Two protocols for the detection of oleaginous bacteria using Oil Red O.

The selection of oleaginous bacteria, potentially applicable to biotechnological approaches, is usually carried out by different expensive and time-consuming techniques. In this study, we used Oil Red O (ORO) as an useful dye for staining of neutral lipids (triacylglycerols and wax esters) on thin-layer chromatography plates. ORO could detect minimal quantities of both compounds (detection limit, 0.0025 mg of tripalmitin or 0.005 mg of cetylpalmitate). In addition, we developed a specific, rapid, and inexpensive screening methodology to detect triacylglycerol-accumulating microorganisms grown on the agar plate. This staining methodology detected 9/13 strains with a triacylglycerol content higher than 20% by cellular dry weight. ORO did not stain polyhydroxyalkanoates-producing bacteria. The four oleaginous strains not detected by this screening methodology exhibited a mucoid morphology of their colonies. Apparently, an extracellular polymeric substance produced by these strains hampered the entry of the lipophilic dye into cells. The utilization of the developed screening methodology would allow selecting of oleaginous bacteria in a simpler and faster way than techniques usually used nowadays, based on unspecific staining protocols and spectrophotometric or chromatographic methods. Furthermore, the use of ORO as a staining reagent would easily characterize the neutral lipids accumulated by microorganisms as reserve compounds. KEY POINTS: • Oil Red O staining is specific for triacylglycerols • Oil Red O staining is useful to detect oleaginous bacteria • Fast and inexpensive staining to isolate oleaginous bacteria from the environment.

Fruit residues as substrates for single-cell oil production by Rhodococcus species: physiology and genomics of carbohydrate catabolism.

Strains belonging to R. opacus, R. jostii, R. fascians, R. erythropolis and R. equi exhibited differential ability to grow and produce lipids from fruit residues (grape marc and apple pomace), as well as single carbohydrates, such as glucose, gluconate, fructose and sucrose. The oleaginous species, R. opacus (strains PD630 and MR22) and R. jostii RHA1, produced higher yields of biomass (5.1-5.6 g L-1) and lipids (38-44% of CDW) from apple juice wastes, in comparison to R. erythropolis DSM43060, R. fascians F7 and R. equi ATCC6939 (4.1-4.3 g L-1 and less than 10% CDW of lipids). The production of cellular biomass and lipids were also higher in R. opacus and R. jostii (6.8-7.2 g L-1 and 33.9-36.5% of CDW of lipids) compared to R. erythropolis, R. fascians, and R. equi (3.0-3.6 g L-1 and less than 10% CDW of lipids), during cultivation of cells on wine grape waste. A genome-wide bioinformatic analysis of rhodococci indicated that oleaginous species possess a complete set of genes/proteins necessary for the efficient utilization of carbohydrates, whereas genomes from non-oleaginous rhodococcal strains lack relevant genes coding for transporters and/or enzymes for the uptake, catabolism and assimilation of carbohydrates, such as gntP, glcP, edd, eda, among others. Results of this study highlight the potential use of the oleaginous rhodococcal species to convert sugar-rich agro-industrial wastes, such as apple pomace and grape marc, into single-cell oils.

Genetic analysis of acyl-CoA carboxylases involved in lipid accumulation in Rhodococcus jostii RHA1.

In actinomycetes, the acyl-CoA carboxylases, including the so-called acetyl-CoA carboxylases (ACCs), are biotin-dependent enzymes that exhibit broad substrate specificity and diverse domain and subunit arrangements. Bioinformatic analyses of the Rhodococcus jostii RHA1 genome found that this microorganism contains a vast arrange of putative acyl-CoA carboxylases domains and subunits. From the thirteen putative carboxyltransferase domains, only the carboxyltransferase subunit RO01202 and the carboxyltransferase domain present in the multidomain protein RO04222 are highly similar to well-known essential ACC subunits from other actinobacteria. Mutant strains in each of these genes showed that none of these enzymes is essential for R. jostii growth in rich or in minimal media with high nitrogen concentration, presumably because of their partial overlapping activities. A mutant strain in the ro04222 gene showed a decrease in triacylglycerol and mycolic acids accumulation in rich and minimal medium, highlighting the relevance of this multidomain ACC in the biosynthesis of these lipids. On the other hand, RO01202, a carboxyltransferase domain of a putative ACC complex, whose biotin carboxylase and biotin carboxyl carrier protein domain were not yet identified, was found to be essential for R. jostii growth only in minimal medium with low nitrogen concentration. The results of this study have identified a new component of the TAG-accumulating machinery in the oleaginous R. jostii RHA1. While non-essential for growth and TAG biosynthesis in RHA1, the activity of RO04222 significantly contributes to lipogenesis during single-cell oil production. Furthermore, this study highlights the high functional diversity of ACCs in actinobacteria, particularly regarding their essentiality under different environmental conditions. KEY POINTS: • R. jostii possess a remarkable heterogeneity in their acyl-carboxylase complexes. • RO04222 is a multidomain acetyl-CoA carboxylase involved in lipid accumulation. • RO01202 is an essential carboxyltransferase only at low nitrogen conditions.

