Fig. 3.

(A) Growth of B. subtilis ∆gcvH strains expressing genes encoding various GcvH and LD proteins. The genes were integrated into the chromosomal amyE site of the B. subtilis ∆gcvH strain under control of Pspac promoter. The B. subtilis ∆gcvH NM20 strain is auxotrophic for lipoic acid and thus growth in the absence of lipoic acid-denoted complementation. Colony formation was scored after 36 h at 37 °C on minimal agar plates with glucose as the sole carbon source in the presence or absence of lipoic acid. The IPTG concentration was 0.5 mM. The wild-type (JH642) and B. subtilis ∆gcvH (NM20) strains served as positive and negative controls, respectively. (B) Western blot analysis of GcvH and LD expression. The strains expressing proteins that failed to complement growth of the B. subtilis ∆gcvH strain were grown for 22 h in minimal medium with lipoic acid and 0.5 mM IPTG. Crude cell extracts were separated on SDS/PAGE gels and immunoblotted with antihexahistidine antibody (Materials and Methods). Crude extracts of the B. subtilis ∆gcvH amyE:: Pspac-E. coli gcvH-His₆ strain (XC173) were used as the positive control. Crude extracts of B. subtilis ∆gcvH derivatives expressing the various gcvH and OADH LD proteins are as indicated.