ranked for R. jostii RHA1 background subtracted growth values at the 96 hour time point. Worksheet 4 presents the values from worksheet 1 ranked for C. glutamicum 13032 background subtracted growth values at the 96 hour time point. Worksheet 5 presents the values from worksheet 1 ranked for R. eutropha H16 background subtracted growth values at the 96 hour time point. (XLS) Table S3 Genebank accession numbers for species used in comparative analysis. Column1_ Species name Column 2) Genbank genome accession numbers for comparative set of species. (XLS) Table S4 TribeMCL analysis of TAGs cycle genes implicated through metabolic reconstruction. Column A) presents the TribeMCL cluster identification number containing all homolo- gous genes for 12 species. Column B) describes the bacterial species. Column C) presents the gene count for the whole TribeMCL analysis of the TAGs cycle. Column D) the EC References 1. McLeod MP, Warren RL, Hsiao WW, Araki N, Myhre M, et al. (2006) The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proc Natl Acad Sci U S A 103: 15582–15587. 2. Alvarez HM, Mayer F, Fabritius D, Steinbuchel A (1996) Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630. Arch Microbiol 165: 377–386. 3. Alvarez HM, Steinbuchel A (2002) Triacylglycerols in prokaryotic microorgan- isms. Appl Microbiol Biotechnol 60: 367–376. 4. Hernandez M, Mohn W, Martinez E, Rost E, Alvarez A, et al. (2008) Biosynthesis of storage compounds by Rhodococcus jostii RHA1 and global identification of genes involved in their metabolism. BMC genomics 9: 600. 5. Kurosawa K, Boccazzi P, de Almeida NM, Sinskey AJ (2010) High-cell-density batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production. Journal of Biotechnology 147: 212–218. 6. Hughes J, Armitage YC, Symes KC (1998) Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture. Antonie van Leeuwenhoek 74: 107–118. 7. Seto M, Kimbara K, Shimura M, Hatta T, Fukuda M, et al. (1995) A Novel Transformation of Polychlorinated Biphenyls by Rhodococcus sp. Strain RHA1. Appl Environ Microbiol 61: 3353–3358. 8. Robrock KR, Mohn WW, Eltis LD, Alvarez-Cohen L (2010) Biphenyl and ethylbenzene dioxygenases of Rhodococcus jostii RHA1 transform PBDEs. Biotechnol Bioeng. 9. Goncalves ER, Hara H, Miyazawa D, Davies JE, Eltis LD, et al. (2006) Transcriptomic assessment of isozymes in the biphenyl pathway of Rhodococcus sp. strain RHA1. Appl Environ Microbiol 72: 6183–6193. 10. Mathieu JM, Mohn WW, Eltis LD, LeBlanc JC, Stewart GR, et al. (2010) 7- ketocholesterol catabolism by Rhodococcus jostii RHA1. Appl Environ Microbiol 76: 352–355. 11. Puglisi E, Cahill MJ, Lessard PA, Capri E, Sinskey AJ, et al. (2010) Transcriptional response of Rhodococcus aetherivorans I24 to polychlorinated biphenyl-contaminated sediments. Microb Ecol 60: 505–515. 12. Peoples OP, Sinskey AJ (1989) Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). Journal of Biological Chemistry 264: 15298– 15303. 13. Slater SC, Voige WH, Dennis DE (1988) Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway. Journal of Bacteriology 170: 4431–4436. 14. Jendrossek D (2009) Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes). J Bacteriol 191: 3195–3202. 15. Rajakumari S, Grillitsch K, Daum G (2008) Synthesis and turnover of non-polar lipids in yeast. Progress in Lipid Research 47: 157–171. 16. Alvarez HM, A S (2010) Biology of Rhodococcus. Microbiology Monographs 16: 263–289. 17. Argyrou A, Vetting M, Blanchard J (2007) New insight into the mechanism of action of and resistance to isoniazid: interaction of Mycobacterium tuberculosis enoyl- ACP reductase with INH-NADP. Journal of the American Chemical Society 129: 9582–9583. 18. Timmins G, Deretic V (2006) Mechanisms of action of isoniazid. Molecular Microbiology 62: 1220–1227. 