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Biosynthesis of storage compounds by Rhodococcus jostii RHA1 and global identification of genes involved… Hernández, Martín A; Mohn, William W.; Martínez, Eliana; Rost, Enrique; Alvarez, Adrián F; Alvarez, Héctor M Dec 12, 2008

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RESEARCH ARTICLE Open AccessBiosynthesis of storage compounds byRhodococcus jostii RHA1 and global identificationof genes involved in their metabolismMartín A Hernández1, William W Mohn2, Eliana Martínez1, Enrique Rost3, Adrián F Alvarez1,4, Héctor M Alvarez1*AbstractBackground: Members of the genus Rhodococcus are frequently found in soil and other natural environments andare highly resistant to stresses common in those environments. The accumulation of storage compounds permitscells to survive and metabolically adapt during fluctuating environmental conditions. The purpose of this study wasto perform a genome-wide bioinformatic analysis of key genes encoding metabolism of diverse storagecompounds by Rhodococcus jostii RHA1 and to examine its ability to synthesize and accumulate triacylglycerols(TAG), wax esters, polyhydroxyalkanoates (PHA), glycogen and polyphosphate (PolyP).Results: We identified in the RHA1 genome: 14 genes encoding putative wax ester synthase/acyl-CoA:diacylglycerol acyltransferase enzymes (WS/DGATs) likely involved in TAG and wax esters biosynthesis; a total of 54genes coding for putative lipase/esterase enzymes possibly involved in TAG and wax ester degradation; 3 sets ofgenes encoding PHA synthases and PHA depolymerases; 6 genes encoding key enzymes for glycogen metabolism,one gene coding for a putative polyphosphate kinase and 3 putative exopolyphosphatase genes. Where possible,key amino acid residues in the above proteins (generally in active sites, effectors binding sites or substrate bindingsites) were identified in order to support gene identification. RHA1 cells grown under N-limiting conditions,accumulated TAG as the main storage compounds plus wax esters, PHA (with 3-hydroxybutyrate and 3-hydroxyvalerate monomers), glycogen and PolyP. Rhodococcus members were previously known to accumulateTAG, wax esters, PHAs and polyP, but this is the first report of glycogen accumulation in this genus.Conclusion: RHA1 possess key genes to accumulate diverse storage compounds. Under nitrogen-limitingconditions lipids are the principal storage compounds. An extensive capacity to synthesize and metabolize storagecompounds appears to contribute versatility to RHA1 in its responses to environmental stresses.BackgroundMembers of the genus Rhodococcus are widely distribu-ted in natural environments, such as soil, water andmarine sediments [1]. They belong to the non-sporulat-ing and mycolic acid-rich group within the actinomy-cetes, together with other related genera, includingMycobacterium, Nocardia, Corynebacterium and Gordo-nia. The frequent occurrence of Rhodococcus sp. in aridsites like deserts around the world may reflect theiradaptation to environments with extreme conditions.These microorganisms developed metabolic strategies tocope with such environments where nutrient-limitationis common. One of these mechanisms may be the accu-mulation of storage compounds that can be utilized bycells as endogenous carbon sources and electron donorsduring periods of nutritional scarcity.Rhodococcus jostii RHA1 is a soil bacterium with theability to degrade and transform polychlorinated biphe-nyls and other aromatic compounds [2]. The completegenome of strain RHA1 is available for screening andidentification of genes and metabolic pathways. For thisreason, R. jostii RHA1 is a good model organism forunderstanding the genetics and physiology of storagecompound metabolism. Strain RHA1 possesses one of* Correspondence: halvarez@unpata.edu.ar1Centro Regional de Investigación y Desarrollo Científico Tecnológico(CRIDECIT), Facultad de Ciencias Naturales, Universidad Nacional de laPatagonia San Juan Bosco, Km 4-Ciudad Universitaria, 9000 ComodoroRivadavia, Chubut, ArgentinaFull list of author information is available at the end of the articleHernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600© 2008 Hernández et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.the largest bacterial genomes sequenced to date,containing 9.7 Mbp arranged in a linear chromosome(7,802,028 bp) and three linear plasmids: pRHL1(1,123,075 bp), pRHL2 (442,536 bp) and pRHL3(332,361 bp) [3].The accumulation of storage lipids by actinomycetes,like members of Mycobacterium, Rhodococcus, Nocardiaand Streptomyces is a well-established feature [4]. Mem-bers of these genera produce variable amounts of tria-cylglycerols (TAG) during growth on different carbonsources, and some species are able to accumulate veryhigh levels of TAG in their cells [4,5]. This is the casefor Rhodococcus opacus PD630, which accumulatesTAG comprising up to 76% of its cellular dry weightafter growth on gluconate [6]. The key enzymesinvolved in TAG and wax ester biosynthesis by bacteriaare the wax ester synthase/acyl-CoA:diacylglycerol acyl-transferase (WS/DGATs) enzymes. Kalscheuer andSteinbüchel identified a bifunctional enzyme from Aci-netobacter baylyi sp. ADP1 that exhibits simultaneouslyboth acyl-CoA:diacylglycerol acyltransferase and acyl-CoA:fatty alcohol acyltransferase (wax ester synthase)activities [7]. WS/DGATs catalyze the final step of TAGor wax ester biosynthesis in prokaryotes, using fatty acidCoA thioesters as substrates for esterification of diacyl-glycerols or long-chain fatty alcohols with the concomi-tant release of CoA [8]. Daniel et al. identified 15putative WS/DGAT genes in M. tuberculosis strainH37Rv, which showed acyltransferase activity whenexpressed in E. coli [9]. In addition, 10 putative WS/DGAT genes were identified in R. opacus PD630, a spe-cies closely related to strain RHA1 [10]. A highly con-served motif HHxxxDG, which may be the catalytic siteresponsible for ester bond formation, is found in WS/DGATs from all known TAG-accumulating bacteria[4,8]. Stored bacterial TAG may be mobilized by cyto-plasmic lipase/esterase enzymes, which may producefree acyl-residues available for generating energythrough