Figure 4. FepB production and cellular localization in strain SET 538 carrying various plasmids.

A: FepB immunodetection in total cell pellets: SET 538 strains harboring pMD1-fepABC, pMD1-fepAB or pMD1-fepB were grown at 37°C in M63 gly supplemented with xyl at two concentrations, 0.2 and 2% as indicated on the figure. Cells were harvested when they reached an OD₆₀₀ = 1. M: FepB precursor proteins purified as inclusion bodies from strain JP313 pBAD24-fepB (the determined amino-terminal sequence was MTNYE); T1: total cell extract of strain SET 538 pMD1-fepABC; T2: total cell extract of strain SET 538 pMD1-fepAB; T3: total cell extract of strain SET 538 pMD1-fepB. Each lane was loaded with 0.5 OD₆₀₀ cell culture equivalent. B: FepB immunodetection in cytoplasmic and periplasmic fractions: SET 538 strains harboring pMD1-fepABC, pMD1-fepAB or pMD1-fepB were grown in M63 gly supplemented with xyl at 2% for the first two strains and 0.2% concentration for the last strain. Cells were harvested when they reached OD₆₀₀ = 1. C1: cytoplasmic fraction of strain SET 538 pMD1-fepABC; C2: cytoplasmic fraction of strain SET 538 pMD1-fepAB; C3: cytoplasmic fraction of strain SET 538 pMD1-fepB. P1: periplasmic fraction of strain SET 538 pMD1-fepABC; P2: periplasmic fraction of strain SET 538 pMD1-fepAB; P3: periplasmic fraction of strain SET 538 pMD1-fepB. FepB is not immunodetected in P1, P2 or P3 periplasmic fractions. Each lane was loaded with 0.5 OD₆₀₀ cell culture equivalent. C: MBP immunodetection in cytoplasmic and periplasmic fractions: The same cytoplasmic (C1, C2, C3) and periplasmic fractions (P1, P2, P3) were immunodetected with anti-MBP antibodies. Each lane was loaded with 0.5 OD₆₀₀ cell culture equivalent. MBP was not immunodetected in C1, C2 or C3 cytoplasmic fractions, but in P1, P2 and P3 periplasmic fractions of the 3 strains.