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 irondependent 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.

Comparative Genomics Reveal a Flagellar System, a Type VI Secretion System and Plant Growth-Promoting Gene Clusters Unique to the Endophytic Bacterium Kosakonia radicincitans

“…Although some of the underlying gene clusters have been studied in detail, the purpose of others, such as the anf operon, is still puzzling (Yang et al, 2014). On the other hand can some capabilities of bacteria not be explained by the presence or absence of genes per se .…”

Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

“…Bacterial strains used in this study are listed in Table S2. Media and antibiotics were used as described (28) and are detailed in SI Materials and Methods.…”

“…The C 2 H 2 reduction method was used to assay nitrogenase activity as described (28). Details are provided in SI Materials and Methods.…”

“…To detect 15 N 2 assimilation, E. coli JM109 derivatives expressing reassembled nitrogenase systems were grown as described (28). More details are provided in SI Materials and Methods.…”

Low‐Temperature N2 Binding to Two‐Coordinate L2Fe0 Enables Reductive Trapping of L2FeN2− and NH3 Generation

“…Building on extensive early Mo and W model work, [3] Schrock [4] and Nishibayashi [5] have reported Mo-containing coordination complexes (A and B in Figure 1, respectively) that facilitate catalytic N 2 reduction to NH 3 in the presence of suitable acids and reductants. Because Fe is (i) the only transition metal known to be essential to enzymatic nitrogenase function, [6] and (ii) the predominant transition metal catalyst used in the Haber-Bosch ammonia synthesis, [7] studying N 2 reduction chemistry at well-defined Fe model complexes is of interest. [8,9] Recently, our lab demonstrated that Fe coordination complexes (C in Figure 1) are capable of modest catalytic N 2 reduction to NH 3 .…”