Genetic and Biochemical Analysis of the Azotobacter vinelandii Molybdenum Storage Protein

Abstract

The N₂ fixing bacterium <i>Azotobacter vinelandii</i> carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron-molybdenum cofactor (FeMo-co). The <i>in vitro</i> FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an <i>A. vinelandii</i> strain lacking the MoSto subunit genes (Δ<i>mosAB</i>) was analyzed. Consistent with its role as storage protein, the Δ<i>mosAB</i> strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased <i>in vivo</i> nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The Δ<i>mosAB</i> strain was found to readily derepress <i>vnfDGK</i> upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The Δ<i>mosAB</i> mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δ<i>vnf</i> Δ<i>anf</i>) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the Δ<i>mosAB</i> and <i>mosAB</i> ⁺ strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to <i>A. vinelandii</i> in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.