The strains WMR15 and WMR58 (Table 1) were used as positive and negative controls, respectively. Plasmid DNA for cloning was isolated with a Genomed plasmid midi kit and further purified by agarose gel electrophoresis. Plasmid DNA was digested with appropriate restriction enzymes and cloned into pBluescriptIIKS+ (Stratagene, La Jolla, CA) cut with the same enzyme or an enzyme forming compatible ends. Both strands were sequenced by primer walking. A complete sequence for each plasmid was

obtained by assembling individual reads with ContigExpress from the VectorNTI package (Invitrogen, Carlsbad, CA). Sequence annotation and phylogenetic analysis Plasmid DNA sequences and predicted open reading frames were used for BLAST, PSI-BALST and FASTA databank searches at the genebank http://​www.​ncbi.​nlm.​nih.​gov and ddbj http://​www.​ddbj.​ac.​jp websites. AlignX VX-680 cost from the VectorNTI package was used to identify further less conserved or short elements e.g. oriV, oriT or ssi sites. The same program was employed to calculate the global identity of plasmid ORFs and sequences retrieved from databases. Phylogentic analyses were

performed with MEGA4 [56]. Neighbour-joining (NJ) trees were constructed using the p-distance model for DNA and the JTT matrix for amino acid sequences. PRI-724 order Positions with gaps were usually completely deleted except for alignments containing highly diverse sequences, where pair wise deletion was chosen. Bootstrap values were calculated from 1000 replicates and indicated at the corresponding nodes. Almost identical tree topologies were obtained with other methods (minimum evolution

MRT67307 and UPGMA) and models (Poisson correction, PAM). G+C contents of plasmids were calculated using ARTEMIS 10 [57]. Detection of ori deletion pHW126 was digested with BglII and HindIII SPTBN5 and the 1463 bp fragment containing the putative rep gene and the upstream intergenic sequences cloned into pBKanT [6] linearised with BamHI/HindIII. The resulting construct, designated pB126ΔBH, was digested with SpeI, the 446 bp fragment isolated and cloned into the same construct digested with XbaI which led to construct pB126-2ori. This construct was used to assay replication origin deletion: pB126-2ori was digested with SalI and the fragment containing the KanR gene and the pHW126 sequences isolated by agarose gel electrophoresis. The purified DNA was diluted to a concentration of 1 ng/μl and self-circularised by incubation with 1 U T4 ligase for 1 h at room temperature in a total reaction volume of 20 μl leading to pHW126-2ori. After transformation into E. coli INVα F’ the cells were plated on MLB-kanamycin (30 μg/ml) plates and incubated overnight at 37°C. Three individual colonies were transferred completely to 100 ml MLB-Kan medium each and grown overnight. Plasmid DNA was isolated from these cultures using a Genomed plasmid midi kit as recommended by the manufacturer.