Powering Nitrogen Fixation:  the Metabolism of the Aerobic Diazotroph,  Azotobacter Vinelandii, As Revealed By 13c-Metabolic Flux Analysis

Diazotrophs, or organisms capable of the enzymatic conversion of atmospheric nitrogen into ammonia, present a metabolic paradox.  On the one hand, nitrogen fixation is an energy-intensive process, requiring 16 moles of ATP and 8 moles of reducing equivalents per mole of N₂ fixed.  On the other hand, the nitrogenase enzyme is highly oxygen-labile, readily inactivated by oxidation of its metal cluster.  Therefore, while an anaerobic lifestyle would circumvent the issue of the oxygen sensitivity of the nitrogenase, low ATP yields from fermentation present a significant limitation to the net ammonium output of anaerobic diazotrophs.  Meanwhile, diazotrophs that are obligate aerobes (e.g. Azotobacter vinelandii, Azorhizobium caulinodans) are thought to cope instead by employing high respiration rates and protective membranes that limit the diffusion of oxygen.  One of the most well studied diazotrophs, Azotobacter vinelandii, is an aerobic, fast-respiring bacterium found in soils globally.  Although the biochemistry of this organism has been extensively documented, the features of its core metabolism that allow it to fix nitrogen and thrive in an aerobic environment have yet to be fully elucidated.

In this contribution, we have constructed a metabolic network model that captures the central carbon metabolism and amino acid biosynthesis pathways of A. vinelandii.  The model was thoroughly validated by performing complementary parallel labeling experiments using all singly labeled glucose tracers, that is, [1-¹³C]glucose through [6-¹³C]glucose and measuring isotopic labeling of biomass by GC-MS. Subsequent ¹³C-metabolic flux analysis (¹³C-MFA) confirmed three key aspects of the metabolism in this organism, namely that A. vinelandii:

(1)    exclusively employs the Entner-Doudoroff pathway for glucose catabolism.

(2)    synthesizes alanine from an unusual pathway involving cysteine instead of pyruvate.

(3)    diverts a significant portion of its carbon flux towards by-product formation and ATP generation rather than cell growth.

Additionally, for the first time, we present conclusive evidence that this diazotroph exhibits high flux through the TCA cycle during active nitrogen fixation, likely to supply reducing equivalents (and subsequently, ATP through the electron transport chain) for nitrogen fixation.  Furthermore, we have observed fairly high growth rates for this microbe during active nitrogen fixation, even in minimal media (0.20 to 0.31 h^-1). Also, by comparing metabolic fluxes for wild-type A. vinelandii and an engineered A. vinelandii strain that overproduces ammonium we obtained additional insights into the metabolism that supports nitrogen fixation. Finally, by performing tracer simulations, we were able to identify [1,6-¹³C]glucose paired with either [1,2-¹³C]glucose or [3,4-¹³C]glucose as the ideal tracers for routine studies of central carbon metabolism in A. vinelandii.

The knowledge gained from studying the core metabolism that enables microbial nitrogen fixation in A. vinelandii offer valuable insight for future efforts to engineer diazotrophs as more environmentally and economically favorable alternatives to man-made sources of fixed nitrogen (e.g. the Haber-Bosch process) for industrial fermentation and agricultural applications.