Insights into the evolutionary history of the virulent factor HBHA of Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, heparin-binding hemagglutinin (HBHAMT) has a relevant role in infection. It is also present in non-virulent mycobacteria and ancient actinobacteria, such as Rhodococcus opacus. To have a better understanding of the underlying mechanisms that shaped the evolutionary divergence of these proteins, we performed a comprehensive phylogenetic analysis of the regulatory sequences that drive the expression of hbha in saprophytic and pathogenic mycobacterial species. The alignment of the hbha loci showed the appearance of intergenic sequences containing regulatory elements upstream the hbha gene; this sequence arrangement is present only in slow-growing pathogenic mycobacteria. The heterologous expression of HBHAMT in oleaginous R. opacus PD630 results in protein binding to lipid droplets, as it happens with HBHA proteins from saprophytic mycobacteria. We hypothesize that mycobacterial hbha gene cluster underwent functional divergence during the evolutionary differentiation of slow-growing pathogenic mycobacteria. We propose here an evolutionary scenario to explain the structural and functional divergence of HBHA in fast and slow-growing mycobacteria.

Rhodococcus as Biofactories for Microbial Oil Production.

Bacteria belonging to the Rhodococcus genus are frequent components of microbial communities in diverse natural environments. Some rhodococcal species exhibit the outstanding ability to produce significant amounts of triacylglycerols (TAG) (>20% of cellular dry weight) in the presence of an excess of the carbon source and limitation of the nitrogen source. For this reason, they can be considered as oleaginous microorganisms. As occurs as well in eukaryotic single-cell oil (SCO) producers, these bacteria possess specific physiological properties and molecular mechanisms that differentiate them from other microorganisms unable to synthesize TAG. In this review, we summarized several of the well-characterized molecular mechanisms that enable oleaginous rhodococci to produce significant amounts of SCO. Furthermore, we highlighted the ability of these microorganisms to degrade a wide range of carbon sources coupled to lipogenesis. The qualitative and quantitative oil production by rhodococci from diverse industrial wastes has also been included. Finally, we summarized the genetic and metabolic approaches applied to oleaginous rhodococci to improve SCO production. This review provides a comprehensive and integrating vision on the potential of oleaginous rhodococci to be considered as microbial biofactories for microbial oil production.

Increasing lipid production using an NADP+-dependent malic enzyme from Rhodococcus jostii.

The occurrence of NADP+-dependent malic enzymes (NADP+-MEs) in several Rhodococcus strains was analysed. The NADP+-ME number in Rhodococcus genomes seemed to be a strain-dependent property. Total NADP+-ME activity increased by 1.8- and 2.6-fold in the oleaginous Rhodococcus jostii RHA1 and Rhodococcus opacus PD630 strains during cultivation under nitrogen-limiting conditions. Total NADP+-ME activity inhibition by sesamol resulted in a significant decrease of the cellular biomass and lipid production in oleaginous rhodococci. A non-redundant ME coded by the RHA1_RS44255 gene located in a megaplasmid (pRHL3) of R. jostii RHA1 was characterized and its heterologous expression in Escherichia coli resulted in a twofold increase in ME activity in an NADP+-dependent manner. The overexpression of RHA1_RS44255 in RHA1 and PD630 strains grown on glucose promoted an increase in total NADP+-ME activity and an up to 1.9-foldincrease in total fatty acid production without sacrificing cellular biomass. On the other hand, its expression in Rhodococcus fascians F7 grown on glycerol resulted in a 1.3-1.4-foldincrease in total fatty acid content. The results of this study confirmed the contribution of NADP+-MEs to TAG accumulation in oleaginous rhodococci and the utility of these enzymes as an alternative approach to increase bacterial oil production from different carbon sources.

