Dissection of the region of Pseudomonas aeruginosa ParA that is important for dimerization and interactions with its partner ParB

Abstract

Pseudomonas aeruginosa ParA belongs to a large subfamily of Walker-type ATPases acting as partitioning proteins in bacteria. ParA has the ability to both self-associate and interact with its partner ParB. Analysis of the deletion mutants defined the part of the protein involved in dimerization and interactions with ParB. Here, a set of ParA alanine substitution mutants in the region between E67 and L85 was created and analysed in vivo and in vitro. All mutants impaired in dimerization (substitutions at positions M74, H79, Y82 and L84) were also defective in interactions with ParB, suggesting that ParA–ParB interactions depend on the ability of ParA to dimerize. Mutants with alanine substitutions at positions E67, C68, L70, E72, F76, Q83 and L85 were not impaired in dimerization, but were defective in interactions with ParB. The dimerization interface partly overlapped the pseudo-hairpin, involved in interactions with ParB. ParA mutant derivatives tested in vitro showed no defects in ATPase activity. Two parA alleles (parA84, whose product can neither self-interact nor interact with ParB, and parA67, whose product is impaired in interactions with ParB, but not in dimerization) were introduced into the P. aeruginosa chromosome by homologous gene exchange. Both mutants showed defective separation of ParB foci, but to different extents. Only PAO1161 parA84 was visibly impaired in terms of chromosome segregation, growth rate and motility, similar to a parA-null mutant.

Molecular Anatomy of ParA-ParA and ParA-ParB Interactions during Plasmid Partitioning

“…These authors mapped the ParB⅐ParA interacting region to within an interval equivalent to residues 89 -105 of ␦ 2 , by yeast two-hybrid and immunoprecipitation assays (40). This is in good agreement with the ⅐␦ interacting region reported here (Fig.…”

“…To reconcile the apparent discrepancy between the crystal structure of ␦ 2 ⅐ATP␥S⅐Mg 2ϩ (15), our data (Fig. 5B), and those published by other groups (39,40), we propose that the bacterial or yeast two-hybrid assays cannot discriminate between the monomer and the dimer interface and that the authors were mapping the dimer-dimer interface (see Fig. 7A, center).…”

“…7, A and C). In contrast, using a bacterial two-hybrid assay, the ␦ monomer-monomer interface was mapped to residues 104 -105 (39), and using yeast two-hybrid and immunoprecipitation assays, the chromosome-encoded Pseudomonas aeruginosa ParA (Sm) monomermonomer interface was mapped to an interval equivalent to residues 89 -105 of ␦ 2 (40). To reconcile the apparent discrepancy between the crystal structure of ␦ 2 ⅐ATP␥S⅐Mg 2ϩ (15), our data (Fig.…”

“…While the functional characterization of the 2 ⅐␦ 2 interacting domains was being carried out during this study, the interacting domain of chromosome-encoded P. aeruginosa ParA (Sm) with ParB (Lg) was published (40). These authors mapped the ParB⅐ParA interacting region to within an interval equivalent to residues 89 -105 of ␦ 2 , by yeast two-hybrid and immunoprecipitation assays (40).…”

Structures of partition protein ParA with nonspecific DNA and ParB effector reveal molecular insights into principles governing Walker-box DNA segregation

“…Previous biochemical studies identified regions in ParB proteins that are involved in binding to their partner ParA proteins (Bartosik et al 2014;Volante and Alonso 2015). Work by Volante and Alonso (2015) revealed that pSM19035 plasmid ParB (also called ω) interacts with residues 88-119 in its partner, ParA (also called δ).…”