oxidation or as precursors for biosynthesis ofother compounds. The lipases involved in endogenousTAG degradation in bacteria have been poorly studied.Deb et al. described a lipase enzyme which is responsi-ble for the utilization of stored TAG during dormancyand reactivation of M. tuberculosis [11].In addition to TAG, members of Rhodococcus are ableto accumulate variable amounts of short chain lengthpolyhydroxyalkanoates (PHA) during cultivation on dif-ferent carbon sources [12-14]. Some Rhodococcus mem-bers accumulate a copolyester; poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)], and others ahomopolyester of 3-hydroxybutyrate monomer units,from unrelated substrates such as gluconate, glucoseand acetate [5,13]. PHA synthases represent the keyenzymes of PHA biosynthesis, which catalyze the stereo-selective conversion of (R)-3-hydroxyacyl-CoA substratesto PHA with the concomitant release of CoA [15,16].The phaCRr gene from R. ruber is the only gene encod-ing a PHA synthase identified and cloned from a mem-ber of Rhodococcus [17]. The PhaCRr enzyme is a shortchain length class I PHA synthase, a class of enzymescomprised of only one type of subunit that utilize CoAthioesters of 3-hydroxy fatty acids with 3 to 5 carbonatoms [15]. Down-stream of the R. ruber phaCRr gene isa gene (ORF4) coding for a putative PHA depolymerase,which is the key enzyme for PHA mobilization [18].Other storage compounds produced by bacteria,besides lipids, are polyphosphates and carbohydrates liketrehalose or glycogen. The latter is a glucose polymerwith a 1,4 and a 1,6 linkages, usually considered astored energy and carbon source [19]. Glycogen accu-mulation by bacteria often occurs during stationaryphase [20]; although some bacterial species synthesizeglycogen mainly during exponential growth phase [21].In addition, glycogen may be accumulated by bacteriaunder different conditions, such as N-limited growth[20] or hyperosmotic stress [22]. The genetic aspects ofglycogen biosynthesis and degradation have been studiedintensively, mainly in E. coli [23]. In addition, there arestudies on the genetics and biochemistry of glycogenmetabolism in some actinomycetes, like Mycobacterium[21] and Corynebacterium [22,24]. So far as we know,there are no previous reports on glycogen biosynthesisby members of Rhodococcus. Key genes encodingenzymes involved in glycogen metabolism in severalbacteria include (1) glgC, encoding an ADP-glucose pyr-ophosphorylase; (2) glgB, encoding a branching enzymethat may introduce a (1–6) linkages during glycogensynthesis [25]; (3) glgX, encoding a glycogen debranch-ing enzyme; (4) glgP, encoding a protein that belongs toa structurally related and ubiquitous group of glucan-degrading enzymes, which catalyze the production ofglucose-1-phosphate by the reversible cleavage of a-1,4bonds at the non-reducing ends of polyglucans, such asmaltodextrins, starch, and glycogen [26]; (5) glgA,encoding a glycogen synthase or glycosyltransferase; and(6) glgE, encoding an alpha amylase which is involved inglycogen degradation [21].Polyphosphates (polyP) are additional storage com-pounds, which may help bacterial cells to respond andadapt to environmental stresses. This polymer is a linearchain of phosphate residues linked by phosphoanhydridebonds and synthesized in bacteria from ATP by poly-phosphate kinases (PPKs), which are the key enzymesfor polyP accumulation [27]. Some PPKs are also able togenerate ATP from polyP and ADP (the reverse reac-tion). In contrast, other PPKs are only involved in thesynthesis of PolyP, making exopolyphosphatases (PPXs)necessary to catalyze PolyP degradation [28]. PolyP mayHernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 2 of 14have multiple functions in bacterial cells, potentiallyenhancing their capacity to respond to oxidative stress,heat shock, osmotic stress, desiccation, and low-phosphate environments [29]. PolyP may also serve asan energy source to replace ATP and may be involvedin the regulation of enzyme activities. The accumulationof polyP has been previously reported for R. opacusPD630 [6], which is a microorganism taxonomicallyrelated to R. jostii RHA1.The purpose of this study was to examine the R. jostiiRHA1 genome for the presence of key genes involved instorage compounds metabolism, like PHA, TAG, waxesters, glycogen and PolyP, and to analyze the physiolo-gical capability of strain RHA1 to accumulate thesereserve substances.MethodsBacterial strain and growth conditionsR. jostii strain RHA1 was cultivated aerobically at 28°Cin nutrient broth medium (NB) or in mineral salts med-ium (MSM) according to Schlegel et al. [30]. Sodiumgluconate (1% w/v) or other substrates were used as solecarbon source. When N-limiting conditions were speci-fied, the concentration of ammonium chloride in theMSM was reduced to 0.1 g/l (MSM0.1) to allow lipidaccumulation [68]. Cells were harvested during expo-nential and stationary growth phases, washed with NaClsolution (0.85%, w/v) and lyophilised for chemicalanalyses.Extraction and analysis of lipidsFreeze-dried cells were extracted with methanol-chloro-form (MeOH-CHCl3, 1:2, v/v). An aliquot of the wholecells extract was analyzed by thin layer chromatography(TLC) on 60F254 silica gel plates (Merck) applying n-hexane-diethyl ether-acetic acid (80:20:1, v/v/v) as a sol-vent system. Lipid fractions were revealed using iodinevapor. Tripalmitin and cetylpalmitate (Merck) were usedas standards.Analysis of fatty acids and PHAFor qualitative and quantitative determination of fattyacids and PHA, 5–8 mg of lyophilised cells were sub-jected to methanolysis in the presence of 15% (v/v)sulphuric acid, and the acyl- and 3-hydroxyacyl-methy-lesters were analyzed by gas chromatography (GC) withan HP 5890 A gas chromatograph equipped with a Win-nowed capillary column (30 m × 0.53 mm × 1 μm) anda flame ionization detector. The injection volume was0.2 μl. Helium (13 mm/min) was used as carrier gas.The temperature of the injector and detector was 270°Cand 320°C respectively. A temperature program wasused for efficient separation of the methyl esters (90°Cfor 5 min, temperature increase of 6°C/min, 240°C for17 min). For quantitative analysis, tridecanoic