Insights into the Metabolism of Oleaginous Rhodococcus spp.

Some species belonging to the Rhodococcus genus, such as Rhodococcus opacus, R. jostii, and R. wratislaviensis, are known to be oleaginous microorganisms, since they are able to accumulate triacylglycerols (TAG) at more than 20% of their weight (dry weight). Oleaginous rhodococci are promising microbial cell factories for the production of lipids to be used as fuels and chemicals. Cells could be engineered to create strains capable of producing high quantities of oils from industrial wastes and a variety of high-value lipids. The comprehensive understanding of carbon metabolism and its regulation will contribute to the design of a reliable process for bacterial oil production. Bacterial oleagenicity requires an integral configuration of metabolism and regulatory processes rather than the sole existence of an efficient lipid biosynthesis pathway. In recent years, several studies have been focused on basic aspects of TAG biosynthesis and accumulation using R. opacus PD630 and R. jostii RHA1 strains as models of oleaginous bacteria. The combination of results obtained in these studies allows us to propose a metabolic landscape for oleaginous rhodococci. In this context, this article provides a comprehensive and integrative view of different metabolic and regulatory attributes and innovations that explain the extraordinary ability of these bacteria to synthesize and accumulate TAG. We hope that the accessibility to such information in an integrated way will help researchers to rationally select new targets for further studies in the field.

The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci.

The regulatory mechanisms involved in lipogenesis and triacylglycerol (TAG) accumulation are largely unknown in oleaginous rhodococci. In this study a regulatory protein (here called NlpR: Nitrogen lipid Regulator), which contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci was identified. Under nitrogen deprivation conditions, in which TAG accumulation is stimulated, the nlpR gene was significantly upregulated, whereas a significant decrease of its expression and TAG accumulation occurred when cerulenin was added. The nlpR disruption negatively affected the nitrate/nitrite reduction as well as lipid biosynthesis under nitrogen-limiting conditions. In contrast, its overexpression increased TAG production during cultivation of cells in nitrogen-rich media. A putative 'NlpR-binding motif' upstream of several genes related to nitrogen and lipid metabolisms was found. The nlpR disruption in RHA1 strain led to a reduced transcription of genes involved in nitrate/nitrite assimilation, as well as in fatty acid and TAG biosynthesis. Purified NlpR was able to bind to narK, nirD, fasI, plsC and atf3 promoter regions. It was suggested that NlpR acts as a pleiotropic transcriptional regulator by activating of nitrate/nitrite assimilation genes and others genes involved in fatty acid and TAG biosynthesis, in response to nitrogen deprivation.

Rhodococcus bacteria as a promising source of oils from olive mill wastes.

The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2-2.7 g/l) and lipids (77-83% of CDW, 1.8-2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1-1.6 g/l of cell biomass and 7.1-14.0% of CDW, 0.1-0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.

Proteome analysis reveals differential expression of proteins involved in triacylglycerol accumulation by Rhodococcus jostii RHA1 after addition of methyl viologen.

Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and ß-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.

Nutrient starvation leading to triglyceride accumulation activates the Entner Doudoroff pathway in Rhodococcus jostii RHA1.

BACKGROUND: Rhodococcus jostii RHA1 and other actinobacteria accumulate triglycerides (TAG) under nutrient starvation. This property has an important biotechnological potential in the production of sustainable oils. RESULTS: To gain insight into the metabolic pathways involved in TAG accumulation, we analysed the transcriptome of R jostii RHA1 under nutrient-limiting conditions. We correlate these physiological conditions with significant changes in cell physiology. The main consequence was a global switch from catabolic to anabolic pathways. Interestingly, the Entner-Doudoroff (ED) pathway was upregulated in detriment of the glycolysis or pentose phosphate pathways. ED induction was independent of the carbon source (either gluconate or glucose). Some of the diacylglycerol acyltransferase genes involved in the last step of the Kennedy pathway were also upregulated. A common feature of the promoter region of most upregulated genes was the presence of a consensus binding sequence for the cAMP-dependent CRP regulator. CONCLUSION: This is the first experimental observation of an ED shift under nutrient starvation conditions. Knowledge of this switch could help in the design of metabolomic approaches to optimize carbon derivation for single cell oil production.

