Galactose Metabolism Distinguishes Rhodococcus opacus PD630 The compound-specific growth assays indicated that galactose and oligogalactoside metabolisms differ between R. jostii RHA1 and R. opacus PD630 (Figure 5b). The ability of R. opacus PD630 to metabolize galactose enables efficient growth on the oligoga- lactosides lactose, lactitol, melibionic acid, melibiose, lactulose, raffinose, and stachyose (Figure 5a). A search of our metabolic reconstruction for the genetic basis of the galactose phenotypic differences between R. opacus PD630 and R. jostii RHA1 led to identification of a galactose-catabolic region of Rhodococcus genomes that is shared between the two R. opacus species B4 and PD630, but not in the closely related R. jostii RHA1 species. Two divergent polycistrons in the galactose catabolic region are fully syntenic between the R. opacus PD630 and R. opacus B4 species that contain the hydrolytic a- and b-galactosidases as well as solute binding protein (SBP), solute binding protein transport- ers (SBPT), and transcription regulatory protein of the DeoR family (Figure 5c) that could collectively hydrolyze and transport mono- and oligo-galactosides supporting growth on these compounds. Genomics of Rhodococcus for Biofuels Rhodococcus Degrades a Diverse Pool of Carboxylic Acids Carboxylic acids appear to be an important carbon source for Rhodococcus and R. eutropha H16. To determine the utilization of carboxylic acids as carbons source, we tested 64 carboxylic acids for growth using the previously described assay (Figure 6). These analyses led to the identification of 39 growth substrates for R. opacus PD630 and R. jostii RHA1 (61% of carboxylates tested), 15 growth substrates for C. glutamicum 13032 (21% of carboxylates tested), and 34 growth substrates for R. eutropha H16 (53% of carboxylates tested)(Figure 6). Acetic and propionic acids support growth in all species tested as did other common intermediates in central metabolism such as pyruvic, succinic, and citric acids. All of the species tested grew on gluconic acid that is degraded by the pentose phosphate pathway. Similar to the observed galacto-saccharide phenotypes, R. opacus PD630 uniquely degrades D-galactonic acid-g-lactone (Figure 6). By contrast, growth on the short chain hydroxy acid glycolic acid is specific to R. jostii RHA1 and R. eutropha H16 (Figure 6). Growth was observed for the longer chain a and b hydroxybutyric acids by both Rhodococcus species and R. eutropha H16, whereas weak growth on c hydroxybutyric acid was again only seen for R. jostii RHA1 Figure 6. Screens of four bacterial species for growth on carboxylate compounds (organic acids). Compounds were clustered as in Figure 5a. doi:10.1371/journal.pgen.1002219.g006 PLoS Genetics | www.plosgenetics.org 10 September 2011 | Volume 7 | Issue 9 | e1002219