A combined metabolomic and bioinformatic approach to investigate the function of transport proteins of the important pathogen Mycoplasma bovis

•Metabolite profiling of mycoplasma mutants investigated putative transport proteins.•The likely substrates of three transport proteins were identified.•The MBOVPG45_0533 protein is most likely a nucleotide sugar transporter.•The MBOVPG45_0568 protein appears to transport biopterin. Mycoplasma bovis is an economically important pathogen of the cattle industry worldwide, and there is an urgent need for a more effective vaccine to control the diseases caused by this organism. Although the M. bovis genome sequence is available, very few gene functions of M. bovis have been experimentally determined, and a better understanding of the genes involved in pathogenesis are required for vaccine development. In this study, we compared the metabolite profiles of wild type M. bovis to a number of strains that each contained a transposon insertion into a putative transporter gene. Transport systems are thought to play an important role in survival of mycoplasmas, as they rely on the host for many nutrients. We also performed 13C-stable isotope labelling on strains with transposon insertions into putative glycerol transporters. Integration of metabolomic and bioinformatic analyses revealed unexpected results (when compared to genome annotation) for two mutants, with a putative amino acid transporter (MBOVPG45_0533) appearing more likely to transport nucleotide sugars, and a second mutant, a putative dicarboxylate/amino acid:cation (Na+ or H+) symporter (DAACS), more likely to function as a biopterin/folate transporter. This study also highlighted the apparent redundancy in some transport and metabolic pathways, such as the glycerol transport systems, even in an organism with a reduced genome. Overall, this study highlights the value of metabolomics for revealing the likely function of a number of transporters of M. bovis.