Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus.

Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterized the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies, and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviors of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.IMPORTANCENitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. The production of ammonia by diazotrophic organisms, harboring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, although we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterized the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centers. In addition, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviors.

Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria.

Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular repABC-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, Dinoroseobacter shibae DFL 12 and Rhodobacter capsulatus B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; Ensifer meliloti 2011 and "Agrobacterium fabrum" C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (Rhodospirillum rubrum S1) as well as in one member of Caulobacterales (Caulobacter vibrioides CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in R. capsulatus.