(Figure 6) suggesting that this species has a more diverse hydroxy- acid metabolism. R. opacus PD630, R. jostii RHA1, and R. eutropha H16 grew well on the C10 dicarboxylate sebacic acid suggesting that b-oxidation of these longer chain dicarboxylates provides rapid growth. b- oxidation could also explain the growth on Tween 40 and Tween 80 compounds by Rhodococcus and Ralstonia (Figure 6). The Tween compounds are converted to fatty acids upon ester-hydrolysis by cutinase proteins encoded for in the genomes of Actinomycetales [50]. Tween 20 supported growth of only R. eutropha H16 likely due to toxicity for the other species resulting from the hydrolytic release of a C12 fatty acid. C. glutamicum 13032 is limited in carboxylates catabolism as seen by the inability to catabolize sebacic acid and the longer chain Tween 40 and Tween 80 (Figure 6). A limited carboxylate catabolic-profile observed for C. glutamicum 13032 in our growth assay is consistent with genes missing for 3-hydroxyacyl-CoA dehydrogenase within the fatty acids b-oxidation pathway (Figure 3a). C. glutamicum Degrades Sialic Acid The identification of the sialic acid or N-acetylneuraminic acid, an abundant component of extracellular glycoproteins, as a growth substrate for the soil bacterium C. glutamicum 13032 (Figure 6) was unexpected because this pathway has only been described for bacteria that colonise animals [51,52]. Examination of the C. glutamicum 13032 genome reveals a likely sialic acid catabolic operon containing genes for a secreted sialidase (cg2935), a sialic acid ABC transporter (cg2937–2940) of the satABCD type seen in Haemophilus ducreyi [53] and a full set of catabolic genes (cg2928–9 and cg2931–3) genes that would allow C. glutamicum to degrade sialic acid to fructose-6-phosphate, pyruvate, and ammonia [51]. A related but incomplete catabolic operon is seen in C. diptheriae NCTC 13129. The sialic acid catabolic genes are not conserved in the other species tested nor were related transporters shown to facilitate uptake of sialic acid [54], consistent with these phenotypic results. Diverse Nitrogen Metabolism in Rhodococcus Rhodococcus displayed the most diverse catabolism of nitrogenous compounds in our species-comparative time course growth assays. Clustered heats maps relating the time course growth of 38 nitrogenous compounds tested. 26 nitrogenous compounds supported growth in at least one of the species tested (Figure S12). A notable feature of the Rhodococcus species nitrogenous metabolism is the ability to catabolize the branched amino acids that result in the formation of propionoyl-CoA thereby establish- ing a cycle of propionoyl-CoA in carbon storage and amino acid catabolism (http://tinyurl.com/propionyl-CoA). A ranked order list of growth values on 190 compounds for each species in the screen is presented in Table S2. Expanded Families of Membrane Transport Proteins in R. opacus PD630 Comparison of the protein domains (Pfam database, Sanger Institute UK) within a set of related Actinomycetales and the Gram-negative outlier R. eutropha H16 revealed that the R. opacus PD630 genome contained 3 membrane transport protein families in the top 9 expanded families of total encoded protein domains (Figure S13). The membrane transport major facilitator super family (MFS) is the most prevalent Pfam domain in R. opacus PD630. There are 229 MFS genes in R. opacus PD630 as compared to 176 in R. jostii RHA1, 104 in R. eutropha H16, and 47 in C. glutamicum 13032 (Figure S14). The high relative number of Genomics of Rhodococcus for Biofuels membrane transporters likely enables the broad catabolism we observed in R. opacus PD630 and R. jostii RHA1. Cholesterol Degradation by a Large Genomic Region The ability to degrade sterols is shared between Rhodococcus and Mycobacterium. Following intravenous injection, M. tuberculosis has been observed to colonize lung tissue that is rich in lipid bodies and cholesterol crystals [55]. Genetic analysis of M. tuberculosis cholesterol catabolic pathways showed this sterol to be an essential carbon source during M. tuberculosis infections in mouse lung models [56,57,58]. Complete sterols degradation requires catab- olism of both of the aliphatic branched side chain as well as the terpene polycyclic rings; however catabolism studies of M. tuberculosis showed that 14C labeling at the fourth position of the steroid A ring was released as CO2; whereas the label at position 26 within the sterol branched side chain was converted into phthiocerol dimycocerosate (PDIM) [57]. This study indicated that part of the sterol was being degraded for energy and the other was being used for assembly of cell wall associated branched-lipids. Sterol A and B ring degradation results in propionoyl-CoA and pyruvate while side chain degradation results in a 2:1 formation of propionoyl-CoA: acetyl-CoA. In M. tuberculosis the the propionoyl- CoA from cholesterol degradation is converted into methylmalo- nyl-CoA then incorporated into branched-fatty acids such as PDIM by the PKS12 MAS-family type 1b synthase [35,37,43,44,59]. A large region (,0.28 Mb) of Rhodococcus chromosomal DNA has been identified through transcriptomic and genetic analysis of R. jostii RHA1 grown on cholesterol as a sole source of carbon [60]. Within this large chromosomal region, there are six clusters of genes that encode for the multiple enzymes dedicated to sterol degradation including: membrane transport, side chain degrada- tion, sterol A and B ring degradation, and sterol C and D ring degradation, for recent review [61]. We performed whole genome alignments between R. opacus PD630, R. jostii RHA1, and R. opacus B4, M. sp JLS, M. vanbaalenii PYR-1, M. smegmatis MC2 155, M. tuberculosis H37Rv, C. glutamicum 13032, and S. avermitilis MA 4680 to evaluate gene conservation within the six gene clusters implicated in cholesterol degradation (Figure S14). We found extensive conservation in the cholesterol degradation genes between Mycobacterium and Rhodococcus as has been reported previously [60]. Our analysis indicates Streptomyces contains many of the key genes for cholesterol degradation but is lacking homologues to the Mce sterol-transport genes [57]. C. glutamicum 13022 did not contain the genes implicated for cholesterol degradation. The only major difference found between the previously described sterol degradation chromosomal region in R. jostii RHA1 and R. opacus PD630 is the presence of a transposase gene in gene cluster 2 (OPAG_09155). Discussion The Rhodococcus genomes encode multiple biosynthetic pathways for making lipids and expanded gene families within those pathways that contribute to the diversity and abundance of lipid products seen with some Actinomycetales. We demonstrated that Rhodococcus TAGs contain the uncommon straight-chain odd- carbon lipids pentadecanoate, heptadecanoate, and heptadeceno- ate by mass and structural determination. The high abundance of the straight-chain odd-carbon lipids are yield-controllable through feeding of the three carbon organic salt propionate. Propionate is converted to propionoyl-CoA, a metabolite that is generated during the catabolism of the branched amino acids isoleucine, valine, and threonine as well as sterols. Propionoyl-CoA levels PLoS Genetics | www.plosgenetics.org 11 September 2011 | Volume 7 | Issue 9 | e1002219