acid wasused as internal standard.Extraction and analysis of cellular polysaccharideThe polysaccharide was isolated from freeze-dried cellsby the classical alkali treatment described previously inseveral works [31-33] and visualized by two differentTLC methods [22,24,34]. Total polysaccharide wasdetermined by the phenol-sulfuric acid method [35].Isolated cellular polysaccharide was boiled in 500 μl of 2M trifluoroacetic acid (TFA) (90 min. 121°C), and theresulting components were analyzed by paper chromato-graphy [36] and TLC methods as described above.Enzyme digestion of cellular polysaccharideSince the isolated polysaccharide may be contaminatedwith other materials, a direct weight was not an accuratemeasurement of glycogen. Thus, samples (1 mg) weredigested with a-amylase (10 UI) and amyloglucosidase(20 UI) in 50 mM sodium acetate buffer (pH 5) at 55°Cfor 2 hours. The amount of glucose under these condi-tions was taken as a measure of glycogen in cells. Glu-cose was determined by a specific glucose oxidasemethod [37].PolyP-staining methodFor staining polyphosphate inclusions, a modification[38] of Methylene-blue staining (Loeffler) was used.Smears of RHA1 cells, cultivated in MSM0.1 with 1%gluconate, were fixed with gentle heat on glass micro-scopic slides and exposed to the following solutions fol-lowed in each case with light washing in distilled water:(1) Loeffler’s methylene-blue solution, for 10 minutes; (2)sulfuric acid 1%, for 5 seconds; (3) Lugol’s iodine solu-tion, for 15 seconds; (4) aqueous safranine, for 2 minutes.For the screening of the different storage compoundmetabolism genes, we used the available RHA1 genomedatabase [39].Database searches and alignments were carried outusing BLAST 2.2.17 [40], CLUSTALW [41] and by com-parison with other studies. Reference protein sequenceswere retrieved from the NCBI database. Identities weredetermined for alignments of full-length sequences.ResultsKey genes for PHA metabolismWe searched the RHA1 genome for genes involved inPHA metabolism. Such genes are often clustered in bac-terial genomes [15,42]. We identified three chromoso-mal loci with genes involved in PHA metabolism, eachcontaining both PHA synthase and PHA depolymerasegenes (Table 1). These genes were not clustered withothers encoding b-ketothiolase and NADPH-dependentacetoacetyl-CoA reductase. In this respect, RHA1 is likeHernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 3 of 14R. ruber and is unlike gram-negative short chain lengthPHA-accumulating bacteria [17]. It is unclear whetherthe small coding sequences located near the PHAsynthase and depolymerase genes of RHA1 (ro00750,ro00752, ro03777 and ro04490) encode phasins, thestructural proteins associated with PHA bodies. Neitherthese, nor any other RHA1 predicted proteins had highsimilarity to known phasins.The three PHA synthases encoded in the genome ofRHA1 have 37% to 39% sequence identity to R. ruberPHA synthase (Table 1). Multiple alignments of the pri-mary structures of 59 PHA synthases from 45 differentbacteria, including R. ruber, showed the presence ofeight highly conserved amino acid residues, which areimportant for the enzyme function [15]. These eighthighly conserved amino acid residues are also present inthe three PHA synthases of RHA1 (Fig. 1). All PHAsynthases additionally contain a putative lipase box, G-X-(S/C)-X-G, in which the essential active-site serine ofthe lipases is replaced with a cysteine in the PHA-synthases [43,44]. The PHA synthases encoded byphaC1 and phaC2 in RHA1 contain the lipase box G-X-C-X-G, while that encoded by phaC3 has a modifiedlipase box, where the first glycine of the motif isreplaced by alanine. Three amino acid residues are pro-posed to be required for catalytic activity of PHATable 1 Key genes encoding synthesis of TAG, wax esters, PHA, glycogen and PolyP by R. jostii RHA1GeneIDGenenameEnzyme name Length(aa)Accessionnumber*Selected ortholog Aminoacid identity(%)ro00753 phaC1 PHA synthase 568 YP_700746 PhaCRrR. ruber 37ro00754 phaZ1 PHA depolymerase 323 YP_700747 ORF4 R. ruber 12ro03778 phaC2 PHA synthase 565 YP_703736 PhaCRrR. ruber 39ro03776 phaZ2 PHA depolymerase 271 YP_703734 ORF4 R. ruber 14ro04491 phaC3 PHA synthase 565 YP_704435 PhaCRrR. ruber 39ro04489 phaZ3 PHA depolymerase 274 YP_704433 ORF4 R. ruber 14ro05974 glgA Probable glycosyltransferase 406 YP_705909 GlgA Mycobacterium sp.MCS73ro05975 glgC ADP glucose Pyrophosphorylase 404 YP_705910 GlgC M. tuberculosis H37Rv 83ro01447 glgP Glycogen phosphorylase 862 YP_701423 GlgP M. tuberculosis H37Rv 69ro01448 glgE Probable alpha amylase 672 YP_701424 GlgE M. smegmatis 66ro01449 glgB Glycogen branching enzyme 732 YP_701425 GlgB Mycobacterium sp.MCS70ro01056 glgX Probable glycogen debranchingenzyme753 YP_701041 GlgX Mycobacterium sp.MCS75ro00023 atf1 WS/DGAT 436 YP_700017 Atf1 A. baylyi ADP1 21ro00024 atf2 WS/DGAT 477 YP_700018 Atf1 A. baylyi ADP1 24ro00039 atf3 WS/DGAT 473 YP_700033 Atf1 A. baylyi ADP1 23ro00087 atf4 WS/DGAT 461 YP_700081 Atf1 A. baylyi ADP1 32ro00583 atf5 WS/DGAT 430 YP_700576 Atf1 A. baylyi ADP1 26ro01601 atf6 WS/DGAT 453 YP_701572 Atf1 A. baylyi ADP1 38ro02966 atf7 WS/DGAT 467 YP_702929 Atf1 A. baylyi ADP1 37ro05356 atf8 WS/DGAT 463 YP_705294 Atf1 A. baylyi ADP1 37ro05649 atf9 WS/DGAT 484 YP_705586 Atf1 A. baylyi ADP1 21ro06332 atf10 WS/DGAT 474 YP_706267 Atf1 A. baylyi ADP1 27ro06855 atf11 WS/DGAT 464 YP_706785 Atf1 A. baylyi ADP1 29ro08369 atf12 WS/DGAT 301 YP_707571 Atf1 A. baylyi ADP1 35ro08645 atf13 WS/DGAT 473 YP_707847 Atf1 A. baylyi ADP1 36ro08660 atf14 WS/DGAT 497 YP_707862 Atf1 A. baylyi ADP1 24ro06503 ppk Polyphosphate kinase 734 YP_706434 Ppk Pseudomonasaeruginosa39ro02065 ppx1 Exopolyphosphatase 317 YP_702030 Ppx Pseudomonasaeruginosa17ro05780 ppx2 Exopolyphosphatase 314 YP_705716 Ppx Pseudomonasaeruginosa17ro06647 ppx3 Exopolyphosphatase 333 YP_706578 Ppx Pseudomonasaeruginosa11* NCRI protein data baseHernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 4 of 14synthase, presumably forming a catalytic triad, which isfound in enzymes belonging to the superfamily ofa/b-hydrolases [15,43]. The conserved residues C-294,D-449 and H-477 of R. ruber PHA synthase were pro-posed to be involved in