19. Raman K, Rajagopalan P, Chandra N (2005) Flux Balance Analysis of Mycolic Acid Pathway: Targets for Anti-Tubercular Drugs. PLoS Comput Biol 1: e46. doi:10.1371/journal.pcbi.0010046. Genomics of Rhodococcus for Biofuels number for the biochemical reactions within the TAGs cycle. Column E) LocusId is the gene identification (Broad) and locustag (NCBI). Column F) gene length. Column G) gene name. (TXT) Acknowledgments We would like to thank Christopher T. Walsh (Harvard), Edward J. Brignole (MIT), Gavin H. Thomas (York University) for helpful discussions and Maithreyan Srinivasan for sequencing project support at (454 Life Sciences). Author Contributions Conceived and designed the experiments: JWH PAG. Performed the experiments: JWH JCU ACD JZ QZ ALBL CAD PAG IG CD TA DG CDK BD JPA. Analyzed the data: JWH JCU ACD JZ QZ ALBL CAD PAG IG CD TA DG CDK BD JPA. Contributed reagents/materials/ analysis tools: BWB AJS. Wrote the paper: JWH. 20. Zimhony O, Cox J, Welch J, Vilcheze C, Jacobs W (2000) Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis. Nature Medicine 6: 1043–1047. 21. Schweizer E, Hofmann J (2004) Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems. Microbiology and molecular biology reviews: MMBR 68: 501–517. 22. Sutcliffe IC (1998) Cell envelope composition and organisation in the genus Rhodococcus. Antonie Van Leeuwenhoek 74: 49–58. 23. Hsu F-F, Soehl K, Turk J, Haas A (2011) Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization. Analytical Biochemistry 409: 112–122. 24. Karp PD, Paley SM, Krummenacker M, Latendresse M, Dale JM, et al. (2010) Pathway Tools version 13.0: integrated software for pathway/genome informatics and systems biology. Brief Bioinform 11: 40–79. 25. Arakaki A, Huang Y, Skolnick J (2009) EFICAz2: enzyme function inference by a combined approach enhanced by machine learning. BMC Bioinformatics 10: 107. 26. Kikuchi S, Rainwater DL, Kolattukudy PE (1992) Purification and character- ization of an unusually large fatty acid synthase from Mycobacterium tuberculosis var. bovis BCG. Archives of Biochemistry and Biophysics 295: 318–326. 27. Vance DE, Mitsuhashi O, Bloch K (1973) Purification and properties of the fatty acid synthetase from Mycobacterium phlei. J Biol Chem 248: 2303–2309. 28. Wu M, Eisen JA (2008) A simple, fast, and accurate method of phylogenomic inference. Genome Biol 9: R151. 29. Kalscheuer R, Steinbuchel A (2003) A novel bifunctional wax ester synthase/ acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278: 8075–8082. 30. Alvarez AF, Alvarez HM, Kalscheuer R, Waltermann M, Steinbuchel A (2008) Cloning and characterization of a gene involved in triacylglycerol biosynthesis and identification of additional homologous genes in the oleaginous bacterium Rhodococcus opacus PD630. Microbiology 154: 2327–2335. 31. Gande R, Dover LG, Krumbach K, Besra GS, Sahm H, et al. (2007) The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis. J Bacteriol 189: 5257–5264. 32. Portevin D, de Sousa-D’Auria C, Montrozier H, Houssin C, Stella A, et al. (2005) The acyl-AMP ligase FadD32 and AccD4-containing acyl-CoA carboxylase are required for the synthesis of mycolic acids and essential for mycobacterial growth: identification of the carboxylation product and determination of the acyl-CoA carboxylase components. J Biol Chem 280: 8862–8874. 33. Gande R, Gibson KJ, Brown AK, Krumbach K, Dover LG, et al. (2004) Acyl- CoA carboxylases (accD2 and accD3), together with a unique polyketide synthase (Cg-pks), are key to mycolic acid biosynthesis in Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis. J Biol Chem 279: 44847–44857. 34. Diacovich L, Peiru S, Kurth D, Rodriguez E, Podesta F, et al. (2002) Kinetic and Structural Analysis of a New Group of Acyl-CoA Carboxylases Found in Streptomyces coelicolor A3(2). J Biol Chem 277: 31228–31236. 35. Rainwater DL, Kolattukudy PE (1985) Fatty acid biosynthesis in Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin. Purification and characterization of a novel fatty acid synthase, mycocerosic acid synthase, which elongates n-fatty acyl-CoA with methylmalonyl-CoA. Journal of Biological Chemistry 260: 616–623. 36. Matsunaga I, Bhatt A, Young DC, Cheng TY, Eyles SJ, et al. (2004) Mycobacterium tuberculosis pks12 produces a novel polyketide presented by CD1c to T cells. J Exp Med 200: 1559–1569. PLoS Genetics | www.plosgenetics.org 17 September 2011 | Volume 7 | Issue 9 | e1002219