Methods Strains and growth conditions A list of bacterial strains used in this study is presented in Table 2. E. coli was grown on YT media overnight (about 16 hours) with 50 μg ml-1 kanamycin sulphate as appropriate. Host dependent, predatory Bdellovibrio

were grown in liquid prey lysate cultures in Ca/HEPES buffer or on YPSC double agar overlays as described elsewhere [20]. Table 2 List of strains used in this study Strain Description Reference E. coli S17-1 thi,pro,hsdR -,hsdM +,recA; integrated VS-4718 cost plasmid RP4-Tc::Mu-Kn::Tn7 [21] E. coli DH5α F’ endA1 hsdR17 (rk -mk -) supE44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacIZYA-argF) U169 deoR (ϕ80dlacΔ(lacZ)M15) [22] E. coli S17-1: pZMR100 Plasmid vector used to confer Kmr on S17-1 & DFB225 that are being used as prey www.selleckchem.com/autophagy.html for Kmr Bdellovibrio strains [23] Bdellovibrio bacteriovorus HD100 Wild-type [4] Bdellovibrio bacteriovorus fliC1 merodiploid Kmr derivative of HD100 merodiploid for fliC1 [24] Bdellovibrio bacteriovorus bd0743 HD100 bd0743::aphII This study Bdellovibrio bacteriovorus bd0881 HD100 bd0881::aphII This study RNA isolation and RT-PCR Total RNA was isolated with modifications of the Promega SV total isolation kit described previously [11]. Heat shock was carried out by incubating 20 ml of prey-dependent Bdellovibrio in 50 ml centrifuge tubes at 29°C, then transferring to a 42°C water bath (with a control transferred

to a 29°C water bath) for 10 minutes before adding 5 ml 5% phenol 95% ethanol (v/v) and proceeding with RNA extraction. Plaque enumeration confirmed that this heat treatment had no significant affect on cell viability. RT-PCR was carried out with the Qiagen one-step RT-PCR kit according to the manufacturer’s OICR-9429 instructions as described elsewhere [25]. Primers used are shown in Table 3. Table 3 List of primers used in this study Primer Sequence Use fliC3RTF ATGCTCAGAGAGTTCTCTGG fliC3 RT-PCR fliC3RTR AATGACTTGTTCAAGAGTCC fliC3 RT-PCR fliC5RTF GCTCAACGTAACTTGGTCGG fliC5 RT-PCR fliC5RTR Oxymatrine AGCCGATCAGCTTAAGAGCC fliC5 RT-PCR bd0881RTF CGCAAGGAAGAAGTCAGTCC bd0881 RT-PCR bd0881RTR CAGGCTTAAACGGGATTTCA

bd0881 RT-PCR bd0743RTF GCTCTTTTTCCGAACTCGTG bd0743 RT-PCR bd0743RTR TACAGCCAATTGCACATCGT bd0743 RT-PCR Bd3314RTF GGATTCGCGGCTATATTCAA bd3314 RT-PCR Bd3314RTR TGGCATCCAGAGCTTCTTTT bd3314 RT-PCR fliC1RTF GCATCTATCGCAGCACAACG fliC1 RT-PCR fliC1RTR CCGTCGAGTCGGCATCAAAT fliC1 RT-PCR Bd743-F GAAATTCTTGAAGCCATGACCAATGCG Cloning bd0743 Bd743-R CGGGATCCGAGTGGCCTCTGGATTCG Cloning bd0743 Bd881-F2 CGGAATTCTGGTCGCAAGAATATCTGCC Cloning bd0881 Bd881-R2 GCTCTAGAATGACTCCAAGCTGGTTGGC Cloning bd0881 Bd3314-F GCTCTAGACAGAAAGGAAACGACGCAC Cloning bd3314 Bd3314-R GCTCTAGAGCTTAGGGGTTCTGTATAA Cloning bd3314 Gene knock-out and luminescent prey assay Kanamycin resistance cassettes were inserted into the rpoE-like sigma factor genes of Bdellovibrio, as described elsewhere [9, 11]. Primers used are listed in Table 3. Luminescent prey assays (with E.