covalent catalysis during PHAbiosynthesis [15]. The three PHA synthases of strainRHA1 all contain the same catalytic triad.The three putative PHA depolymerase proteins ofRHA1 (Table 1) have high identities (45% to 46%) toP. putida KT2440 PHA depolymerase, whose functionand characteristics have been studied previously [45].Interestingly, the RHA1 enzymes have relatively lowidentity (12% to 14%) to R. ruber PHA depolymerase.Two of the RHA1 genes (phaZ2 and phaZ3) are pre-dicted to encode intracellular PHA depolymerases,whereas phaZ1 seems to encode an extracellular PHA-depolymerase. This protein is longer than the other twoproteins, with approximately 50 additional amino acidresidues, which presumably correspond to a signal pep-tide necessary for secretion of the protein across thecytoplasmic membrane prior to its removal by signalpeptidases [46]. Such a secreted PHA-depolymeraselikely functions in catabolism of exogenous PHAs.The three PHA depolymerases of strain RHA1 containthe lipase-box pentapeptide G-X-S-X-G in theirsequences and the proposed catalytic triad (correspond-ing to S102, A221, H248 in the P. putida ortholog), withthe serine residue within the lipase-box motif. Therespective lipase boxes of the RHA1 PHA depoly-merases (PhaZ1, GYSWG; PhaZ2, GLSWG; PhaZ3,GLSWG) aligned well with those of the PHA depoly-merases of R. ruber (encoded by ORF4, GGSQG) andP. putida KT2440 (Pp5004, GVSWG). Thus, R. jostiiRHA1 is equipped with the necessary genes/proteins forthe biosynthesis, accumulation and mobilization ofPHA. Figure 1 Alignment of PHA-synthase genes of R. jostii RHA1 and R. ruber. Amino acid residues that are conserved in all the known PHAsynthases are indicated below the sequences (*). Conserved residues probably involved in catalysis are shown (●). Putative lipase box isindicated with a rectangle.Hernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 5 of 14Key genes for TAG metabolismThe key enzymes for the biosynthesis and mobilizationof TAG in bacteria are WS/DGATs and lipase/esterases,respectively [4,8]. We used WS/DGAT genes from A.baylyi ADP1 [7], and M. tuberculosis [9] to screen theRHA1 genome for related genes. We identified 14 atfgenes in the genome of RHA1 (Table 1). Some or all ofthese genes are likely involved in TAG biosynthesis inRHA1. All predicted RHA1 WS/DGATs have a lengthranging from 430 to 497 amino acid residues, exceptatf12 product, which possesses 301 amino acid residues.Eleven of these genes contain the putative active sitemotif of WS/DGATs (HHxxxDG) (Fig. 2), while in atf4,atf10 and atf14, the second histidine of the motif isreplaced by lysine, serine and proline; respectively. Ele-ven atf genes are located in the RHA1 chromosome,whereas atf12, atf13 and atf14 are located on plasmidpRHL1. The WS/DGAT genes of strain RHA1 are notlocated in operons with other genes involved in TAGmetabolism, and they are widely distributed throughoutthe genome, which seems to be common in TAG-accu-mulating actinomycetes [8,9]. However, some of the 14RHA1 WS/DGAT genes are adjacent or proximal toother genes likely involved in TAG or lipid metabolism.The atf6 gene is located up-stream of a probable ester-ase gene and the atf9 gene is located down-stream of agene coding for a putative glycerol-3-phosphate acyl-transferase, which is involved in the biosyntheticpathway of phospholipids and TAG. The atf11 gene islocated down-stream of a gene encoding a lipase/ester-ase enzyme. The atf10 gene is located proximal to fattyacid desaturase genes. The predicted WS/DGAT pro-teins found in the genome database of RHA1 are 21% to38% identical to Atf1 of A. baylyi ADP1.The RHA1 genome encodes a broad repertoire forlipid degradation. We identified a total of 54 genes cod-ing for putative lipase/esterase proteins (34 lipases and20 esterases) in the genome (Table 2). These geneslikely involved in neutral lipid degradation are widelydistributed throughout the genome. Twelve of thesegenes are located on plasmids, nine on pRHL1 andthree on pRHL2, while the rest of the putative lipase/esterase genes are located in the RHA1 chromosome.This is the first report of the occurrence of WS/DGATgenes and lipase/esterase genes on plasmids. Overall,analysis of the RHA1 genome indicated that this bacter-ium is endowed with broad capacity for TAG biosynth-esis and degradation, with potentially redundant genesand enzymes.Accumulation of storage lipidsWe investigated the accumulation of PHA, TAG andwax esters during cultivation of RHA1 in the presenceof different carbon sources under nitrogen-limiting con-ditions. Nitrogen-limitation promotes the biosynthesisand accumulation of lipids by bacteria [5]. R. jostiiFigure 2 Alignment of WS/DGAT genes of R. jostii RHA1. The putative active site motif of WS/DGATs is shown with a bar below thesequences.Hernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 6 of 14RHA1 was able to synthesize and accumulate mainlyTAG and minor amounts of a short-chain length copo-lyester, containing 3-hydroxybutyric acid (3HB) and3-hydroxyvaleric acid (3HV) as monomer units (Fig. 3and Table 3). When the cells were cultivated on hexade-cane or on a mixture of hexadecane-hexadecanol, theyproduced wax esters in addition to TAG, as revealed byTLC (Fig. 3). The main fatty acids produced by hexade-cane- and hexadecane-hexadecanol-grown cells wererelated to the chain length of those substrates, as well asto b-oxidation products of those substrates (Table 3).During cultivation on glucose, sodium gluconate,sodium acetate and 3-hydroxibutyric acid, which allhave to be degraded to acetyl-CoA before they enterother metabolic pathways, RHA1 accumulated TAG, butno wax esters were detected by TLC analysis (Fig. 3).Hexadecanoic acid (C16:0) and octadecenoic acid (C18:1)were always the predominant fatty acids occurring inthe accumulated lipids after cultivation on the abovefour carbon sources (Table 3). In addition, the propor-tion of fatty acids with odd numbers of carbon atomswas relatively high after cultivation on those four sub-strates, ranging from 11.1% to 31.0% of the total fattyacids. The copolyester accumulated by strain RHA1;(poly-3HB-co-3HV), contained higher relative amountsof the C5 monomer (3HV) than