Ninety-eight nanograms of the 5′ HEX-labelled TN or BN suicide substrates were incubated in 20 μL-reaction volumes containing TDMNG buffer (50 mM Tris pH 7.5, 5 mM DTT, 75 mM MgCl2, 25 mM NaCl and 25% glycerol) in the presence of 1.5 mM MBP-XerS each in the presence of 1 μg poly dI-dC. After a 60 min incubation at 37 °C, reactions were stopped with 5 μL

of 2% SDS and 5 μL of Orange loading dye (NEB), incubated at 100 °C for 10 min and then electrophoresed in a 6% TBE gel in the presence of 0.1% SDS, and scanned with a EPZ5676 mouse Typhoon imager. The difSL cleavage site was determined using 98 ng of the 3′ FITC-labelled TN or BN suicide substrate and was incubated in 20 μL of TDMNG buffer in the presence of variable concentrations of MBP-XerS in the presence of 1 μg Entinostat chemical structure poly dI-dC. After a four-hour incubation at 37 °C, reactions were stopped with 20 μL of formamide, incubated at 75 °C for 2 min and then electrophoresed in

a 20% polyacrylamide TBE gel with 6 M urea, and scanned with a Typhoon imager. Molecular weight ladders were prepared by chemical degradation of the 3′ FITC-labelled oligonucleotides following the G+A chemical sequencing protocol (Bencini et al., 1984). Under the conditions used, cleavage was observed at each nucleotide position, generating a ladder of fragments differing by a single nucleotide. The thermosensitive plasmid pBEA756 was used to inactivate the xerS gene of S. suis (Fittipaldi et al., 2007). An internal sequence of the

xerS gene was amplified by PCR and cloned into the EcoRI site of pBEA756, forming the plasmid pBEAXerCint. Plasmids were then electroporated into S. suis from (prepared according to Pulliainen et al., 2003) using a Bio-Rad gene pulser using 0.2-cm cuvettes at 2.5 kV. Immediately after the pulse, 1 mL of cold THY medium supplemented with 0.3 M sucrose was added, and the samples were incubated at 28 °C for 3 h, and spread on a THA plate containing 1% yeast, 400 μg mL−1 kanamycin and incubated at 28 °C. The resulting transformants were then grown in THY broth overnight with kanamycin selection at 28 °C. Aliquots of overnight cultures were spread on selective THA plates and incubated at 37 °C to inactivate the gram-positive origin. Cells which remained kanamycin resistant, presumably had integrated the plasmid into the chromosome by homologous recombination at the xerS locus, inactivating the gene. This was confirmed by Southern blot analysis, using genomic DNA prepared from kanamycin-resistant cultures using the DNeasy tissue kit. Complementation of the xer− phenotype was observed after re-introducing a cloned xerS gene with its promoter into pGhost9, and electroporating the construct into xerS mutant cells.