Fig. 3.

(A) Requirement of K78 for in vivo pupylation of Ftn. C. glutamicum ΔpupΔftn carrying pVWEx1-pup-ftn or pVWEx1-pup-ftn-K78A was cultivated in BHI complex medium and harvested in the exponential growth phase. Crude cell extracts were subjected to Ni²⁺-NTA affinity chromatography, and 3.3 µg protein of each eluate was analyzed by SDS/PAGE and Coomassie staining. Pupylation was confirmed by immunoblot analysis (Lower) using polyclonal anti-Pup antiserum. Protein bands visible in the Coomassie-stained gel were excised, trypsinized, and subjected to MS analysis to determine protein identity or the pupylated lysine residue. (B–D) Growth of C. glutamicum WT and the indicated mutant strains in glucose minimal medium under iron limitation (1 µM FeSO₄). The chromosomal ftn-K78A mutation prevents pupylation of Ftn. Mean values and SDs calculated from three independent biological replicates are shown.