Mercury(II)-resistance transposons Tn502 and Tn512, from Pseudomonas clinical strains, are structurally different members of the Tn5053 family.

The mercury(II)-resistance transposons Tn502 and Tn512 were sequenced and shown to be members of the Tn5053 family. They are currently the sole representatives from the clinical setting and were obtained from geographically disparate Pseudomonas aeruginosa strains. The family is comprised of six novel transposons that display genetic and structural variability that has arisen in different ways. The mer and tni arms of the transposons can be differently combined, suggesting that chimeric interchanges have occurred, possibly mediated by the TniR resolvase. The mer modules within the mer arms are remnants of Tn21/Tn501-like transposons that inserted into a tni-containing ancestral transposon. The mer modules themselves are polymorphic and that of Tn502 is a new type. It includes the putative urf2M gene, the 3'-end of which expresses a protein and hence is a bone fide gene (tniM). Homologues of tniM occur beyond the Tn5053 family and include Tn21 tnpM. Other studies have implicated tniM and tnpM in transposition. Based on sequence and compositional differences, members of the environmentally widespread Tn5053 family constitute a different lineage from the related and clinically successful intI-containing Tn402 family.

DNA interaction and phosphotransfer of the C4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli.

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C(4)-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C(4)-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-(32)P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C(4)-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 micro M for untreated DcuR and < or =1 to 2 microM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.