Figure 1.

Tyrosine phosphorylation of different SSBs. (A) In vivo phosphorylation of bacterial SSBs. The odd-numbered lane contains purified 6xHis tagged protein (50 ng) separated on SDS–PAGE, electroblotted on PVDF membrane, and probed with anti-PY antibody, and the even-numbered lane contains the same sample treated with alkaline phosphatase. B.subtilis SSB purified from B.subtilis is in lanes 1 and 2, BsSSB purified from E.coli in lanes 3 and 4, EcSSB purified from E.coli in lanes 5 and 6, ScSSB purified from E.coli in lanes 7 and 8, and the eluate from the Ni-NTA column obtained from a crude extract of wild-type B.subtilis is in lanes 9 and 10. (B) In vitro phosphorylation of B.subtilis SSBs. Autoradiography of in vitro phosphorylation assays separated on SDS–PAGE. The lanes contained the products of reactions with the following proteins: YwpH (lane 1), YwpH and YwqC-NCter (lane 2), YwpH and YwqD (lane 3), YwpH, YwqC-NCter and YwqD (lane 4), BsSSB (lane 6), BsSSB and YwqC-NCter (lane 7), BsSSB and YwqD (lane 8), BsSSB, YwqC-NCter and YwqD (lane 9). Lanes 5 and 10 were the same as lanes 4 and 9, respectively, only YwqE and MnCl₂ were added after 60 min, and the reactions were left for an additional 120 min before loading on SDS–PAGE. (C) In vivo labeling of B.subtilis SSB. 6xHis SSB was purified from SPSSBHT, SPSSBHT-ΔywqD and SPSSBHT-ΔywqE grown in [³³P]phosphate. Purified BsSSB was separated by SDS–PAGE; the Coomassie-staining of the gel is shown in the right panel; and autoradiography signals in the left: SPSSBHT in lanes 1 and 4, SPSSBHT-ΔywqD in lanes 2 and 5, and SPSSBHT-ΔywqE in lanes 3 and 6.