RA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group isolated originally from Aeromonas spp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable replicon in Escherichia coli. This fragment specifies the korA-incC-korB-orf11 operon coding for an active partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation. IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative ؊35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set of unrelated repeat motifs distributed over ϳ300 bp. Dissection of this region identified the centromere sequence that is vital for partitioning. The ϳ300-bp fragment also encompasses the origin of conjugative transfer, oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set of cis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary origin of segregation and conjugative transfer modules in these plasmids and members of the IncU group.Plasmids from the IncU incompatibility group have been isolated from the fish pathogens Aeromonas salmonicida and Aeromonas hydrophila in worldwide locations (4,5,6,12,19,20,44), as well as from clinical isolates of Escherichia coli and A. hydrophila (40,53). The archetypal IncU plasmid is the low-copy-number, conjugative RA3 plasmid (45,909 bp) isolated from A. hydrophila in Japan in 1979 (5), which confers resistance to sulfonamides, streptomycin, and chloramphenicol. RA3 transfers with high frequency from E. coli to Pseudomonas putida (Gammaproteobacteria) and Ralstonia eutropha (Betaproteobacteria) and with three-magnitude-lower efficiency between E. coli and Agrobacterium tumefaciens (Alphaproteobacteria). RA3 is stably maintained in all tested representatives of these three classes (31). The nucleotide sequence of RA3 (GenBank accession no. DQ401103) (31) revealed the existence of putative functional modules involved in replication, maintenance, and conjugative transfer that are almost identical to those of the tetracycline-resistant IncU plasmid pFBAOT6 (41). The sequence confirms a high degree of conservation in the backbone functions of IncU plasmids suggested previously, on the basis of restriction enzyme analysis (47). Particularly interesting features of the RA3/pFBAOT6 backbone include a replication module with similarity to several unrelated environmental plasmids from a wide diversity of beta-and gamm...

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.

The mupirocin trans-AT polyketide synthase pathway, provides a model system for manipulation of antibiotic biosynthesis. Its final phase involves removal of the tertiary hydroxyl group from pseudomonic acid B, PA-B, producing the fully active PA-A in a complex series of steps. To further clarify requirements for this conversion, we fed extracts containing PA-B to mutants of the producer strain singly deficient in each mup gene. This additionally identified mupM and mupN as required plus the sequence but not enzymic activity of mupL and ruled out need for other mup genes. A plasmid expressing mupLMNOPVCFU + macpE together with a derivative of the producer P. fluorescens strain NCIMB10586 lacking the mup cluster allowed conversion of PA-B to PA-A. MupN converts apo-mAcpE to holo-form while MupM is a mupirocin-resistant isoleucyl tRNA synthase, preventing self-poisoning. Surprisingly, the expression plasmid failed to allow the closely related P. fluorescens strain SBW25 to convert PA-B to PA-A.