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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\n                  <jats:italic>Vibrio natriegens<\/jats:italic>\n                  , a \u03b3-proteobacterium of growing interest for synthetic biology. Little is known about\n                  <jats:italic>V. natriegens<\/jats:italic>\n                  nitrogenase regulation, which could be used to exploit inexpensive N\n                  <jats:sub>2<\/jats:sub>\n                  for various applications, including NH\n                  <jats:sub>4<\/jats:sub>\n                  <jats:sup>+<\/jats:sup>\n                  production. Here, we characterized the roles of several annotated\n                  <jats:italic>V. natriegens<\/jats:italic>\n                  nitrogenase regulatory proteins in response to NH\n                  <jats:sub>4<\/jats:sub>\n                  <jats:sup>+<\/jats:sup>\n                  versus N\n                  <jats:sub>2<\/jats:sub>\n                  . 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 \u03b3-proteobacteria, NifA was sufficient to activate nitrogenase gene expression, as a mutant lacking NtrBC grew normally with N\n                  <jats:sub>2<\/jats:sub>\n                  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\n                  <jats:sub>II<\/jats:sub>\n                  proteins GlnB and GlnK; disruption of both P\n                  <jats:sub>II<\/jats:sub>\n                  proteins led to NtrBC-dependent nitrogenase overactivity, marked by NH\n                  <jats:sub>4<\/jats:sub>\n                  <jats:sup>+<\/jats:sup>\n                  excretion. The redundant repression of NtrBC by GlnB and GlnK more closely resembles that of non-diazotrophic\n                  <jats:italic>E. coli<\/jats:italic>\n                  than other diazotrophic \u03b3-proteobacteria. Together, our findings provide a framework for\n                  <jats:italic>V. natriegens<\/jats:italic>\n                  nitrogenase regulation that can be leveraged for applications like NH\n                  <jats:sub>4<\/jats:sub>\n                  <jats:sup>+<\/jats:sup>\n                  production.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>HIGHLIGHTS<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>\n                        A genetic examination of\n                        <jats:italic>Vibrio natriegens<\/jats:italic>\n                        nitrogenase regulation is performed\n                      <\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>NtrBC is important for early nitrogenase gene expression but is not essential<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>NifA autoactivation is sufficient for nitrogenase expression<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>\n                        P\n                        <jats:sub>II<\/jats:sub>\n                        proteins GlnB and GlnK are redundant negative regulators of nitrogenase\n                      <\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>\n                        Genetic targets are identified that result in excretion of NH\n                        <jats:sub>4<\/jats:sub>\n                        <jats:sup>+<\/jats:sup>\n                      <\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>GRAPHICAL 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