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. 2019 Jun 18;10(3):e01138-19. doi: 10.1128/mBio.01138-19

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Copyright © 2019 Vang Nielsen et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 3 — HipT_O127 phosphorylates TrpS at S197 in vitro, and HipT_O127 is autophosphorylated in vitro. (A) Phosphorylation of TrpS and autophosphorylation of HipT_O127 and HipA in vitro. HipT_O127 or HipA (0.2 μM), [γ-³²P]ATP (0.1 μM), and ATP (66 μM) were incubated with (+) or without (−) TrpS [purified from strain C41(DE3)/pSVN46], as well as with (+) or without (−) a mixture of all E. coli tRNAs (0.5 μg) per microliter of reaction mixture as indicated. (B) Phosphorylation of GltX and autophosphorylation of HipT_O127 and HipA in vitro. HipT_O127 or HipA (0.2 μM), [γ-³²P]ATP (0.1 μM), and ATP (66 μM) were incubated with (+) or without (−) GltX (purified from strain JW2395), as well as with (+) or without (−) a mixture of E. coli tRNAs (0.5 μg) per microliter of reaction mixture as indicated. (C) Phosphorylation of TrpS at S197 and autophosphorylation of HipT_O127 in vitro. HipT_O127 (1.5 μM) [purified from strain C41(DE3)/pSVN42] was added to the reaction mixtures as indicated, in addition to [γ-³²P]ATP (0.1 μM) and ATP (66 μM) with (+) or without (−)TrpS (1.5 μM), TrpS^S197A (1.5 μM), or TrpS^S197D (1.5 μM) as indicated.