DNA of pIGMS31, pIGMS32, and pIGRK, prepared using a silica–guanidinium thiocyanate DNA isolation method (Boom

et al., 1999), was subjected to in vitro transposition with transposon EZ::TN , bearing a kanamycin resistance cassette, according to the manufacturer’s instructions (EZ::TN™ Insertion kit; Epicentre Biotechnologies). buy AZD6244 Relevant DNA regions were amplified by PCR using appropriate template DNAs, specific oligonucleotide primers, dNTPs and Pfu polymerase (Qiagen, with supplied buffer) in a Mastercycler (Eppendorf). The primers used are listed in Table 1. Amplified DNA fragments were separated by 0.8% agarose gel electrophoresis, purified using the Gel Out kit (A&A Biotechnology), and cloned into appropriate plasmid vectors. The nucleotide sequences of pIGMS31, pIGMS32, and pIGRK were determined in the DNA Sequencing and Oligonucleotide Synthesis Laboratory at the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, using a dye terminator sequencing kit and an automated sequencer (ABI 377 Perkin Elmer). The obtained nucleotide sequences were assembled using the program Sequencher 4.1.4 (Gene Codes

Corporation, AnnArbor, MI) and were further analyzed using the click here VectorNTI 8 software package (Invitrogen, Frederick, MD) and Artemis (Rutherford et al., 2000). Similarity searches were performed using the blast programs (Altschul et al., 1997) available at the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The mating procedure (between E. coli strains) was performed in liquid medium using E. coli S17-1 carrying a mobilizable kanamycin-resistant plasmid (as the donor strain) and rifampicin-resistant E. coli DH5αR (as the recipient). The mating mixture was incubated for 2 h at 37 °C (without agitation). The cell suspension Tacrolimus (FK506) was then diluted, and 100 μL of appropriate

dilutions was plated on selective media containing rifampicin and kanamycin to select for transconjugants. The inter-species matings were carried on solid media as previously described (Dziewit et al., 2007). Spontaneous resistance of the recipient strains to the antibiotics used in selection was not observed under these experimental conditions. The plasmid content of transconjugants was verified by screening several colonies using a rapid alkaline extraction procedure and agarose gel electrophoresis. All matings were repeated at least three times. The nucleotide sequences of pIGMS31, pIGMS32, and pIGRK have been annotated and deposited in the GenBank database under accession numbers AY543072, DQ298019, and AY543071, respectively. The initial screening of plasmids carried by K. pneumoniae strain 287-w, performed using a classical alkaline lysis procedure, revealed the presence of two replicons, designated pIGMS31 (c. 2.5 kb) and pIGMS32 (c. 9 kb).