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Nitrogenase-dependent H 2 production by photosynthetic bacteria, such as Rhodobacter capsulatus , has been extensively investigated. An important limitation to increase H 2 production using genetic manipulation is the scarcity of high-throughput screening methods to detect possible overproducing mutants. Previously, we engineered R. capsulatus strains that emitted fluorescence in response to H 2 and used them to identify mutations in the nitrogenase Fe protein leading to H 2 overproduction. Here, we used ultraviolet light to induce random mutations in the genome of the engineered H 2 -sensing strain, and fluorescent-activated cell sorting to detect and isolate the H 2 -overproducing cells from libraries containing 5 × 10 5 mutants. Three rounds of mutagenesis and strain selection gradually increased H 2 production up to 3-fold. The whole genomes of five H 2 overproducing strains were sequenced and compared to that of the parental sensor strain to determine the basis for H 2 overproduction. No mutations were present in well-characterized functions related to nitrogen fixation, except for the transcriptional activator nifA2 . However, several mutations mapped to energy-generating systems and to carbon metabolism-related functions, which could feed reducing power or ATP to nitrogenase. Time-course experiments of nitrogenase depression in batch cultures exposed mismatches between nitrogenase protein levels and their H 2 and ethylene production activities that suggested energy limitation. Consistently, cultivating in a chemostat produced up to 19-fold more H 2 than the corresponding batch cultures, revealing the potential of selected H 2 overproducing strains., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Barahona, Isidro, Sierra-Heras, Álvarez-Melcón, Jiménez-Vicente, Buesa, Imperial and Rubio.)

Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four Arabidopsis thaliana proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In A. thaliana , import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes., (Copyright © 2020 Eseverri, Baysal, Medina, Capell, Christou, Rubio and Caro.)

The N 2 fixing bacterium Azotobacter vinelandii carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron-molybdenum cofactor (FeMo-co). The in vitro FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an A. vinelandii strain lacking the MoSto subunit genes (Δ mosAB ) was analyzed. Consistent with its role as storage protein, the Δ mosAB strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased in vivo nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The Δ mosAB strain was found to readily derepress vnfDGK upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The Δ mosAB mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δ vnf Δ anf ) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the Δ mosAB and mosAB + strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to A. vinelandii in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.

One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (single-domain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation ( nif ) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (Δ nifB ) mutant restoring the Nif + phenotype and thus demonstrating their functionality in vivo . In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.

Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O 2 -sensitive S -adenosyl methionine (SAM)-radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium Azotobacter vinelandii and the thermophilic methanogen Methanocaldococcus infernus in Saccharomyces cerevisiae and in Nicotiana benthamiana . NifB proteins were targeted to the mitochondria, where O 2 consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe-S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the A. vinelandii version appeared to form aggregates. Similarly, NifB from M. infernus was expressed at higher levels in leaves of Nicotiana benthamiana and accumulated as a soluble protein while A. vinelandii NifB was mainly associated with the non-soluble cell fraction. Soluble M. infernus NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the in vitro FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening nif genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.