acetonitrile (MeCN) and 2% DCM, the program ramps to 60% MeCN linearly by 100 minutes, holds at that ratio until 105 minutes, then ramps to 100% DCM by 110 minutes holding until 115 min, and returns to the starting conditions by 120 minutes. Methyl 12-methyltetradecanoate (C15:0) and Methyl 16-methylheptadecanoate (C18:0) branched FAMEs standards (Sigma) were used to determine the HPLC retention times of analytes of interest. Fractions were collected for each FAME species: 17:1 eluted at 12.8 minutes, C 15:0 eluted at 13.5, C17:0 eluted at 16.6 minutes for subsequent GC-EI-MS and 1H-NMR analysis. TLC purification of TAGs was performed as previously described for single solvent system [5] followed by scraping of the TAG species that were detected by water staining. Scraped TAGs were extracted with 1:1 (vol/vol) chloroform methanol for 1 Hr prior to filtration with 0.2 mM PTFE membrane (VWR International). Extracts were dried under nitrogen then subjected to transesterification and analyzed by (GC-FID) [5]. TAGs Cycle Comparative Analysis R. opacus PD630 TAGs cycle genes were identified through metabolic reconstruction and literature-based curation. The genes assigned to biochemical reactions within the TAGs cycle were analyzed to identify all significant gene pairs (BLASTP, E, = 1e-5) followed by Markov clustering as implemented by TribeMCL [42]. Candidate gene families were then aligned using MAFFT [77] and manually curated in Jalview 2.6.1 [79] using neighbor joining trees and curation of alignments. Because we were classifying by EC number which is a broad classification of function and not necessarily homology, a strict cutoff for percent overlap or percent similarity was not used to retain as many members of particular EC category as annotated in KEGG (http://www.genome.jp/kegg/). A full list of curated EC # related Gene families implicated at each biochemical step of the TAGs cycle are represented with 11 other bacterial species in Table S4. Multiexperiment viewer [87] was used to hierarchically cluster the species in the TAGs cycle and visualize in a heat map. Clustering was pearson correlated with average linkage. The threshold for color-saturation was set to 15 genes per reaction category. Phenotype Growth Assays on 190 Compounds Chemical compounds capable of supporting growth as a sole source of carbon were identified using a tetrazolium-based growth assay developed by Biolog Incorporated; wherein growth of cells and aerobic respiration were measured by Dye D (Biolog, Inc) reduction resulting in purple color and turbidity read at 590 nm absorbance in Fluorostar plate reader. Innoculation cultures were grown for 1 day on LB agar plates at 30uC from which 3.8E23 ODu/well were transferred from 0.5 ml water resuspensions after measurement in nanodrop (Thermo Scientific) at 600 nm. During growth, plates were wrapped in aluminum foil and measured at 44, 72, and 96 hours. The measured A590 values were normalized by first subtracting values from uninoculated plates. The average of normalized values were background subtracted with average values of inoculated wells that lacked a carbon source (negative control). The background subtracted growth values were separated into chemical categories by filtering compound classifications in Excel. The normalized growth values for each compound were clustered using Cluster 3.0 [88] with centered correlation. Heat maps were generated in Java TreeView 1.1.3 [88]. Growth substrates were identified at 96 hours incubation for compounds that had A590 values .0.2. Genomics of Rhodococcus for Biofuels Supporting Information Dataset S1 Multiple sequence alignment of Rhodococcus FAS type 1a protein. To visualize multiple sequence alignments of FAS type 1a protein 1) download jalview http://www.jalview.org/ 2) open Jalview 3) input alignment file: FAS_type1a_MSA.fa (Dataset S1) 4) load features features/annotations FAS_protein_domains_ jalview.txt (Dataset S2). (TXT) Dataset S2 Protein domain annotation of FAS type 1a synthase. Load onto multiple sequence alignment according to instructions presented in Dataset S1. (TXT) Figure S1 Extensive synteny between three Rhodococcus species. Alignment of the assembled and stitched R. opacus PD630 draft genome with R. jostii RHA1 (top panel) and R. opacus B4 (bottom panel). Green lines indicate the end of completed genome replicons and purple lines show the ends of R. opacus PD630 assembled scaffolds that were aligned and orientated manually. (EPS) Figure S2 Outline of metabolic reconstructions steps used to generate the Opacuscyc14.5_working model. (EPS) Figure S3 Genomic and metabolic features of a comparative set of bacterial species. Left panel is a single-copy phylogenetic tree built from a common set of 701 genes present in all of the genomes of these select Actinomycetales. The 701 genes were compared at the DNA sequence level to establish phylogenetic relationships for each species. The bootstrap values are represented adjacent to the nodes. The branch length (scale bar) reflects the average number of substitutions/nucleotide. In the right panel, the predicted proteome from each organism was functionally categorized by protein homology according to KEGG orthology. The heat map represents hierarchical clustering of the number of genes in each category with the set of species being compared. Color scale bar on bottom indicates number of different genes in each category with a range from 0 to .400 genes. The bottom panel chart presents general genomic features of a comparative set of species. (EPS) Figure S4 Phylogenetic trees of acetyl- and propionoyl-CoA carboxylases. Closest matches of R. opacus PD630 proteins to AccA and AccD proteins that have been functionally character- ized in Mycobacterium are designated with the number of each isozyme (AccA1,2,3 and AccD1,2,3,4,5,6). AccA proteins detect- ed in each species represented as (species, number of homologous proteins): (R. opacus B4, 11), (R. opacus PD630, 12), (R. jostii RHA1, 11), (M. vanbaalenii PYR-1, 5), (M. sp JLS, 6), (M. tuberculosis H37Rv, 4), (M. leprae Br4923, 1). AccD proteins detected in each species represented as (species, # of homologous proteins): (R. opacus B4, 20), (R. opacus PD630, 21), (R. jostii RHA1, 21), (M. vanbaalenii PYR-1, 12), (M. sp JLS, 13), (M. tuberculosis H37Rv, 9), (M. leprae Br4923, 4). (EPS) Figure S5 The biochemical reactions of the TAGs cycle. The TAG metabolic cycle consists of 5 phases: 1) initiation to make acyl primers, 2) elongation by FAS to make C16–C26 fatty acyl- CoAs, 3) transesterification of fatty acyl-CoAs with glycerol, 4) hydrolysis of fatty acids from glycerol, and 5) b-oxidation of the released fatty acyl-CoAs. The EC numbers are used to describe the biochemical reactions and the color scheme, similar to Figure 3, indicates the number of genes implicated through PLoS Genetics | www.plosgenetics.org 15 September 2011 | Volume 7 | Issue 9 | e1002219