005, 0.025, 0.05, 0.1, 5, 20 or 100 mM. To test for specificity of induction, additional cultures were incubated in the presence of 0, 0.5, 5 and 50 μM CX-5461 supplier PbNO3 in mXBM; 0, 0.5, 5 and 50 mM Na2HAsO4·7 H2O in 0.2X NB; and 0, 0.5, 5, 50 mM hydrogen peroxide (H2O2) in 0.2X NB. Cells were incubated for 2.5 hours at 30°C with agitation. Induction experiments with Cr(VI)-sensitive strain D11 transformed with pKH22, pKH23 and pKH24 were carried out in the same manner with the following exceptions:

kanamycin was added to a concentration of 30 μg ml-1 and chromate was added to one culture at a concentration of 0.025 mM. Generation of chromate-sensitive FB24 derivative The lead- and chromate-sensitive mutant, D11, was generated from the resistant wild-type strain FB24 by growing cells in LB without chromate. AZ 628 manufacturer Cultures were transferred daily by diluting cells 1:1000 into fresh media. Transfers were maintained for approximately 90 generations

at 30°C with shaking at 200 rpm and then screened for cells sensitive to 75 μM lead on mXBM agar plates. Lead-sensitive colonies were then tested for Cr(VI) sensitivity on 0.1X nutrient agar (NA) plates supplemented with 0.5, 1, 2 and 5 mM K2CrO4. Loss of plasmid DNA in strain D11 was assessed by Southern hybridization and rep-PCR. Loss of the CRD genes was confirmed by PCR using gene-specific primers. Total genomic DNA was extracted from cultures grown overnight in NB with appropriate selection. Cells were harvested by centrifugation, suspended in TE buffer, and treated with

lysozyme (1 mg ml-1) for one hour followed by treatment with proteinase K (10 mg ml-1). Cells were lysed using a FastPrep instrument (Qbiogene, Carlsbad, CA) at a setting of 4 for Carnitine palmitoyltransferase II 30 s with 0.64 cm ceramic beads. Genomic DNA was purified by phenol: chloroform: isoamyl alcohol extraction and precipitated with isopropanol [50]. DNA was digested with restriction enzymes (SacI and XcmI) and separated on a 0.7% agarose gel and transferred to Hybond-N+ membrane (Amersham Pharmacia, Pisscataway, NJ) using a Trans-blot semi dry transfer cell (Bio-Rad, Hercules, CA) following the manufacturer’s Belnacasan research buy recommendations for voltage and transfer time. A digoxigenin-labeled probe targeting the 10.6-kb CRD on Arthrobacter sp. strain FB24 pFB24-104 [GenBank: NC_008539] was generated by PCR with primers C42/F and C42/R (Table 4) using the TripleMaster PCR system (Eppendorf North America, Inc., Westbury, NY) according to the manufacturer’s reaction mixture and cycling specifications for long-range PCR. Hybridization and chromogenic detection was carried out under high stringency conditions as described in the DIG Application Manual for Filter Hybridization (Roche Applied Science, Indianapolis, IN). Table 4 PCR and qRT-PCR primers used in this study.