λ25 spectrophotometer

Strain PK14639 (PK14623 carrying pPK14637 plasmid) was grown in 1 L of Terrific broth (RPI) with 50 μg/mL ampicillin and 10 mM arabinose, inoculated to 0.5% from an overnight culture to an OD_600nm of 1.0, and induced with 0.4 μM IPTG along with the addition of 2 mM cysteine and 0.2 mg/mL of Ferric ammonium citrate. After 1 h, the culture was transferred to 4°C and sparged with argon overnight. All subsequent steps were performed under anaerobic conditions in an anaerobic chamber (Coy Laboratory Products). The cells were harvested, resuspended in lysis buffer (50 mM potassium phosphate buffer, 100 mM NaCl, 10% glycerol, 1 mM DTT, pH 7.2), and lysed using a French press at 20,000 lb/in². The lysate was centrifuged for 1 h at 4°C at 45,000 rpm in a Beckman 70.1 Ti rotor. The supernatent was then loaded onto a 5-mL StrepTrap HP (Cytiva) column equilibrated in the same lysis buffer and connected to a FPLC AKTA Pure system. The protein was eluted using a step gradient with lysis buffer containing 2.5 mM desthiobiotin (EMD Millipore). For measurement of the visible spectrum, the N terminus tagged protein was transferred under anaerobic conditions into a screw top quartz cuvette (Starna). The spectrum was recorded in a Perkin-Elmer λ25 spectrophotometer.

The thermal denaturation method of Marmur and Doty [108 (link)], performed basically as described by Smith et al. [109 (link)], was used to estimate G+C contents of DNA from P. fijiensis isolate CIRAD86 (CBS120258) plus that from the closely related banana pathogens P. musae (isolate UQ430; CBS121371) and P. eumusae (isolate CBS122457) as well as the previously sequenced Z. tritici isolate IPO323 (CBS115943) [17 (link)]. Genomic DNA was isolated from cultures grown in PD broth at 25°C on a rotary shaker (150 rpm) following the procedure described by Raeder and Broda [110 ] and was dissolved in 0.1X SSC. Melting curves were obtained on a Perkin-Elmer λ25 spectrophotometer equipped with a thermal programmer. The G+C contents were calculated from the T_m values (melting/transition temperature) derived from the peaks of the first derivatives of the melting curves [111 (link)]. DNA from Candida parapsilosis isolate CBS604 (T_m in 0.1 x SSC, 70.6°C) [3 ] was used as a calibration control. Determinations were performed at least twice for each isolate.