Wild type domestication: loss of intrinsic metabolic traits concealed by culture in rich media

Background: Bacteria are typically isolated on rich media to maximise isolation success, removing them from their native evolutionary context. This eliminates selection pressures, enabling otherwise deleterious genomic events to accumulate. Here we present a cautionary tale of these 'quiet mutations' which can persist unnoticed in bacterial culture lines. Methods: We used a combination of microbiological culture (standard and minimal media conditions), whole genome sequencing and metabolic modelling to investigate putative Klebsiella pneumoniae L-histidine auxotrophs. Additionally, we used genome-scale metabolic modelling to predict auxotrophies among completed public genomes (n=2,637). Results: Two sub-populations were identified within a K. pneumoniae frozen stock, differing in their ability to grow in the absence of L-histidine. These sub-populations were the same 'strain', separated by eight single nucleotide variants and an insertion sequence-mediated deletion of the L-histidine biosynthetic operon. The His- sub-population remained undetected for >10 years despite its in inclusion in independent laboratory experiments. Genome-scale metabolic models predicted 0.8% public genomes contained greater than or equal to 1 auxotrophy, with purine/pyrimidine biosynthesis and amino acid metabolism most frequently implicated. Discussion: We provide a definitive example of the role of standard rich media culture conditions in obscuring biologically relevant mutations i.e. nutrient auxotrophies, and estimate the prevalence of such auxotrophies using public genome collections. While the prevalence is low, it is not insignificant given the thousands of K. pneumoniae that are isolated for global surveillance and research studies each year. Our data serve as a pertinent reminder that rich-media culturing can cause unnoticed wild type domestication.