mallei SR1 ATCC 23344 sucrose-resistant

derivative [40] DDA0742 SR1 derivative harboring a deletion of the 156 bp NarI–SfuI fragment internal to hcp1; Δhcp1 [25] B. thailandensis DW503 E264 derivative; Δ(amrR-oprA) (Gms) rpsL (Smr) [41] DDII0868 DW503::pGSV3-0868; Gmr; hcp1 – This study Plasmids pCR2.Nocodazole datasheet 1-TOPO 3,931-bp TA vector; pMB1 oriR; Kmr Invitrogen pCR2.1-0868 pCR2.1-TOPO containing 342-bp PCR product generated with II0868-up and II0868-dn This study pGSV3 Mobilizabile Gmr suicide click here vector [42] pGSV3-0868 pGSV3 derivative containing EcoRI insert from pCR2.1-0868 This study a r, resistant; s, susceptible. PCR The two deoxyribonucleotide primers used for PCR amplification of an internal gene fragment of B. thailandensis BTH_II0868 (hcp1) were purchased from Invitrogen (Frederick, MD) and designated II0868-up (5’-AGGGCAAGATTCTCGTCCAG-3’) and II0868-dn (5’-TCTCGTACGTGAACGATACG-3’).

The PCR product was sized and isolated using agarose gel electrophoresis, cloned using the pCR2.1-TOPO TA Cloning Kit (Invitrogen), and transformed into chemically competent E. coli TOP10. PCR amplification was performed in a final reaction volume of 100 μl containing 1X Taq PCR Master Mix (Qiagen), 1 μM oligodeoxyribonucleotide MI-503 concentration primers, and approximately 200 ng of B. thailandensis DW503 genomic

DNA. PCR cycling was performed using a PTC-150 MiniCycler with a Hot Bonnet accessory (MJ Research, Inc.) and heated HAS1 to 97°C for 5 min. This was followed by 30 cycles of a three-temperature cycling protocol (97°C for 30 s, 55°C for 30 s, and 72°C for 1 min) and one cycle at 72°C for 10 min. DNA manipulation and plasmid conjugation Restriction enzymes, Antarctic phosphatase, and T4 DNA ligase were purchased from Roche Molecular Biochemicals and were used according to the manufacturer’s instructions. DNA fragments used in cloning procedures were excised from agarose gels and purified with a GeneClean III kit (Q · BIOgene). Bacterial genomic DNA was prepared by a previously described protocol [29]. Plasmids were purified from overnight cultures by using Wizard Plus SV Minipreps (Promega). Plasmid pGSV3-0868 (Table 2) was electroporated into E. coli S17-1 (12.25 kV/cm) and conjugated with B. thailandensis for 8 h, as described elsewhere [30]. Pm was used to counterselect E. coli S17-1 (pGSV3-0868).