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. Author manuscript; available in PMC: 2008 Sep 23.
Published in final edited form as: J Biol Chem. 2003 Jan 15;278(14):12120–12129. doi: 10.1074/jbc.M300379200

Fig. 2. Transfer of the R. leguminosarum cosmids expressing the lipid A oxidase activity into S. meliloti.

Fig. 2

The active cosmids identified in Fig. 1 and the empty vector pLAFR-1 were transferred into S. meliloti 1021 via tri-parental mating (27, 28). Cell-free extracts and membranes derived from late log phase cells containing these cosmids were prepared and assayed for oxidase activity using 10 μM [14C]B (600 cpm/reaction tube) as the substrate. Lanes 2–4 show the results with 0.5 mg/ml crude extracts, whereas lanes 5–7 show assays with 0.5 mg/ml washed membranes. Lane 1, no enzyme control; lane 2, pLAFR-1; lane 3, p1S11D; lane 4, p1U12G; lane 5, pLAFR-1; lane 6, p1S11D; lane 7, p1U12G. The reaction mixtures were incubated overnight at 30 °C. Similar results were obtained when [4′-32P]1-dephospholipid IVA was used as the substrate (data not shown).