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

Figure 1.

Figure 1

Production of rhizoferrin by the Ralstonia pickettii NIS synthetase in E. coli and in vitro.A, gene clusters containing the NIS synthetase and major facilitator superfamily (MFS) transporter in Ralstonia pickettii and homologs in Francisella novicida. GenBank protein accession numbers are shown below each ORF, and length of each putative encoded protein product is shown in parentheses. DAPDC, a PLP-dependent decarboxylase annotated by GenBank as meso-diaminopimelate decarboxylase. B, CAS activity in liquid culture supernatant of E. coli BL21 containing pETDuet-1 expressing either the R. pickettii NIS synthetase (R. pic synth, WP_012761004), MFS transporter (R. pic trans, WP_012761003), or both (R.pic synth + R. pic trans), or the R. pickettii NIS synthetase and F. novicida figB MFS transporter WP_003041320 (R. pic synth + F. nov trans). C, CAS activity of the strains shown in Figure 1B after growth on CAS plates. D, mass spectrum of partially purified siderophore from LB growth medium culture supernatant of E. coli BL21 expressing the R. pickettii NIS synthetase and MFS transporter from pETDuet-1. A mass corresponding to rhizoferrin (m/z 437) and its sodium adduct (m/z 459) are detected. E, in vitro siderophore biosynthesis and substrate preference of the purified recombinant R. pickettii NIS synthetase. CAS siderophore detection from in vitro assays of 1.5 μM NIS synthetase with 10 mM indicated substrates. The relevant masses of rhizoferrin and homorhizoferrin detected in these assays are shown in Figure S1. F, siderophore detection on CAS plates after growth of E. coli containing pETDuet-1 co-expressing the Legionella pneumophila figA homolog NIS synthetase (NIS, WP_010947056, first 19 amino acids removed) with R. pickettii MFS transporter (WP_012761003) or empty vector (replicates). The corresponding L. pneumophila NIS synthetase gene cluster is shown.