The published sequences from the genomes of three strains of Stenotrophomonas, five strains of Xanthomonas spp. and two strains of Pseudomonas aeruginosa were included in the study. Bacterial cell lysates, containing genomic DNA, were prepared as previously described (Moore et al., 1999).

Region 1, of two gyrB gene regions analysed, covering nucleotide positions 360–1275 (S. maltophilia strain k279a gyrB sequence; accession no. AB194327), was amplified Verteporfin by PCR, using the primers UgyrBF and UgyrBR (Yamamoto et al., 2000). Region 2, which covers nucleotide positions 1509–2370 (S. maltophilia strain k279a gyrB sequence; AB194327), was amplified, using the primers XgyrB1F and XgyrB1R (Young et al., 2008). PCR was carried out with the Taq PCR Mastermix Kit (Qiagen, Hilden, Germany). Ten-fold dilutions of cell lysate supernatants (5 μL), containing the template DNA, were added to each amplification reaction mix. All samples were run, in duplicate, in 25-μL (final volume) reactions. PCR was performed

as follows: initial denaturation at 95 °C for 2 min; followed by 35 cycles of 95 °C for 30 s (denaturation); 55 °C for 1 min (hybridization); selleck inhibitor and 72 °C for 2 min (extension); with a final extension step of 72 °C for 10 min (Peltier Thermal Cycle, MJ Research Inc., Waltham, MA). The duplicate PCR products were combined and purified, using the Qiaquick PCR Purification Kit (Qiagen), and stored at −20 °C. The reactions for sequencing of Region 1 of gyrB included the PCR amplification primer pair, as well as internal sequencing primers, Smal-gyrB-seq-F (5′-SAGYTTCGTSGARCAYCTGGC-3′), hybridizing at positions 717–737 and Smal-gyrB-seq-R (5′-TGGCCTGCTTGGCGATGCCG-3′), hybridizing selleckchem at positions 948–967. The gyrB Region 2 was sequenced with the same primers as were used for the PCR amplification. Sequencing reactions were performed with the

Big Dye Terminator 3.1 Kit (Applied Biosystems, Carlsbad, CA) under the following conditions: 25 cycles of 96 °C for 30 s; 55 °C for 15 s; and 60 °C for 4 min. The sequencing reaction products were purified by alcohol precipitation. The samples were denatured by heating at 95 °C for 2 min immediately before the addition of deionized formamide (Applied Biosystems). The denatured sequencing reaction products were analysed in the ABI Prism® 3100-Avant Genetic Analyzer (Applied Biosystems). Sequencing of 16S rRNA genes were done, using the PCR amplification and sequencing protocols described above and with primers described previously (Hauben et al., 1997). The DNA sequences were edited to remove the PCR primer sequences and to generate uniform lengths for each gene region sequence. For each strain, the sequences of the gyrB Region 1 were compiled from the individual, overlapping sequences derived using the four primers, while the sequences of the gyrB Region 2 were compiled from two sequencing reactions.