the C4 one (3HB), asrevealed by GC analysis (Table 3). When cells weregrown on acetate or 3-hydroxybutyric acid as sole car-bon sources, they produced higher relative amounts of3HB units in the copolyester in comparison with thosecells cultivated on glucose or gluconate (Table 3).Key genes for glycogen metabolismSix glg genes encoding key enzymes for glycogen meta-bolism were identified in RHA1 (Table 1). Three ofthese genes, glgP, glgE and glgB, are in a cluster, whichis conserved in glycogen-accumulating M. smegmatis[21] and M. tuberculosis. Another two of the genes, glgAand glgC, are adjacent at a different locus and diver-gently oriented. The sixth gene, glgX, is at a third locus,adjacent to another carbohydrate metabolism gene,which encodes a 1–4 a D-glucan 1-a-D-glucosylmutase.Several other genes associated with carbohydrate meta-bolism occur in the RHA1 genome, encoding glycosyl-transferases, glycosidases, sugar transporters, glucosedehydrogenases, sugar kinases, glycohydrolases andaldolases.The glgC gene of RHA1 encodes a 404-aa ADP-glucose pyrophosphorylase. This protein is 43%, 73%,83% and 91% identical to orthologs from E. coli APECTable 2 Lipases and esterases enzymes from RHA1 andtheir cellular localizationLipases* Subcelular localization *Esterases Subcelular localizationro00140 Unknown ro00968 cytoplasmicro00410 extracelular ro01280 cytoplasmicro01215 Unknown ro01602 cytoplasmicro01244 cytoplasmic ro01804 cytoplasmicro01897 Unknown ro03129 cytoplasmicro02361 cytoplasmic ro03795 cytoplasmicro02486 cytoplasmic ro03905 cytoplasmicro02663 Unknown ro04327 cytoplasmicro03099 cytoplasmic ro04513 Unknownro03436 cytoplasmic ro04768 cytoplasmicro04027 Unknown ro04911 cytoplasmicro04081 cytoplasmic ro05142 cytoplasmicro04106 Unknown ro06006 cytoplasmicro04422 Unknown ro06374 cytoplasmicro04722 Unknown ro06680 cytoplasmicro05038 Unknown ro06687 cytoplasmicro05138 cytoplasmic ro06780 cytoplasmicro05310 cytoplasmic ro07106 cytoplasmicro05456 Unknown ro08559 cytoplasmicro06108 Unknown ro10311 cytoplasmicro06372 Unknown - -ro06856 cytoplasmic - -ro06995 Unknown - -ro07162 Unknown - -ro08131 Unknown - -ro08132 extracelular - -ro08421 Unknown - -ro08422 Unknown - -ro09010 cytoplasmic - -ro09026 cytoplasmic - -ro09036 cytoplasmic - -ro09094 Unknown - -ro10223 cytoplasmic - -ro10264 cytoplasmic - -* Gene IDFigure 3 Accumulation of TAG and wax esters by R. jostii RHA1as revealed by TLC analyses. A, TLC of lipids extracted fromgluconate and hexadecane-grown cells; B, Occurrence of TAG andwax esters in cells after growth on different carbon sources.Hernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 7 of 14O1, C. glutamicum, M. tuberculosis and N. farcinicaIFM10152, respectively. In the E. coli ADP-glucose pyro-phosphorylase, Tyr-114 and Lys-195 residues were iden-tified as important in ATP and glucose-1-phosphatebinding, respectively [47], and Asp-142 and Arg-32 resi-dues were proposed to be involved in catalysis [48,49].These four residues are conserved in GlgC of RHA1(Tyr-99, Lys-180, Asp-128 and Arg-19, respectively). Inaddition, we identified a sequence motif, RAKPAV (resi-dues 27–32 in GlgC of RHA1), which is present in allADP-glucose pyrophosphorylases and is consideredimportant for activator binding [24,50,51]. This enzymeis considered essential for glycogen synthesis and itsregulation [20].The glgB gene of RHA1 encodes a branching enzymethat introduces a (1–6) linkages during glycogen synth-esis. In only a few bacteria, has this glucan branchingenzyme been studied in detail. Alignment of RHA1GlgB with orthologs allowed identification of four con-served regions as well as the conserved amino acidstypical for members of family 13 glycoside hydrolases(alpha amylase family), whose properties have been stu-died intensively. The RHA1 GlgB catalytic residues wereidentified as Asp-407, Glu-461 and Asp-529, corre-sponding to the amino acids Asp-308, Glu-351 andAsp-419 of the Bacillus stearothermophilus enzyme, pre-viously shown to constitute the catalytic triad [25,52].These residues are also present in the orthologousenzymes of E. coli and Mycobacterium sp. MCS, the lat-ter 70% identical to that of RHA1. Moreover, four otherconserved amino acids, Asp-338, His-343, Arg-406 andHis-528, likely involved in substrate binding, were iden-tified in RHA1 GlgB, based on the amino acid sequencealignment.The glgX gene of RHA1 encodes a glycogen debranch-ing enzyme. This 753-aa protein has a predicted molecu-lar mass of 83 kDa. The protein has high identities withdebranching enzymes from other actinomycetes, such asMycobacterium sp. MCS (75%) and C. glutamicumATCC 13032 (66%), as well as the E. coli debranchingenzyme GlgX (43%), which has been functionally charac-terized [53].The glgP gene of RHA1 encodes an 862-aa glycogenphosphorylase with high identity to orthologs from othermicroorganisms, such as M. tuberculosis H37Rv (69%).GlgP of RHA1 contains conserved features of other gly-cogen phosphorylases (Fig. 4), including proposed activesites and a signature sequence (EACGTSGMKSALNG)consistent with a pyridoxal-phosphate cofactor bindingsite [54].The glgA gene of RHA1 encodes a 406-aa glycosyl-transferase with a calculated molecular weight of 42.9kDa. This protein has 73% identity to a glycogensynthase from Mycobacterium sp. MCS and 25% identityto one from E. coli APEC O1. We were not able to findthe proposed active site described for the well-studiedglycogen synthase of E. coli [55]. However, based on aprevious multiple sequence alignment of selected glyco-syltransferases [56], we identified an E-X7-E motif inGlgA of RHA1 that is shared among glycogen synthasesof prokaryotes and eukaryotes (data not shown). ThisE-X7-E motif is part of the active site of eukaryotic gly-cogen synthases and both conserved glutamic acid resi-dues (E) are involved in catalysis [56].Finally, glgE of RHA1 encodes a 672-aa alpha amylase.The primary sequence of this protein has 66% identityand 77% similarity to that of a putative glucanase (GlgE)of M. smegmatis. An M. smegmatis temperature-sensi-tive glgE mutant showed an abnormal accumulation ofglycogen during exponential growth, in comparison tothe wild-type strain [21]. A sequence alignment (notshown) shows that the histidine residue that wasmutated (His349) in M. smegmatis is conserved in GlgEof RHA1 (His322).Table 3 PHA and fatty acid content (%) of R. jostii RHA1 after cultivation in MSM0.1 with different carbon sources*Glucose Gluconate Acetate 3HB Hexadecane Hexadecane + hexadecanolPHA 2.2 7.6 3.5 5.8 tr tr3HB 20.0 15.3 32.9 27.3 nd nd3HV 80.0 84.7 67.1 72.7 tr trFatty acids 48.4 56.9 21.2 32.5 30.4 7.0C14:0 1.3 1.3 1.6 1.5 5.6 6.3C15:0 4.4 3.7 2.3 1.6 nd ndC16:0 31.0 35.5 34.6 38.4 62.6 76.5C16:1 10.1 9.1 13.3 13.1 29.5 17.2C17:0 11.8 11.0 4.9 4.0 nd ndC17:1 14.8 9.8 9.4 5.5 1.0 ndC18:0 9.3 13.4 9.1 12.3 nd ndC18:1 17.3 16.2 24.8 23.6 1.3 nd* Total PHA and Fatty acids are expressed as % of cellular dry weight. Relative proportions of individual PHAs and fatty acids are expressed as % (w/w).Abbreviations: nd, not detected; tr, traces.Hernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 8 of 14Figure 4 Partial view of amino acid sequence alignment of glucan phosphorylases. The identical amino acids in the three sequences areindicated by black background. The deduced active sites are indicated by lines, and the putative pyridoxal-phosphate cofactor binding site isindicated by asterisks.Hernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 9 of 14Glycogen accumulation by RHA1After cultivation of cells on both, nutrient broth andminimal salts medium with gluconate as sole carbonsource, a phenol-sulphuric acid reactive material wasdetected in RHA1 using two different TLC methods witha commercial glycogen as standard (Fig. 5A and 5B).Samples of isolated polysaccharide from RHA1 digestedwith TFA were analyzed by paper chromatography andTLC methods. These analyses showed that glucose wasthe sole sugar component of the polysaccharide. Identicalresults were obtained with hydrolyzed glycogen standard(Fig. 5C). These results indicate that the phenol-sulphuricacid reactive material isolated from strain RHA1 is a glu-cose polymer.The glucose polymer (glucan) was also characterizedand quantified by enzymatic digestion and comparison tothe commercial glycogen. Hydrolysis with only with aamylase, which catalyzes the endohydrolysis of a 1,4 D-glucosidic linkages, released glucose, maltose plus oligo-saccharides that were identified by TLC and paperchromatography (data not shown). Hydrolysis with aamylase plus amyloglucosidase, which catalyzes hydroly-sis of terminal a 1,4 D-glucose residues and a 1,6 D-glu-cosidic bonds, converted total carbohydrate to freeglucose (Fig. 5D), indicating the presence of both a 1,4and a 1,6 bonds. Taken together, these results indicatethat the polysaccharide accumulated by RHA1 isglycogen.Glycogen was detected in both media (NB andMSM0.1) during exponential and stationary growthphases (Table 4). In both media, glycogen accumulationduring exponential growth phase was higher than duringstationary phase, especially in MSM0.1 medium with 1%gluconate as the sole carbon source.Key genes for PolyP metabolism and accumulation ofPolyPA single ppk gene was identified in RHA1 (Table 1).This gene was adjacent to other functionally unrelatedgenes. The ppk gene product has three regions and twohistidine residues (H485 and H504) (data not shown)that may be involved in the enzymatic activity and areconserved in orthologs from P. aeruginosa [57] andE. coli [58].On the other hand, three ppx genes encoding putativecytoplasmic exopolyphosphatases (Ppx-related proteins)were found widely distributed in the RHA1 genome.Exopolyphosphatase enzymes are normally involved inPolyP degradation. Ppx enzymes from E. coli [59] andP. aeruginosa (28, 60) have been cloned, sequenced andwell-characterized. The RHA1 Ppx proteins range from11% to 31% identical to that of P. aeruginosa PPX(Table 1).The occurrence of putative a ppk gene in the genomeof strain RHA1 suggests that this bacterium has thepotential to synthesize PolyP, like other members ofRhodococcus [6]. In addition, PolyP is likely mobilizedby RHA1 cells using PPX-related proteins. In order toconfirm the ability of strain RHA1 to accumulate PolyP,we analyzed cells by microscopy after cultivation on glu-conate as the sole carbon source under N-limitingFigure 5 Glycogen chromatographic analysis. TLC analysis ofglycogen in cells grown on Gluconate (A) and NB (B), withglycogen standard (1) and cellular glycogen in stationary (2) andexponential (3) growth phases. Analytical paper chromatography ofRHA1 carbohydrates hydrolyzed with TFA (C), with maltose standard(1), hydrolyzed RHA1 sample (2), glucose standard (3), xylosestandard (4) and hydrolyzed glycogen standard (5), or hydrolyzedwith alpha amylase/amyloglucosidase (D), with stationary- andexponential-phase RHA1 grown on NB (1 and 2), glucose standard(3), stationary- and exponential-phase RHA1 grown on gluconate(4 and 5).Table 4 Glycogen accumulation by R. jostii RHA1 grownon NB and MSM0.1 with gluconate during differentgrowth phases*Media and growth phase Glycogen(% CDW)NB Exponential growth phase 2.3 ± 0.4NB Stationary growth phase 1.9 ± 0.2MSM0.1 + Gluconate 1% Exponential growthphase3.5 ± 0.01MSM0.1 + Gluconate 1% Stationary growth phase 2.05 ± 0.3*All data were recorded as means of three separate assays ± standarddeviation.Hernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 10 of 14conditions. PolyP-bodies were observed in RHA1 cells asdark granules in contrast to a light red cell backgroundby means of a modification of the methylene-blue stain-ing method (Fig. 6). Identical results were observed withcells of R. opacus PD630 used as positive control, whichwas previously shown to accumulate PolyP [6].DiscussionIn this study, we report the ability of R. jostii RHA1 tosynthesize and accumulate different storage compounds,including poly(3HB-co-3HV), TAG, wax esters, glycogenand polyP. We found that the RHA1 genome is remark-ably rich in genes involved in storage lipid metabolism,with genes encoding 14 WS/DGATs, 54 lipases/esterasesand three sets of PHA synthases/depolymerases. Theseresults agree with previous observations in other actino-mycetes, like M. tuberculosis, which devotes a large por-tion of its genome to genes involved in lipid metabolism[61].Similarly to RHA1, M. tuberculosis