Metabolic mechanisms of nitrogen substrate utilisation in three rhizosphere bacterial strains investigated using quantitative proteomics

Nitrogen metabolism in the rhizosphere microbiome plays an important role in mediating plant nutrition, particularly under low inputs of mineral fertilisers. However, there is relatively little mechanistic information about which genes and metabolic pathways are induced by rhizosphere bacterial strains to utilise diverse nitrogen substrates. Here we investigate nitrogen substrate utilisation in three taxonomically diverse bacterial strains previously isolated from Arabidopsis roots. The three strains represent taxa that are consistently detected as core members of the plant microbiome: Pseudomonas, Streptomyces and Rhizobium. We use phenotype microarrays to determine the nitrogen substrate preferences of these strains, and compare the experimental results versus computational simulations of genome-scale metabolic network models obtained with EnsembleFBA. Results show that all three strains exhibit generalistic nitrogen substrate preferences, with substrate utilisation being well predicted by EnsembleFBA. Using label-free quantitative proteomics, we document hundreds of proteins in each strain that exhibit differential abundance values following cultivation on five different nitrogen sources: ammonium, glutamate, lysine, serine and urea. Proteomic data show that the three strains use different metabolic strategies to utilise specific nitrogen sources. One diverging trait appears to be their degree of proteomic flexibility, with Pseudomonas sp. Root9 utilising lysine nutrition via widespread protein-level alterations to its flexible metabolic network, whereas Rhizobium sp. Root491 shows relatively stable proteome composition across diverse nitrogen sources. Our results give new protein-level information about the specific transporters and enzymes induced by diverse rhizosphere bacterial strains to utilise organic nitrogen substrates.\n\nImportanceNitrogen is the primary macronutrient required for plant growth. In contemporary agriculture, the vast majority of nitrogen is delivered via mineral fertilisers, which have undesirable environmental consequences such as waterway eutrophication and greenhouse gas production. There is increasing research interest in designing agricultural systems that mimic natural ecosystems, where nitrogen compounds are cycled between plants and soil, with the mineralisation of recalcitrant soil organic-N molecules mediated via microbial metabolism. However, to date there is little mechanistic information about which genes and metabolic pathways are induced by rhizosphere bacterial strains to metabolise organic-N molecules. Here, we use quantitative proteomics to provide new information about the molecular mechanisms utilised by taxonomically diverse rhizosphere bacterial strains to utilise different nitrogen substrates. Furthermore, we generate computational models of bacterial metabolism from a minimal set of experimental information, providing a workflow that can be easily reused to predict nitrogen substrate utilisation in other strains.