metabolic reconstruction and TribeMCL gene-family analysis for each reaction. (EPS) Figure S6 A phylogenetic tree of the diacylglycerol acyl transferases (DGATs). The closest match in R. opacus PD630 to the genes defined previously in R. jostii RHA1 are labeled with an atf gene number outside of the circular phylogenetic-tree [30] (originally named through identification in Acinetobacter baylyi). DGAT/WS proteins detected in each species represented as (species, number of homologous proteins): (R. opacus B4, 9), (R. opacus PD630, 14), (R. jostii RHA1, 16), (M. vanbaalenii PYR-1, 12), (M. sp JLS, 14), (M. tuberculosis H37Rv, 12), (M. leprae Br4923, 1). (EPS) Figure S7 Glucose fermentation analytics for R. opacus PD630 and R. jostii RHA1. Top panel plots shows decreases in ammonium sulfate in the culture media (blue) and increases in residual cell dry weight (red) (total cell weight – total lipids) during fermentation on glucose. Bottom panel plots show depletion of media glucose concentration and increasing total fatty acids (green). (EPS) Figure S8 GC and EI coupled mass spectra of pentadecanoic acid. GC-MS of C15:0 purified from Rhodococcus (GC-MS top panel, GC-EI-MS bottom panel). (EPS) Figure S9 GC and EI coupled mass spectra of heptadecanoic acid. GC-MS of C17:0 purified from Rhodococcus (GC-MS top panel, GC-EI-MS bottom panel). (EPS) Figure S10 GC and EI coupled mass spectra of heptadecenoic acid. GC-MS of C17:1 purified from Rhodococcus (GC-MS top panel, GC-EI-MS bottom panel). (EPS) Figure S11 1H-NMR of FAMEs of C15:0, C17:0, and cis-C17:1 purified from Rhodococcus. Control lipids were FAMEs of branched 16-methyl-heptadecanoate and linear pentadecanoate. (EPS) Figure S12 Screens of four bacterial species for growth on nitrogenous compounds. Nitrogen-containing compounds were clustered according to how R. opacus PD630, R. jostii RHA1, C. glutamicum 13032, and R. eutropha H16 were able to grow on them from 2–4 days. Yellow indicates evidence of growth. 38 Nitrogenous compounds including many amino acids were tested for growth. R. opacus PD630 catabolized 25 nitrogenous com- pounds as the sole carbon source (66%), R. jostii RHA1 catabolized 28 compounds (74%), C. glutamicum 8 (23%), and R. eutropha H16 11 (38%). The cyclical amino acid L-pyroglutamic acid was able to support growth in all species tested whereas the zwitterion L- glutamic acid was a weaker growth substrate. A notable feature of amino acid catabolism was observed for both Rhodococcus species and R. eutropha H16 contained the catabolic pathways for the branched amino acids, L-isoleucine, L-threonine, and L-valine catabolism. The catabolic pathways for branched amino acids result in the formation of propionoyl-CoA a substrate in the biosynthesis of stored odd-carbon lipids containing as presented here. (EPS) Figure S13 ORFs from genome annotation were searched for recognizable Pfam protein domains. The number of protein domains found within each species genomes are presented in a Genomics of Rhodococcus for Biofuels heat map with hierarchical-clustering clustering of species and rank-order of the most abundant domains in R. opacus PD630 (top to bottom). (EPS) Figure S14 The cholesterol degradation region of the R. opacus PD630 genome. The chromosomal region of R. opacus PD630 genome containing 6 clusters of cholesterol degradation genes were mapped from the R. jostii RHA1 genome [61]. At the top of each cluster there is a nucleotide ruler for the stitched R. opacus PD630 genome. Below that are the genes with name and an orientation. Below the genes is a contig marker in blue that shows one contig without gaps contains the cholesterol degradation region for all six gene clusters. Below the contig marker there are multiple sequence alignments showing blocks of sequence homology in green (top strand) and purple (bottom strand). The names of the species containing the homology sequence are shown within the block of homology. (EPS) Table S1 Correlation table for growth of R. opacus PD630 on 190 chemical compounds between predictions from metabolic reconstruction and observed phenotypes. Correlation categories are named with respect to predictions from the metabolic reconstruction model of R. opacus PD630. A true positive encodes a complete compound degradation pathway in the metabolic reconstruction and the chemical compound supports growth of R. opacus PD630. A true negative lacks a complete compound degradation pathway and growth is not observed on that compound. A false negative lacks a complete degradation pathway but growth on the compound was observed. A false positive predicted growth but no growth was observed. The calculations and values for precison, recall, false-positive-rate, false-negative rate, specificity, and accuracy are detailed within the figure. The initial comparicyc_model metrics were compared to the more advanced opacus_working model metabolic reconstruction by calculating a delta-by-refinement; wherein the opacus_comparicyc values were subtracted from the opacus_working values. (PDF) Table S2 Four species of bacteria were screened for growth on 190 chemical compounds. Worksheet 1 compound normalized and background subtracted growth values. Column A) compounds used as a sole source of carbon. Column B) Chemical Abstract Service (CAS) chemical identification number for compounds tested as growth substrates. Column C) the chemical category for the compound tested. Column D) the average values after subtraction of the chemicals in the wells without cells 44 hours after inoculation of R. opacus PD630. Column E) the standard deviation of the compound subtracted values after 44 hours of growth. Column F) background subtracted growth wherein the average values of six negative controls that contain inoculated cells but no carbon growth substrate were used to further subtract 590 nm absorbance that is independent of growth on each carbon source. Columns (G–I) are processed in the same way for the 72 hour time point as was done for the 44 hour time points (Columns D–F). The 96 hour time points are represented in columns (J–L). R. jostii RHA1 growth value measurement for time points 44, 72, and 96 hours are presented in Columns (O–U). C. glutamicum 13032 growth value measurement for time points 44, 72, and 96 hour time points are presented in (Columns V-AD). R. eutropha H16 growth value measurement for time points 44, 72, and 96 hour time points are presented in Columns (AE–AM). Worksheet 2 presents the values from worksheet 1 ranked for R. opacus PD630 background subtracted growth values at the 96 hour time point. Worksheet 3 presents the values from worksheet 1 PLoS Genetics | www.plosgenetics.org 16 September 2011 | Volume 7 | Issue 9 | e1002219