresis, the gels were stained with ethidium bromide, DNA was visualized in UV light and photographed. (B) Graphic summary of DNA-binding affinity of ParB mutants. Localization of analyzed mutations is indicated by arrows. Three types of arrow correspond to distinct DNA binding abilities: highly impaired mutants are indicated by gray arrows, partially impaired mutants are indicated by black V-type arrows, and mutants binding to DNA with WT ParB activity are indicated by black solid arrows. For comparison, the results for oligomerization ability of mutant derivatives are shown below the ParB scheme.

Fig. 5.

Growth inhibition of P. aeruginosa parB-null mutant by overproduction of ParB mutant derivatives. (A) Growth of PAO1161parB_null (pBBR1MCS1 tacp-parB mutants). Overnight cultures were diluted 100-fold into L broth supplemented with chloramphenicol or chloramphenicol and 0.5 mM IPTG. The cultures were incubated with shaking at 37°C, and the OD₆₀₀ was measured at hourly intervals. As controls, PAO1161parB_null(pBBR1MCS1) (vector) and PAO1161parB_null(pJMB500) (WT parB) were used. For clarity, only the results for IPTG-induced cultures for three representative mutant ParB derivatives are shown. (B) Graphic summary of inhibition effect of ParB derivatives on PAO1161parB_null growth. The ParB derivatives retaining a growth inhibition effect when overproduced are indicated either by black solid arrows (high toxicity) or black V-type arrows (slightly lower toxicity), whereas modifications abolishing growth inhibition are indicated by gray solid arrows.

Fig. 6.

Characterization of PAO1161 parB mutants defective in spreading. (A) ParB subcellular localization in PAO1161 parB spreading mutants. Fixed cells were prepared from exponential phase of culture growth (OD₆₀₀ = 0.4) on L broth at 37°C. Overlaid images of immunofluorescence (green) signal and DAPI (blue) staining show ParB foci in the cells of PAO1161 (WT ParB), PAO1161parB₃ (ParB A97T), PAO1161parB₇ (ParB A158V), PAO1161parB₉ (ParB R94C), PAO1161parB₅₅ (ParB G71S), and PAO1161parB₆₂ (ParB T165I) mutants. Magnifications of single cells are shown for clarity. (B) Swimming motility of PAO1161 parB mutants defective in spreading. Arrows demonstrate the diameters of the zones of swimming (extending beyond visible growth zones on the surface). (C) Swarming motility of PAO1161 parB mutants defective in spreading.

Fig. 7.

Alignment of ParB from P. aeruginosa PAO1 with selected representatives of ParB family. Dark gray shadowing indicates the similar residues in more than three representatives. Light gray shadowing indicates similarities between two proteins. Structural motifs from crystallographic studies on KorB of plasmid RP4/RK2 (above the alignment in green) and Spo0J/ParB from T. thermophilus (indicated below in yellow) are drawn with rectangles corresponding to α-helices and arrows corresponding to β-sheets. Labeling of the helices corresponds to the original nomenclature (26, 32). H-T-H motifs are striped. Red-shaded residues in ParB P. aeruginosa sequence correspond to substituted residues, and pink-shaded residues correspond to deleted residues in the course of this study. A red arrow indicates the localization of stop codon in the ParB 1-249 derivative. Green residues marked in the KorB sequence correspond to DNA-binding determinants outside of the H-T-H sequence (26). Blue residues in Spo0J of B. subtilis correspond to mutations analyzed previously (3).

Fig. 8.

Model of N-terminal part of ParB from P. aeruginosa interacting with DNA. (A and B) Different projections of two ParB monomers (the N-terminal P114-T229 fragment) modeled on the basis of the KorB RK2/RP4 structure (26). (C and D) Different projections of two ParB monomers (the N-terminal Q37-L224 fragment) modeled on the basis of Spo0J from the T. thermophilus structure (32). Monomer subunits are indicated in green and dark blue, with regions of the H-T-H highlighted in light blue. Residues mutated in the present study are colored red (impaired in DNA binding) and yellow (impaired in polymerization) in both monomers but numbered only in the dark blue one.