All constructs, except for pKH62 and pKH72, were prepared by subcloning into pBluescript SK+ (Stragene, La Jolla, CA) prior to cloning into pART2 [55]. Recombinant SB431542 cell line plasmid DNA was transformed into strain D11 by electroporation as described elsewhere [56]. Ampicillin was used for selection at a concentration of 100 μg ml-1 for pBluescript-derived transformants, and kanamycin was used at a concentration of 40 μg ml-1 for pART2-derived transformants. Plasmids were submitted to the Purdue University Core Genomics Center for validation of insert sequences. Plasmid pKH11 was generated by amplifying a 10.6 kb fragment bearing bases 72880 to 83464 of pFB24-104 using

the TripleMaster PCR system (Eppendorf North America, Inc., Westbury, NY) according to the manufacturer’s specifications and primers C42/F and C42/R. The PCR product was digested with HindIII and XbaI and ligated into pBluescript SK+ to give pKH11. Plasmid pKH21 contains a 7.3 kb insert bearing bases 74642 to 81771 from FB24-104; the insert was isolated by digesting pAOWA10128 (obtained from DOE-JGI) with XbaI and HindIII. The remaining constructs

(Table 3) were generated by restriction digestion of either pKH11 or pKH21 using standard cloning procedures [50]. Expression analysis by quantitative reverse transcriptase PCR (qRT-PCR) Primer sequences for qRT-PCR are listed in Table 4. Total RNA was extracted from this website Arthrobacter cell pellets using the FastRNA PRO Blue Kit (MP Biomedical, Solon, OH) and treated with Turbo DNA-Free DNAse (Ambion, Austin, TX) to remove contaminating DNA. RNA concentrations were quantified by measuring the A260 on a Smart Spec 3000 spectrophotometer (Bio-Rad, Hercules, CA). cDNA was synthesized from 100 ng total RNA using ImProm II reverse transcriptase (RT) (Promega, Madison, WI) following the manufacturer’s reaction conditions. PCR was performed using the following conditions: 98°C for 5 min, followed by 30 cycles of 94°C for 30 s, 56-58°C (depending on the primer pair) dipyridamole for 30 s, 72°C for 1 min, with a final extension step at 72°C for 10 min. For real-time

PCR, 1 μl of the reverse transcription reaction mixtures prepared as described above was used as the template. The PCR mixture contained 1 U of HotMaster Taq (Eppendorf North America, Inc., Westbury, NY), 1× HotMaster Taq PCR buffer with 25 mM MgCl2, 1% bovine serum albumin, 0.2 mM each of dNTPs, 0.25 mM each of a forward and reverse primer, SYBR Green (1:30,000; Molecular Bcl-2 inhibitor Probes, Eugene, OR) and 10 nM FITC (Sigma, St. Louis, MO) in a final volume of 25 μl. Reactions were carried out using a Bio-Rad MyIQ single-color real time PCR detection system, and data were analyzed using the MyIQ Optical System software version 2.0. Transcript copy numbers were calculated from a standard curve using known concentrations of pKH11.