Effects of synthetic large-scale genome reduction on metabolism and metabolic preferences in a nutritionally complex environment

The soil bacterium Sinorhizobium meliloti forms nodules on the roots of leguminous plants, where N 2 is reduced to ammonia. Its genome includes a 3.65 Mb chromosome, a 1.35 Mb pSymA megaplasmid, and a 1.68 Mb pSymB chromid. pSymA and pSymB constitute ~45 % of the genome and here a non-targeted approach was used to identify the metabolic consequences of the removal of these replicons. Polar and non-polar metabolites from wild-type, ∆pSymA, ∆pSymB, and ∆pSymAB cells and supernatants across a growth curve were analyzed by LC–HILIC–TOF–MS. 2008 metabolite features were identified in the extracellular metabolome of cells grown in LBmc containing yeast extract and casein hydrolysate. 1474 features were found from the intracellular metabolites of cells grown in minimal M9-sucrose medium. Analysis revealed both time and genotype influenced the metabolome, with the removal of pSymB having a much greater effect than the loss of pSymA. Strains lacking pSymB showed an increase in sugar, amino acid, and nucleotide metabolites in the intracellular metabolome, and the loss of pSymB clearly impaired the cell’s ability to catabolize exogenous amino acids. We conclude that despite the ability of wild-type, ∆pSymA, ∆pSymB, and ∆pSymAB strains to grow in both M9-sucrose and LBmc media, the removal of pSymA, and particularly pSymB, had clear and dramatic effects on the S. meliloti metabolome. The larger effect associated with the pSymB chromid is consistent with the large number of metabolic genes on this replicon and the greater genetic and metabolic integration of this replicon with the S. meliloti chromosome.