Nitrogen (N2) fixation, or diazotrophy, supports a large part of primary production in oceans. Cultureindependent approaches highlighted the presence in abundance of marine non-cyanobacterial diazotrophs (NCD) but their ecophysiology remains elusive, mostly because of the low number of isolated NCD and because of the lack of available genetic tools for these isolates. Here, a dual genetic and functional approach allowed unveiling the ecophysiology of a marine NCD affiliated to the species Vibrio diazotrophicus. Physiological characterization of the first marine NCD mutant obtained so far was performed using a soft-gellan assay, demonstrating that a ΔnifH mutant in not able to grow in nitrogenfree media. Furthermore, we demonstrated that V. diazotrophicus produces a thick biofilm under diazotrophic conditions, suggesting biofilm production as an adaptive response of this NCD to cope with the inhibition of nitrogen-fixation by molecular oxygen. Finally, the genomic signature of V. diazotrophicus is essentially absent from metagenomic data of Tara Ocean expeditions, despite having been isolated from various marine environments. We think that the genetically tractable V. diazotrophicus strain used in this study may serve as an ideal model to study the ecophysiology of these overlooked procaryotic group.

Nitrogen (N2) fixation, or diazotrophy, supports a large part of primary production in oceans. In these environments, diazotrophic cyanobacteria are known to largely contribute to N2 fixation. However, culture-independent approaches highlighted the presence in abundance of non-cyanobacterial diazotrophs (NCD) in all open oceans and in estuarine environments. Unfortunately, the ecophysiology of marine NCD remains elusive, mostly because of the low number of isolated NCD and because of the lack of available genetic tools for these isolates. Here, a dual genetic and functional approach allowed unveiling the ecophysiology of a marine NCD affiliated to the species Vibrio diazotrophicus. We developed genetic tools for this strain, leading to the obtention of the first genetic mutant of a marine NCD, deleted in its nifH gene. Physiological characterization of the mutant was performed using a soft-gellan assay, demonstrating that the nifH mutant in not able to grow in nitrogen-deprived media, while in trans complementation restores the wild type phenotype. Furthermore, we demonstrated here that V. diazotrophicus produces a thick biofilm under diazotrophic conditions, suggesting biofilm production as an adaptive response of this NCD to cope with the inhibition of nitrogen-fixation by molecular oxygen. Finally, the genomic signature of V. diazotrophicus is essentially absent from metagenomic data of Tara Ocean expeditions, despite having been isolated from various marine environments. This suggests that a hidden NCD diversity occurs in those environments, but unveiling this diversity requires additional sampling strategies. Given the increasing evidence that marine NCD play a major role in the biogeochemical cycle of nitrogen and carbon (by fueling the biological carbon pump), an in depth characterization of their ecophysiology is needed. We think that the genetically tractable V. diazotrophicus strain used in this study may serve as an ideal model to study the ecophysiology of these overlooked procaryotic group.