appropriate. Discrepancies in the final ORFs were resolved via manual review. Ribosomal RNAs (rRNAs) were identified with RNAmmer [70]. The tRNA features were identified using tRNAScan [71]. Other non-coding features were identified with RFAM [72]. Every annotated gene in the Rhodococcus opacus PD630 genome is assigned a locus number of the form OPAG_##### both at the Broad Institute web site and in GenBank with accession ABRH01000000. Stitching the Genome Assembly Based on Synteny The R. opacus PD630 V3 genome assembly contained 293 (contigs), containing 16 scaffolds longer than 2,000 residues. The 16 large scaffolds of the R. opacus PD630 genome, 10 of which were largely syntenic with R. jostii RHA1 and R. opacus B4 linear chromosomes, were ordered and oriented to reflect the observed synteny followed by the smaller contigs. Nucmer and mummerplot, components of the mummer 3.0 software package [67], were used to align the genomes and create graphical representations of those alignments. The mummerplot graph files were annotated to show boundaries between each of the scaffolds in the mummerplot graphs. Through manual inspection of these graphs, the syntenic order and orientation of these contigs were assembled into a stitched FASTA file with the addition of 500 N nucleotides at scaffold termini. Metabolic Reconstruction Enzymes for nine comparative species in Opacuscyc14.5_com- pare were computationally predicted using the EFICAz2 algo- rithm, enzyme prediction using gene-name matching, pathway prediction [24], transcription unit prediction [73], transporter prediction [74], and pathway hole filling [75] was performed with the Pathway Tools 14.5 software [24]. These databases can be accessed at (http://rhodocyc.broadinstitute.org). In addition, Opacuscyc14.5_working also predicted enzymes using homology to proteins with an EC# assignment in the database at Kyoto Encyclopedia of Gene and Genomes KEGG (http://www. genome.jp/kegg/). Subsequent literature-based manual curation was used to refine Opacuscyc14.5 TAGs biosynthesis and degradation cycle. Phylogenetic Analysis of FAS, MAS, PKS12, and DGAT Gene Families A set of directed gene pairs was generated by performing an all- against-all BLASTP search (min % aligned = 10 and e-va- lue,1e25) between a comparative set of genomes (all accession numbers available in Table S3). Genes were clustered using OrthoMCL [76] with a Markov inflation index of 1.5 and a maximum e-value of 1e25. Gene clusters were identified to which the M. tuberculosis FAS, MAS, PKS12, and DGAT genes belonged to. These clusters had all members plotted on the AMPHORA phylogenetic tree of the Actinobacteria [28]. In the phylogenetic analysis of FAS genes, we included three fungal FAS genes and two stramenopile FAS genes, while in the phylogenetic analysis of MAS-family genes we added four animal FAS genes. Amino acid sequences were aligned using MAFFT [77] using the E-INS-i method. A maximum likelihood phylogeny was estimated using the PROTGAMMABLOSUM62 model in RAxML [78] with 1000 bootstrap replicates. FAS type 1a protein alignments were visualized using Jalview [79] and domains were annotated based on predictions from the Conserved Domain Database [80]. Phylogenetic Resolution of Acc and DGAT Proteins A custom blast database containing seven Rhodococcus & Mycobacterium genomes, including R. opacus B4, R. opacus PD630, Genomics of Rhodococcus for Biofuels R. jostii RHA1, M. tuberculosis H37Rv, M. vanbaalenii PYR-1, M. sp JLS & M. leprae Br4923 was generated. Protein sequences annotated as either AccA or AccD within Mycobacterium tuberculosis H37Rv were identified and accessions extracted into separate lists, whereas the WS/DGAT protein accessions were identified from the R. jostii RHA1 genome annotation. Each set of amino acid sequences were blasted against the database using BLASTP with an expect threshold of 1E230. Unique matches were identified and whole sequences extracted for alignment with MUSCLE v3.7 [81], using a maximum of 24 iterations. Alignments were manually checked with ClustalX v2.0 [82], at which point WS/ DGAT sequences without any residues aligning to the proposed active site motif (H[L/S/P]xxxDG) [4] were rejected. The multiple sequence alignments were converted to phylip format for passing to ProtTest v2.4 [83], which determines the best-fit substitution model and produces a phylogenetic tree with maximum likelihood estimation, using PhyML v3.0 [84]. Newick formatted trees were represented with iTOL [85]. Mass and Structural Analysis of Odd-Carbon Lipids GC-MS analyses were conducted on a TraceGC Ultra DSQ mass spectrometer (Thermo Scientific) equipped with an AT-5 ms column from Alltech (60 m60.25 mm i.d. 60.25 mm df). The injector and transfer line were maintained at 280uC while the ion source was set at 180uC in electron ionization (EI) mode. High purity helium was used as carrier gas at a flow rate of 1 mL/ minute. The sample was injected onto the column in split mode and heated at 40uC for a minute. The GC oven temperature was increased to 280uC at 20uC per minute. The NMR spectra were recorded on a Varian Inova instrument, operating at 500 MHz for 1H and 125 MHz for 13C, equipped with a three channel, 5 mm, indirect detection probe, with z-axis gradients. The solvent was chloroform-d, and the temperature was 25uC. The chemical shifts for 1H were referenced to the residual solvent signal, 7.27 ppm on the tetramethylsilane scale. Proton spectra were acquired in 4 transients, with a 30u pulse, an acquisition time of 5 s and no relaxation delay. The intensity of the signals was referenced to the signal of the terminal methyl in the alkyl chain, at 0.88 ppm, 3H. Purifications of Odd-Carbon Lipids 100 ml cultures of R. opacus PD630 and R. jostii RHA1 were grown in defined media [5] with substitution of 1% propionate/ 0.056% NH4SO4 for 1 week at 30uC with agitation followed by addition of 1 gram of propionate from sterile 20% stock solution. 50 mls of culture was collected after 2 weeks by pelleting and freeze dried prior to lipid extraction. 50 mg of dried cells were transesterified in a 2 ml volume of 50% CHCl3/42.5% methanol/ 7.5% H2SO4 for 2.5 hours at 100uC in sealed 166125 ml glass tubes (Kimble Glass Co.). Rhodococcus fatty acid methyl esters (FAMEs) preps were then concentrated under a stream of N2, resuspended in dichloromethane (DCM), and analyzed (10-mL injections) using an Agilent 1050 High Performance Liquid Chromatography (HPLC; Agilent Technologies, Inc, Wilmington, DE USA) coupled to a Wakosil II RS-Prep C18 column (5 mm, 20 mm6250 mm; Wako Chemicals USA, Inc., Richmond, VA USA) and a Sedere Sedex 75 Evaporative Light Scattering Detector (ELSD; SEDERE, Alfortville Cedex, France) for determination of relevant fractionation range. The ELSD drift tube was set at 50uC, and the air nebulization pressure at 3.5 bar. The dual solvent system used is a linear gradient program based on the method published by Mansour [86], with an extended run time, and substitutes (DCM) for chloroform. Starting with 98% PLoS Genetics | www.plosgenetics.org 14 September 2011 | Volume 7 | Issue 9 | e1002219