Comparative metabolomics of Mycoplasma bovis and Mycoplasma gallisepticum reveals fundamental differences in active metabolic pathways and suggests novel gene annotations

Mycoplasmas are simple, but successful parasites that have the smallest genome of any free-living cell and are thought to have a highly streamlined cellular metabolism. Here we have undertaken a detailed metabolomic analysis of two species, Mycoplasma bovis and Mycoplasma gallisepticum, which cause economically important diseases in cattle and poultry, respectively. Untargeted GC/MS and LC/MS analyses of mycoplasma metabolite extracts revealed significant differences in the steady state levels of many metabolites in central carbon metabolism, while 13C stable isotope labelling studies revealed marked differences in carbon source utilization. These data were mapped onto in silico metabolic networks predicted from genome wide annotations. The analyses elucidated distinct differences, including a clear difference in glucose utilisation, with a marked decrease in glucose uptake and glycolysis in M. bovis compared to M. gallisepticum, which may reflect differing host nutrient availabilities. The 13C-labeling patterns also revealed several functional metabolic pathways that were previously unannotated in these species, allowing us to assign putative enzyme functions to the products of a number of genes of unknown function, especially in M. bovis. This study demonstrates the considerable potential of metabolomic analyses to assist in characterising significant differences in the metabolism of different bacterial species, and in improving genome annotation.