Low Levels of Amino Acid Auxotrophy Among Environmental Pseudomonas Isolates.

Auxotrophy, the inability of bacteria to synthesize one or multiple essential metabolites, is thought to be common among bacteria. However, studies often rely on either bioinformatic genome-based prediction of auxotrophies or on experiments with low strain numbers. Here, we combine experimental and bioinformatic approaches to assess amino acid auxotrophies among 315 co-isolated natural Pseudomonas strains from pond and soil habitats. Experiments revealed that Pseudomonas isolates are predominantly prototrophs. We identified one histidine auxotroph, one auxotroph with complex intertwined dependencies and four 'fragile' phenotypes showing delayed growth without supplemented amino acids. Applying three bioinformatic pipelines largely confirmed experimental data but yielded specific biases in auxotrophy over- or underestimation. Moreover, none of the pipelines could resolve the genetic basis of the slow-growing phenotypes or of more complex dependencies. Our analysis further revealed the existence of multiple alternative biosynthesis pathways for methionine, proline and phenylalanine, with significant pathway enrichments being linked to phylogeny and habitat. We conclude that combining experiments with bioinformatics is a powerful approach to assess the metabolic potential of environmental bacteria. Moreover, taxa like Pseudomonas can be predominantly prototrophic possibly owing to their generalist lifestyle, thus calling for nuanced ecological concepts predicting auxotrophy levels based on lifestyle and habitat.