contains 15 atfgenes encoding WS/DGATs, [9]. Some of these genes,like rv3130c, show the highest induction and activityduring hypoxia [9,62]. Multiple atf genes were also iden-tified in the genomes of other actinomycetes, includingthree in S. coelicolor [63], 10 in R. opacus [10], 12 inM. bovis AF2122/97, 8 in M. smegmatis mc2155, 5 inN. farcinica IFM 10152 and 4 in Nocardioides sp. JS614[8]. By contrast, TAG/wax esters accumulating gram-negative bacteria seem to have single atf genes, as is thecase for A. baylyi sp. ADP1, Psychrobacter sp. andPolaromonas sp. [8]. In general, actinomycetes accumu-late higher amounts of TAG than gram-negative bac-teria, and they have a high lipid content in differentcellular structures, such as cell envelope or lipid inclu-sions [4,13].The multiplicity of atf genes in Rhodococcus and otheractinomycetes, and their ability to synthesize TAGs asmain storage lipids, suggest an important role of theselipids in the physiology of these organisms and their abil-ity to cope with adverse environments. TAGs are excel-lent reserve materials for several reasons. Their extremehydrophobicity allows their accumulation in largeamounts in cells without changing the osmolarity of thecytoplasm. Oxidation of TAGs produces the relativelyhigh yields of energy in comparison with other storagecompounds such as PHA and carbohydrates, since thecarbon atoms of TAG acyl residues are in a very reducedstate [5]. Moreover, TAGs may play other importantfunctions in cells of actinomycetes, including (1) regulat-ing the fatty acid composition of lipid membranes, (2) asa sink for reducing equivalents in cells when the terminalacceptor is not sufficiently supplied under low oxygenconditions, (3) as source of precursors for biosynthesisand turnover of mycolic acids during adaptation to chan-ging environmental conditions, (4) as a reservoir of meta-bolic water for cells under water stress conditions, (5) asan agent to detoxify free fatty acids or unusual fatty acidsthat may disturb membrane fluidity during catabolism ofhydrocarbons, or (6) as a source of precursors for thebiosynthesis of antibiotics [4,64-67].The RHA1 genome contains three sets of genesinvolved in PHA metabolism. However, PHAs representonly minor components of RHA1 storage lipids underthe conditions used in this study. This is a common fea-ture of all Rhodococcus members so far investigated,except for R. ruber, which is able to accumulate consid-erable amounts of both PHA and TAG (approx. 1:1)[14,68]. PHA may also serve as an endogenous source ofcarbon and energy for cells, although strain RHA1 pos-sesses higher amounts of TAG, which are energeticallymore efficient than PHA, as discussed above. Thus, theimportance of PHA for this function is relative. On theother hand, PHA, with C more oxidized than in TAG,may help cells to balance their redox state in environ-ments with fluctuating conditions like soil.In addition to TAG, wax esters were detected in hexa-decane- and hexadecane/hexadecanol-grown cells. It wasevident that fatty acid plus alcohol intermediates wereavailable in these cells for the biosynthesis of wax estersin addition to TAG by the WS/DGATs, which are nor-mally bifunctional enzymes involved in the synthesis ofboth storage lipids. It is known that fatty alcohols occuras intermediates during the degradation of alkanes. Incontrast, no wax esters but TAG were detected aftergrowth of cells on glucose, gluconate, acetate and 3-hydroxybutyric acid. These results suggest that strainRHA1 is not capable of providing fatty alcohols asFigure 6 Modified Loeffler’s methylene blue staining ofRhodococcus jostii RHA1 grown on MSM0.1 with gluconate 1%(w/v). (A) General view of cells by optic microscopy (1,000 ×). (B)and (C) Magnified views of cells showing dark stained polyPinclusions and lightly stained cytoplasm. These photographs havebeen digitally processed to increase magnification (Originalmagnification: 1,000 ×).Hernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 11 of 14substrates during growth on these carbon sources,which have to be degraded to acetyl-CoA before theyenter other metabolic pathways.The high proportion of odd-numbered fatty acids andthe 3HV monomer in the stored TAG and PHA, respec-tively, suggest that strain RHA1 possesses an efficientmechanism for production of the intermediate propio-nyl-CoA, which was presumably utilized as precursorfor the biosynthesis of fatty acids containing an odd-number of carbon atoms and the 3HV units of thecopolyester. In addition, the growth of cells on acetateor 3-hydroxybutyric acid as sole carbon sources inducedhigher relative amounts of 3HB units in the copolyesterin comparison with those cells cultivated on glucose orgluconate. Acetate may favor the production of 3HBunits, since this monomer is normally synthesized bycondensation of two acetyl-CoA residues. On the otherhand, 3-hydroxybutyric acid used as carbon source maybe mainly degraded before entering the biosynthesispathway for PHA, although a small part may be useddirectly as monomer unit for the copolyester, likely afteractivation as 3-hydroxybutyryl-CoA.The results of this study indicate that R. jostii RHA1 alsopossess the ability to synthesize and accumulate glycogenas an additional storage compound. To our knowledgethis is the first report on glycogen accumulation by a Rho-dococcus member. However, glycogen accumulation hasbeen reported to occur in other actinomycetes, such asmembers of Mycobacterium [21,31] and Corynebacterium[24]. The RHA1 genome contains all necessary structuralgenes for the biosynthesis and degradation of glycogen. Inaddition, in this study, we chemically characterized theglycogen accumulated by strain RHA1. In contrast tomany bacterial species which accumulate glycogen onlyduring stationary phase or limited growth conditions [20],RHA1 accumulated more glycogen during exponentialgrowth phase. Glycogen accumulation during exponentialgrowth phase has been observed in other actinomycetes,such as M. smegmatis [21,31] and C. glutamicum [24].The glycogen content in strain RHA1 decreased duringstationary phase relative to exponential growth phase,under the culture conditions used in this study. The totalcontent