The Type VI secretion systems (T6SSs) allow bacteria to translocate effector proteins to other bacteria or to eukaryotic cells. However, little is known about the role of T6SS in endosymbiotic bacteria. In this work we describe the T6SS of Rhizobium etli Mim1, a bacteria able to effectively nodulate common beans. Structural genes and those encoding possible effectors have been identified in a 28-gene DNA region of R. etli Mim1 pRetMIM1f plasmid. Immunodetection of Hcp protein, a conserved key structural component of T6SS systems, indicates that this secretion system is active at high cell densities, in the presence of root exudates, and in bean nodules. Rhizobium etli mutants affected in T6SS structural genes produced plants with lower dry weight and smaller nodules than the wild-type strain, indicating for the first time that the T6SS plays a positive role in Rhizobium–legume symbiosis.

Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products.

The genes of the type VI secretion system (T6SS) from Rhizobium etli Mim1 (ReMim1) that contain possible effectors can be divided into three modules. The mutants in them indicated that they are not required for effective nodulation with beans. To analyze T6SS expression, a putative promoter region between the tssA and tssH genes was fused in both orientations to a reporter gene. Both fusions are expressed more in free living than in symbiosis. When the module-specific genes were studied using RT-qPCR, a low expression was observed in free living and in symbiosis, which was clearly lower than the structural genes. The secretion of Re78 protein from the T6SS gene cluster was dependent on the presence of an active T6SS. Furthermore, the expression of Re78 and Re79 proteins in E. coli without the ReMim1 nanosyringe revealed that these proteins behave as a toxic effector/immunity protein pair (E/I). The harmful action of Re78, whose mechanism is still unknown, would take place in the periplasmic space of the target cell. The deletion of this ReMim1 E/I pair resulted in reduced competitiveness for bean nodule occupancy and in lower survival in the presence of the wild-type strain.

Biological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo- R -homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism Saccharomyces cerevisiae . NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated in vitro using only proteins expressed in S. cerevisiae . FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms.

Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo‐dependent, V‐dependent, or Fe‐only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N₂ binding and reduction, are, respectively, designated as FeMo‐cofactor, FeV‐cofactor, and FeFe‐cofactor. Fe‐only nitrogenase is a poor catalyst for N₂ fixation, when compared to the Mo‐dependent and V‐dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe‐only nitrogenase a gene product designated AnfO preserves the fidelity of Fe‐only nitrogenase by preventing the misincorporation of FeV‐cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N₂. These results are interpreted to indicate that AnfO controls the fidelity of Fe‐only nitrogenase maturation during the physiological transition from conditions that favor V‐dependent nitrogenase utilization to Fe‐only nitrogenase utilization to support diazotrophic growth.

The free-living diazotroph Azotobacter vinelandii produces three genetically distinct but functionally and mechanistically similar nitrogenase isozymes, designated as Mo-dependent, V-dependent, and Fe-only. They respectively harbor nearly identical catalytic cofactors that are distinguished by a heterometal site occupied by Mo (FeMo-cofactor), V (FeV-cofactor), or Fe (FeFe-cofactor). Completion of FeMo-cofactor and FeV-cofactor formation occurs on molecular scaffolds prior to delivery to their catalytic partners. In contrast, completion of FeFe-cofactor assembly occurs directly within its cognate catalytic partner. Because hybrid nitrogenase species that contain the incorrect cofactor type cannot reduce N₂ to support diazotrophic growth, there must be a way to prevent misincorporation of an incorrect cofactor when different nitrogenase isozyme systems are produced at the same time. Here, we show that fidelity of the Fe-only nitrogenase is preserved by blocking the misincorporation of either FeMo-cofactor or FeV-cofactor during its maturation. This protection is accomplished by a two-domain protein, designated AnfO. It is shown that the N-terminal domain of AnfO binds to an immature form of the Fe-only nitrogenase and the C-terminal domain, tethered to the N-terminal domain by a flexible linker, has the capacity to capture FeMo- and FeV-cofactor. AnfO does not prevent the normal activation of Fe-only nitrogenase because completion of FeFe-cofactor assembly occurs within its catalytic partner and, therefore, is never available for capture by AnfO. These results support a post-translational mechanism involving the molecular sorting of structurally similar metallocofactors that involve both protein-protein interactions and metallocofactor binding while exploiting differential pathways for nitrogenase associated catalytic cofactor assembly.