Identifying paths to ammonium production in Vibrio natriegens by characterizing nitrogenase regulation

Regulation of nitrogenase, which converts nitrogen gas (N₂) into ammonium (NH₄⁺), typically involves a conserved set of regulatory proteins across diverse N₂-fixing (diazotrophic) bacteria. However, the interactions and relative influence of these regulators can vary between species. Thus, one cannot make assumptions about nitrogenase regulation when working with uncharacterized diazotrophs like Vibrio natriegens, a γ-proteobacterium of growing interest for synthetic biology. Little is known about V. natriegens nitrogenase regulation, which could be used to exploit inexpensive N₂ for various applications, including NH₄⁺ production. Here, we characterized the roles of several annotated V. natriegens nitrogenase regulatory proteins in response to NH₄⁺ versus N₂. Using functional genomics, targeted mutations, and reporter assays, we identified a typical regulatory hierarchy where the two-component system NtrBC governs a nitrogen-scavenging regulon that includes NifA, the transcriptional activator of nitrogenase genes. Unlike other diazotrophic γ-proteobacteria, NifA was sufficient to activate nitrogenase gene expression, as a mutant lacking NtrBC grew normally with N₂ after a lag phase. Thus, NtrBC was dispensable, but still important for timely nitrogenase expression. Furthermore, NtrBC was negatively regulated by the nitrogen-responsive P_II proteins GlnB and GlnK; disruption of both P_II proteins led to NtrBC-dependent nitrogenase overactivity, marked by NH₄⁺ excretion. The redundant repression of NtrBC by GlnB and GlnK more closely resembles that of non-diazotrophic E. coli than other diazotrophic γ-proteobacteria. Together, our findings provide a framework for V. natriegens nitrogenase regulation that can be leveraged for applications like NH₄⁺ production.