Carbon substrate re-orders relative growth of a bacterium using Mo-, V-, or Fe-nitrogenase for nitrogen fixation

Abstract

Biological nitrogen fixation is catalyzed by the molybdenum (Mo), vanadium (V) and iron (Fe)-only nitrogenase metalloenzymes. Studies with purified enzymes have found that the 'alternative' V- and Fe-nitrogenases generally reduce N₂ more slowly and produce more byproduct H₂ than the Mo-nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotroph Rhodopseudomonas palustris, the type of carbon substrate influences the relative rates of diazotrophic growth based on different nitrogenase isoforms. The V-nitrogenase supports growth as fast as the Mo-nitrogenase on acetate but not on the more oxidized substrate succinate. Our data suggest that this is due to insufficient electron flux to the V-nitrogenase isoform on succinate compared with acetate. Despite slightly faster growth based on the V-nitrogenase on acetate, the wild-type strain uses exclusively the Mo-nitrogenase on both carbon substrates. Notably, the differences in H₂ :N₂ stoichiometry by alternative nitrogenases (~1.5 for V-nitrogenase, ~4-7 for Fe-nitrogenase) and Mo-nitrogenase (~1) measured here are lower than prior in vitro estimates. These results indicate that the metabolic costs of V-based nitrogen fixation could be less significant for growth than previously assumed, helping explain why alternative nitrogenase genes persist in diverse diazotroph lineages and are broadly distributed in the environment.

Figure 1

Electron balancing processes used by R. palustris during diazotrophic, photoheterotrophic growth. During photoheterotrophic growth, R. palustris consumes carbon substrates and converts them into biomass precursors and CO₂ (‘central metabolism,’ arrows A and C, respectively). This process reduces cellular electron carriers (arrow B), which must be re‐oxidized to maintain intracellular redox homeostasis and thereby enable continued carbon substrate assimilation. The electron sinks that stabilize the cellular electron pool are re‐fixation of some of the CO₂ produced during substrate assimilation (arrow E), synthesis of certain biomass precursors (arrow D), and Nase activity (arrow F). To achieve redox homeostasis, substrate‐derived electrons must be fully allocated between biomass, H₂ (arrow I) and CO₂ (arrow H). The cellular electron pool can also be re‐oxidized by extracellular electron acceptors like dimethyl sulfoxide (DMSO, arrow G).

Figure 2

A. R. palustris growth rates. The wild type strain, which uses the Mo‐Nase isoform, grows fast in all treatments. The V‐Nase strain grows faster than the Mo‐Nase utilizing wild type strain when provided with the more reduced carbon substrate, acetate, but not with the more oxidized substrate, succinate. It also grows faster than the Mo‐Nase mutant strain on acetate and butyrate + bicarbonate. B. Growth rate differences are linked to electron balance. The addition of an external electron acceptor, dimethyl sulfoxide (DMSO), diminishes the growth advantage of the V‐Nase strain on the more reduced carbon substrate acetate. In the absence of DMSO, the growth rates are significantly different within each carbon treatment (p < 0.05). However, in the presence of DMSO, the growth rates of the Mo‐ and V‐Nase strains are not significantly different. The error bars show the mean ± SE. C. R. palustris protein abundance dendrograms. Hierarchical clustering of all detected proteins and of the proteins annotated to COG H (coenzyme transport and metabolism) mimics the observed growth rate trends, demonstrating that, physiologically, on acetate, the wild‐type strain is more similar to the V‐Nase strain than the Mo‐Nase strain. From an energy acquisition perspective (COG C), the wild‐type strain is more similar to the V‐Nase strain than the Mo‐Nase strain on both succinate and acetate.

Figure 3

R. palustris biomass composition. The fastest‐growing strains are also the most nitrogen rich. The wild type and V‐Nase strains adjust their growth rates depending on the carbon substrate, whereas the Fe‐ and Mo‐Nase strains grow at similar rates in all treatments. Error bars show the mean ± SE.

Figure 4

A. Rates of N₂ and CO₂ fixation and H₂ production of R. palustris strains growing on succinate (left) and acetate (right). For H₂ production, the error bars represent the standard error of the slope when data from multiple biological replicates (n ≥ 2) are plotted together. For CO₂ and N₂ fixation rates, the error bars represent the standard error of multiple biological replicates (n ≥ 2). Different letters represent treatments that are significantly different at p < 0.05. B. Electron flux through Nase and Rubisco. The fraction of the electron flux through Nase versus Rubisco remains approximately constant despite differences in total flux and in the partitioning of electrons through Nase to proton versus N₂ reduction. C. Growth rate versus nitrogen fixation rate and total electron flux. The nitrogen fixation rate and the reductive electron flux through Nase and Rubisco scale with the growth rate. Error bars show the mean ± SE. Data for the Fe‐Nase strain are provided in the Supporting Information.