Fig. 4.

EsaB is present in cells at low amounts. (a) Titration of α-EsaB antibodies. The indicated amounts of purified EsaB, alongside 30 µl of OD₆₀₀5 adjusted cells were loaded on a SDS-PAGE as indicated and blotted using α-EsaB antibodies. Two exposures of the blot are shown. (b) RN6390 and the isogenic ΔessC strain (top) or strain Newman (bottom panel) were grown aerobically in TSB medium with or without hemin, as indicated, until an OD₆₀₀ of 2 was reached, at which point cells were harvested as described in Methods. In each case, for detection of EsaB, 25 µl of OD₆₀₀2 adjusted cells were loaded and 25 ng of purified EsaB protein was loaded as a positive control. 5 µl of OD₆₀₀2 adjusted cells were probed against anti-TrxA antisera as a cytoplasmic control. (c) RN6390 harbouring empty pRAB11 (labelled RN6390), and the isogenic esaB deletion strain harbouring pRAB11 (labelled ΔesaB), or pRAB11 encoding native EsaB or EsaB-YFP was cultured aerobically in TSB medium until an OD₆₀₀ of 2 was reached. Samples were fractionated to give cells and supernatant (sn), and supernatant proteins were precipitated using TCA. For each gel, 10 µl of OD₆₀₀1 adjusted cells and 15 µl of culture supernatant were loaded. Blots were probed with anti-EsxA, anti-EsxB or anti-EsxC antisera, alongside anti-TrxA (cytoplasmic control). Cell and supernatant samples have been blotted on the same gel but intervening lanes have been spliced out. (d) EsaB-YFP can be detected in whole cells. RN6390 harbouring empty pRAB11 (labelled RN6390), and the isogenic esaB deletion strain harbouring pRAB11 (labelled ΔesaB), or pRAB11 encoding EsaB-YFP was cultured aerobically in TSB medium until an OD₆₀₀ of 2 was reached. Whole cell samples (20 µl of OD₆₀₀2 adjusted cells) were loaded and blots were probed with anti GFP antibodies. Two exposures of the blot are shown.