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. 2020 Dec 10;296:100146. doi: 10.1074/jbc.RA120.016738

Figure 2.

Figure 2

F. novicidia FigC is required for rhizoferrin production by FigA. A, CAS reactive siderophore production in culture supernatant by E. coli BL21 expressing either the F. novicida NIS synthetase figA (WP_003041317), PLP-dependent decarboxylase figC (WP_003041325) or figA + figC, from pETDuet-1. A positive control of E. coli BL21 coexpressing the R. pickettii figA homolog NIS synthetase and MFS transporter from pETDuet-1 is shown (R. pic syn + trans). B, the same strains shown in (A) grown on CAS plates. C, mass spectrum of partially purified siderophore from the culture supernatant of E. coli BL21 coexpressing the F. novicida figA + figC from pETDuet-1. The prominent peak at 437.2 corresponds to rhizoferrin. D, the effect of coexpressing the F. novicida figB MFS transporter (F.n. trans) or R. pickettii MFS transporter (WP_012761003, R.p. trans) from pACYCDuet-1 (pAC), on siderophore production from expression of F. novicida figA + figC (pETDuet-1, pET) in E. coli BL21. Presence of siderophore in the culture supernatant was detected by CAS reactivity. E, CAS siderophore detection from in vitro assays with purified recombinant F. novicida FigA, FigC, FigA + FigC or R. pickettii NIS synthetase (R. pic syn). Enzymes are present at 2 μM with 3 mM Na-citrate and 10 mM L-ornithine (10 mM putrescine for the R. pickettii NIS synthetase). F, CAS siderophore detection from in vitro assays with purified recombinant F. novicida FigA + FigC (2 μM) with 3.0 mM Na-citrate and 10 mM indicated amines or amino acids. CAS, Chrome Azurol S; MFS, major facilitator superfamily.