of glycogen in cells of strain RHA1 was rather lowin comparison with the amount of accumulated TAG. Wesuggest that the biosynthesis pathways of PHA, TAG andglycogen compete for common precursors, which are usedby cells preferentially for TAG biosynthesis rather than forPHA and glycogen accumulation. However, the metabolicrelationship in strain RHA1 between these storage com-pounds must be investigated in further detail. Moreover,more studies are necessary to determine the role of glyco-gen in the RHA1 physiology. The content of glycogen incells may be the result of a well coordinated process ofsynthesis and degradation as occur in M. smegmatis inwhich, glycogen has been proposed as a carbon capacitorfor glycolysis during exponential growth [21]. Glycogenmay have a role as metabolic intermediate since it is accu-mulated mainly during the exponential growth phase bycells and is mobilized later in the stationary phase, or mayact as part of a sensing/signalling mechanism. Interest-ingly, Persson et al. [69] proposed that the expression ofsome genes involved in the response of E. coli to carbonstarvation or stationary phase, like that encoding the uni-versal stress protein (uspA), is regulated by glycolytic inter-mediates such as fructose-6-phosphate. Alteration in thepool size of phosphorylated sugars of the upper glycolyticpathway may ensure expression of stress proteins preced-ing the complete depletion of the external carbon sourceand growth arrest [69]. Thus, glycogen formation may actto attenuate phosphorylated sugar signals and to protectcells from sudden increases in fluxes of sugars.The occurrence of one gene encoding Ppk and threeputative Ppxs in the RHA1 genome and the presence ofabundant intracellular metachromatic granules indicatedthat strain RHA1 possesses the ability to accumulatepolyP. The occurrence of polyP has been described inother actinomycetes [6], such as members of Corynebac-terium and Mycobacterium after cultivation on nitro-gen-limited [70] or phosphate-rich media [71]. Theavailability of this high-energy phosphate polymer mayenhance the capacity of strain RHA1 to survive in soilenvironments by providing phosphate for biosynthesis,maintenance energy or an osmoprotectant.ConclusionThis study evaluates the global capacity for storage com-pound metabolism by RHA1 and demonstrates the func-tionality of the biosynthetic pathways. This knowledgeconstitutes a framework for additional studies to deter-mine the physiological and ecological functions and sig-nificance of these storage compounds for RHA1 andrelated bacteria. The occurrence of diverse storage com-pounds in RHA1 emphasizes the complexity of the phy-siology and biochemistry of this heterotrophic soilbacterium. Strain RHA1 is endowed with diverse sets ofgenes and enzymes for the metabolism of storage com-pounds, which seem to be redundant for storage lipidmetabolism and polyP synthesis but not for glycogenmetabolism or polyP degradation. Individual isoforms ofenzymes potentially have different substrate specificity,may play distinct functional roles in the pathways of gly-cerolipid biosynthesis or may be differentially expressedunder various environmental conditions. Additionalinformation about gene or protein expression andenzyme activities will be required to distinguish specificroles of isoenzymes in strain RHA1. This complexityreflects the richness, diversity and versatility of lipidmetabolism in RHA1 and related lipid-rich bacteria.Hernández et al. BMC Genomics 2008, 9:600http://www.biomedcentral.com/1471-2164/9/600Page 12 of 14The ability of these bacteria to accumulate diverse sto-rage compounds may permit cells to rapidly respond tostress and to maintain active its metabolism under fluc-tuating environmental conditions, providing them withan adaptive advantage over less versatile bacteria.AcknowledgementsThis study was financially supported by the Agencia Nacional de PromociónCientífica y Tecnológica, Argentina (Project PME N° 216), SCyT of theUniversity of Patagonia San Juan Bosco (PI31) and the Oil M&S company(agreement with CRIDECIT-FCN-UNPSJB). The authors are grateful to RoxanaSilva, Paola Haro, María S. Villalba and María Luján Flores for technicalassistance and helpful discussions on the topic. H.M. Alvarez is a careerinvestigator and M.A. Hernández a scholarship holder of the ConsejoNacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.Author details1Centro Regional de Investigación y Desarrollo Científico Tecnológico(CRIDECIT), Facultad de Ciencias Naturales, Universidad Nacional de laPatagonia San Juan Bosco, Km 4-Ciudad Universitaria, 9000 ComodoroRivadavia, Chubut, Argentina. 2Department of Microbiology andImmunology, Life Science Institute, University of British Columbia, Vancouver,BC, V6T 1Z3, Canada. 3Facultad de Ingeniería, Universidad Nacional de laPatagonia San Juan Bosco, Km 4-Ciudad Universitaria, 9000 ComodoroRivadavia, Chubut, Argentina. 4Departamento de Genética Molecular,Instituto de Fisiología Celular, Universidad Nacional Autónoma de México,04510 México D.F, México.Authors’ contributionsMAH carried out the experimental studies, participated in the sequenceanalysis and alignment, helped in the design of the study and drafted themanuscript. EM and ER participated in the experimental studies on storagelipids. AFA participated in the acquisition of data, sequence analysis andalignment, and interpretation of data. WWM participated in coordination ofthe study, interpretation of data and helped to draft the manuscript. HMAconceived the study and participated in its design and coordination,interpretation of data, and helped to draft manuscript. All authors read andapproved the final manuscript.Received: 18 July 2008 Accepted: 12 December 2008Published: 12 December 2008References1. Martínková L, Uhnáková B, Pátek M, Nésvera J, Krén V: Biodegradationpotential of the genus Rhodococcus. Environ Int 2009, 35(1):162-177.2. Masai E, Yamada A, Healy JM, Hatta T, Kimbara K, Fukuda M, Yano K:Characterization of biphenyl catabolic genes of gram-positivepolychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. ApplEnviron Microbiol 1995, 61:2079-2085.3. 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