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

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.

Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2.

A novel shuttle entrapment vector, pMMB2, was used to identify a large transposable element, TnPpa1 (44.3 kb), of Paracoccus pantotrophus DSM 11072. TnPpa1 has a composite structure with divergently oriented copies of a cryptic transposon, Tn3434 (Tn3 family), located at both termini. The core region of the element contains a large set of putative genes, whose products show similarity to enzymes involved in central intermediary metabolism (e.g. tricarboxylic acid cycle or 2-methylcitrate cycle), transporters, transcriptional regulators and conserved proteins of unknown function. A 4.2 kb DNA segment of TnPpa1 is homologous to a region of chromosome cII of Rhodobacter sphaeroides 2.4.1, which exemplifies the mosaic structure of this element. TnPpa1 is bordered by 5 bp long directly repeated sequences and is located within a mega-sized replicon, pWKS5, in the DSM 11072 genome. Spontaneous inversion of the core region of TnPpa1 was detected in the host genome. Analysis of the distribution of TnPpa1 in three other strains of P. pantotrophus revealed that this element was present exclusively within DSM 11072, which suggests its relatively recent acquisition by lateral transfer. The identification of TnPpa1 (which may be considered a transposable genomic island) provides evidence for the transposition and lateral transfer of large DNA segments of chromosomal origin (carrying various housekeeping genes), which may have a great impact on the evolution of bacterial genomes.