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. 2021 Jun 10;17(6):e1009617. doi: 10.1371/journal.pgen.1009617

Fig 4. Reciprocal amino acid changes (related to nifAE356K in A. vinelandii) yield constitutively active NifA in Proteobacteria.

Fig 4

(A) Diagram of the A. vinelandii NifA domains. (B) Alignment of residues close to E356 (black arrow) in the central AAA+ domain of NifA proteins regulated by NifL. Sequence numbers refer to A. vinelandii NifA. Sequences used in the alignment are Av: A. vinelandii DJ, Ps: Pseudomonas stutzeri A1501 (Gammaproteobacteria), Ao: Azoarcus olearius DQS4, Sl: Sideroxydans lithotrophicus ES-1 (Betaproteobacteria), Me: Martelella endophytica YC6887 (Alphaproteobacteria) and Mf: Mariprofundus ferrooxydans M34 (Zetaproteobacteria). Panels C to E show β-galactosidase activities in the E. coli ET8000 chassis resulting from activation of a nifH::lacZ fusion (plasmid pRT22) by wild type and variant NifL-NifA systems from three different diazotrophs. Plasmids used to express NifL-NifA variants are as follows. (C) pPR34: Av-NifL-NifA, pPMA: Av-NifL-NifA-E356K; (D) pMB1804: Ps-NifL-NifA, pMB1805: Ps-NifL-NifA-E356K; (E) pMB1806: Ao-NifL-NifA, pMB1807: Ao-NifL-NifA-E351K. The assays were performed in NFDM media supplemented with 2% glucose in either nitrogen-limiting (200 μg/ml of casein hydrolysate, -N) or nitrogen excess (7.56 mM ammonium sulphate, +N) conditions.