For confirmation, both bands were cut out, extracted with a Macherey-Nagel gel extraction kit and used as a template for PCR amplification with the primer pair pHW126-11/Kan rev. The amplification product was cleaned and directly sequenced employing the same primers as used for PCR. As a control pHW15-2ori, which possesses two pHW15 origins of replication in tandem repeat, was tested in the same way. pB15In(NsiI) was constructed by inserting pHW15 [6] linearised with NsiI into pBKanT. Subsequently, this construct was linearised with HindIII and PstI and ligated with the 1218 bp fragment obtained by digesting pBKanT-pHW15Δ(ORF1+2+3)

[6] with HindIII and NsiI. This led to construct pB15-2ori which was finally digested with SalI and self-circularised to Selleckchem JSH-23 ARS-1620 molecular weight obtain pHW15-2ori. Southern blot analysis Approximately 3 μg

genomic DNA were digested with an appropriate restriction enzyme and separated by agarose gel electrophoresis. After denaturation with 0.5 M NaOH, neutralisation with 5× TBE and equilibration with 1× TBE the DNA was transferred to a Hybond-N+ membrane (GE Healthcare, Buckinghamshire, UK) by semi-dry electroblotting using 1× TBE as transfer buffer. Cross linking was achieved by irradiation with 120 mJ/cm2 UV of 254 nm. Subsequently, the membrane was pre-hybridised with Church buffer [58] containing 100 μg/ml freshly denaturated herring sperm DNA. The probe was prepared by PCR: a 50 μl reaction contained 1 U GoTaq (Promega, Madison, WI), 10 μl 5× buffer containing Mg2+, 1 ng pHW4594 as template, 1 μl primer mix (pHW4594-fwd/pHW4595-rev; each 5 μM), 1 μl nucleotide mix (0.5 mM each of dATP, dGTP and dTTP

and 0.05 mM dCTP) and 30 μCi [α-32P]-dCTP (3000 Ci/mmol; PerkinElmer, Waltham, MA). After an initial denaturation step at 94°C for 5 min 35 cycles of 94°C for 30 sec, 50°C for 1 min and 72°C for 2 min were performed prior a final extension step at 72°C for 10 min. The denaturated amplicon (95°C, 10 min) was added to the blocked membrane Etofibrate and hybridised for 18 h at 60°C. The membrane was washed 5 times with 0.05% SDS in 1× SSC [51] at 60°C and once with distilled water at room temperature. Signals were detected by autoradiography. Determination of genomic G+C contents The genomic DNA G+C contents of selected strains were determined by HPLC analysis as described previously [6]. Nucleotide BAY 1895344 chemical structure sequence accession numbers Plasmids sequences obtained in this study were deposited in the EMBL nucleotide sequence database with the following accession numbers: [EMBL:FN429021], pHW42; [EMBL:FN429022], pHW114A; [EMBL:FN429023], pHW114B; [EMBL:FN429024], pHW120; [EMBL:FN429025], pHW4594; [EMBL:FN429026], pHW30076; [EMBL:FN429027], pHW66; [EMBL:FN429028], pHW121; [EMBL:FN429029], pHW104; [EMBL:FN429030], pHW126. Accession numbers retrieved from databases are listed in Additional file 5.