Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Abstract

All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium- or iron-dependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

Fig. 1.

Construction of the artificial FeFe nitrogenase system and measurement of nitrogenase activity. (A) Schematic map of the construction process of the artificial FeFe system. Nitrogenase activity of the artificial FeFe system was compared with nitrogenase activity of the MoFe system, as measured by the C₂H₂ reduction assay (B) or the ¹⁵N assimilation assay (C), respectively. Bars marked as MoFe represent the MoFe nitrogenase system (pKU7017), and bars marked as FeFe represent the artificial FeFe nitrogenase system (pKU7801). Error bars indicate the SD observed from at least three independent experiments.

Fig. 2.

Deletion analysis of the genes present in the artificial FeFe nitrogenase system. (A) Scheme showing the genetic organization of the artificial FeFe nitrogenase and its deletion derivatives. The black and white rectangles represent the deleted genes, and the colored rectangles represent the remaining genes. (B and C) Nitrogenase activity of the deletion mutants compared with the artificial FeFe nitrogenase system (pKU7801, bars marked as Parental) and the empty vector (pACYC184, bars marked as Vector) in E. coli JM109. Strains were grown anaerobically in nitrogen-deficient conditions and assayed for C₂H₂ reduction (B) or ¹⁵N assimilation (C), respectively. Error bars indicate the SD observed from at least three independent experiments.

Fig. 3.

Genetic analysis of the requirement for the nifENX or vnfENX gene. The C₂H₂ reduction activity of the minimal FeFe nitrogenase system and constructions in which either nifENX or vnfENX was also present were compared. The minimal FeFe nitrogenase system (pKU7815) is marked as “minimal.” The minimal FeFe nitrogenase system with three different kinds of additional ENX genes is represented as KpnifENX⁺ (pKU7819), AvnifENX⁺ (pKU7820), and AvvnfENX⁺ (pKU7821), respectively. The nitrogenase activity observed from the minimal system represents 100% activity. Error bars indicate the SD observed from at least three independent experiments.

Fig. 4.

Genetic analysis of the requirement for nifM. (A) Comparison of the C₂H₂ reduction activity of MoFe nitrogenase and the minimal FeFe system in E. coli strain JM109, in the presence or absence of nifM (solid and open bars, respectively). Activity on the y axis is represented as a percentage of either the nifM⁺ MoFe system (pKU7017, bars indicated as MoFe) or the nifM⁺ derivative of the minimal FeFe system (pKU7822, bars marked as “minimal”). The nifM⁻ equivalents of these constructs are pKU7017-ΔnifM and pKU7815, respectively (Table S1). (B) Influence of nifM on the expression of NifH and AnfH as measured by Western blotting of the cultures indicated in A. Sup, supernatant; WCL, whole cell lysate. (C) Relative nitrogenase activity of the minimal FeFe system (expressed from plasmid pKU7815) in different E. coli mutants. WT indicates strain JM109; ΔsurA, ΔppiC, ΔppiD, and ΔsurA/ΔppiD indicate deletion mutants of strain JM109. Error bars indicate the SD observed from at least three independent experiments.

Fig. 5.

Genetic analysis of nifF and nifJ orthologs in E. coli. Comparison of the C₂H₂ reduction activity of the minimal FeFe nitrogenase and its nifF/nifJ deletion derivatives in different genetic backgrounds. (A) Nitrogenase activity observed from the minimal FeFe system (pKU7815) and its nifF deletion derivative (pKU7823, marked as minimal, ΔnifF) in WT (JM109) and its fldB deletion derivative (represented by the solid and open bars), respectively. (B) Nitrogenase activity of the minimal FeFe system and its nifF deletion derivative (marked as minimal, ΔnifF) and its nifF deletion derivative with additional fldA or fldB carried on a plasmid (pKU7824, marked as minimal, ΔnifF, pfldA, and pKU7825, marked as minimal, ΔnifF, pfldB, respectively). (C) Nitrogenase activity observed from the minimal FeFe system, its nifJ deletion derivative (pKU7826, marked as minimal, ΔnifJ) and its nifJ deletion derivative with an additional plasmid carrying the ydbK gene (pKU7827, marked as minimal, ΔnifJ, pydbK) in WT (solid bars) and the ydbK deletion strain (open bars).