Metabolic Footprinting of Mutant Libraries to Map Metabolite Utilization to Genotype

The discrepancy between the pace of sequencing and functional
characterization
of genomes is a major challenge in understanding complex microbial
metabolic processes and metabolic interactions in the environment.
Here, we identified and validated genes related to the utilization
of specific metabolites in bacteria by profiling metabolite utilization
in libraries of mutant strains. Untargeted mass spectrometry based
metabolomics was used to identify metabolites utilized by Escherichia coli and Shewanella oneidensis MR-1. Targeted high-throughput metabolite profiling of spent media
of 8042 individual mutant strains was performed to link utilization
to specific genes. Using this approach we identified genes of known
function as well as novel transport proteins and enzymes required
for the utilization of tested metabolites. Specific examples include
two subunits of a predicted ABC transporter encoded by the genes SO1043 and SO1044 required for the utilization
of citrulline and a predicted histidase encoded by the gene SO3057 required for the utilization of ergothioneine by S. oneidensis. In vitro assays with purified
proteins showed substrate specificity of SO3057 toward
ergothioneine and histidine betaine in contrast to substrate specificity
of a paralogous histidase SO0098 toward histidine.
This generally applicable, high-throughput workflow has the potential
both to discover novel metabolic capabilities of microorganisms and
to identify the corresponding genes.