Formation of indigoidine derived-pigments contributes to the adaptation of Vogesella sp. strain EB to cold aquatic iron-oxidizing environments.

We investigated previously under explored cold aquatic environments of Andean Patagonia, Argentina. Oily sheens similar to an oil spill are frequently observed at the surface of water in creeks and small ponds in these places. Chemical analysis of a water sample revealed the occurrence of high concentrations of iron and the presence of a free insoluble indigoidine-derived pigment. A blue pigment-producing bacterium (strain EB) was isolated from the water sample and identified as Vogesella sp. by molecular analysis. The isolate was able to produce indigoidine and another derived-pigment (here called cryoindigoidine) with strong antifreeze properties. The production of the pigments depended on the cell growth at cold temperatures (below 15 °C), as well as on the attachment of cells to solid surfaces, and iron limitation in the media. The pigments produced by strain EB showed an inhibitory effect on the growth of diverse microorganisms such as Candida albicans, Escherichia coli and Staphylococcus aureus. In addition, pigmented cells were more tolerant to freezing than non-pigmented cells, suggesting a role of cryoindigoidine/indigoidine as a cold-protectant molecule. The possible roles of the pigments in strain EB physiology and its interactions with the iron-rich environment from which the isolate was obtained are discussed. Results of this study suggested an active role of strain EB in the investigated iron-oxidizing ecosystem.

Physiological and genetic differences amongst Rhodococcus species for using glycerol as a source for growth and triacylglycerol production.

We analysed the ability of five different rhodococcal species to grow and produce triacylglycerols (TAGs) from glycerol, the main byproduct of biodiesel production. Rhodococcus fascians and Rhodococcus erythropolis grew fast on glycerol, whereas Rhodococcus opacus and Rhodococcus jostii exhibited a prolonged lag phase of several days before growing. Rhodococcus equi only exhibited poor growth on glycerol. R. erythropolis DSMZ 43060 and R. fascians F7 produced 3.9-4.3 g cell biomass l(-1) and 28.4-44.6% cellular dry weight (CDW) of TAGs after 6 days of incubation; whereas R. opacus PD630 and R. jostii RHA1 produced 2.5-3.8 g cell biomass l(-1) and 28.3-38.4% CDW of TAGs after 17 days of growth on glycerol. Genomic analyses revealed two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2) amongst rhodococci. Those species that possessed cluster 1 (glpFK1D1) (R. fascians and R. erythropolis) exhibited fast growth and lipid accumulation, whereas those that possessed cluster 2 (glpK2D2) (R. opacus, R. jostii and R. equi) exhibited delayed growth and lipid accumulation during cultivation on glycerol. Three glycerol-negative strains were complemented for their ability to grow and produce TAGs by heterologous expression of glpK2 from R. opacus PD630. In addition, we significantly reduced the extension of the lag phase and improved glycerol assimilation and oil production of R. opacus PD630 when expressing glpK1D1 from R. fascians. The results demonstrated that rhodococci are a flexible and amenable biological system for further biotechnological applications based on the reutilization of glycerol.

Label-free and redox proteomic analyses of the triacylglycerol-accumulating Rhodococcus jostii RHA1.

The bacterium Rhodococcus jostii RHA1 synthesizes large amounts of triacylglycerols (TAGs) under conditions of nitrogen starvation. To better understand the molecular mechanisms behind this process, we performed proteomic studies in this oleaginous bacterium. Upon nitrogen starvation, we observed a re-routing of the carbon flux towards the formation of TAGs. Under these conditions, the cellular lipid content made up more than half of the cell's dry weight. On the proteome level, this coincided with a shift towards non-glycolytic carbohydrate-metabolizing pathways. These pathways (Entner-Doudoroff and pentose-phosphate shunt) contribute NADPH and precursors of glycerol 3-phosphate and acetyl-CoA to lipogenesis. The expression of proteins involved in the degradation of branched-chain amino acids and the methylmalonyl-CoA pathway probably provided propionyl-CoA for the biosynthesis of odd-numbered fatty acids, which make up almost 30 % of RHA1 fatty acid composition. Additionally, lipolytic and glycerol-degrading enzymes increased in abundance, suggesting a dynamic cycling of cellular lipids. Conversely, abundance of proteins involved in consuming intermediates of lipogenesis decreased. Furthermore, we identified another level of lipogenesis regulation through redox-mediated thiol modification in R. jostii. Enzymes affected included acetyl-CoA carboxylase and a ß-ketoacyl-[acyl-carrier protein] synthase II (FabF). An integrative metabolic model for the oleaginous RHA1 strain is proposed on the basis of our results.

Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains.

Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.

Identification of a novel ATP-binding cassette transporter involved in long-chain fatty acid import and its role in triacylglycerol accumulation in Rhodococcus jostii RHA1.

Members of the genus Rhodococcus are specialists in the biosynthesis and accumulation of triacylglycerols (TAGs). As no transport protein related to TAG metabolism has yet been characterized in these bacteria, we used the available genomic information of Rhodococcus jostii RHA1 to perform a broad survey of genes coding for putative lipid transporter proteins in this oleaginous micro-organism. Among the seven genes encoding putative lipid transporters, ro05645 (now called ltp1: lipid transporter protein) coding for an ATP-binding cassette protein was found clustered with others genes encoding enzymes catalysing the three putative acylation reactions of the Kennedy pathway for TAG synthesis. Overexpression of ltp1 in the RHA1 strain led to an increase of approximately sixfold and threefold in biomass and TAG production, respectively, when cells were cultivated on palmitic acid and oleic acid. Moreover, overexpression of ltp1 also promoted a significant increase in the uptake of a fluorescently labelled long-chain fatty acid (LCFA), as compared with the WT strain RHA1, and its further incorporation into the TAG fraction. Gluconate-grown cells showed increasing amounts of intracellular free fatty acids, but not of TAG, after overexpressing ltp1. Thus, for the first time to our knowledge, a transporter functionally related to TAG metabolism was identified in oleaginous rhodococci. Our results suggested that Ltp1 is an importer of LCFAs that plays a functional role in lipid homeostasis of R. jostii RHA1.

Triacylglycerol accumulation and oxidative stress in Rhodococcus species: differential effects of pro-oxidants on lipid metabolism.

In general, members of Rhodococcus genus are highly resistant to desiccation. Desiccation is a complex process which includes the formation of reactive oxygen species that results in significant damage to cells. In this study, we demonstrate that extremophile actinobacterial strains isolated from diverse environments, mainly belonging to Rhodococcus genus, exhibited high tolerance to the pro-oxidants hydrogen peroxide (H2O2) and methyl viologen (MV). In addition, we investigated the possible interconnections between the responses of the oleaginous Rhodococcus opacus PD630 to oxidative stress and lipid metabolism, since both processes demand a metabolic reorganization of cells. Experiments with metabolic inhibitors showed differential effects of both pro-oxidants on lipid metabolism in PD630 cells. The inhibition of carotenoid biosynthesis by the addition of diphenylamine to the media negatively affected the tolerance of cells to H2O2, but not to MV. The inhibition of triacylglycerol (TAG) biosynthesis and accumulation in PD630 did not affect the tolerance of cells to H2O2 and MV; whereas, the blockage of lipolysis decreased the tolerance of cells to H2O2 (but not MV) under carbon-starvation conditions. Interestingly, the addition of MV to the media (but not H2O2) induced a reduction of TAG accumulation by cells. Resuming, results of this study revealed metabolic connections between lipid metabolism and oxidative stress responses in R. opacus PD630, and probably in other extremophile TAG-accumulating rhodococci.

MLDSR, the transcriptional regulator of the major lipid droplets protein MLDS, is controlled by long-chain fatty acids and contributes to the lipid-accumulating phenotype in oleaginous Rhodococcus strains.

Microorganism lipid droplet small regulator (MLDSR) is a transcriptional regulator of the major lipid droplet (LD)-associated protein MLDS in Rhodococcus jostii RHA1 and Rhodococcus opacus PD630. In this study, we investigated the role of MLDSR on lipid metabolism and triacylglycerol (TAG) accumulation in R. jostii RHA1 at physiological and molecular levels. MLDSR gene deletion promoted a significant decrease of TAG accumulation, whereas inhibition of de novo fatty acid biosynthesis by the addition of cerulenin significantly repressed the expression of the mldsr-mlds cluster under nitrogen-limiting conditions. In vitro and in vivo approaches revealed that MLDSR-DNA binding is inhibited by fatty acids and acyl-CoA residues through changes in the oligomeric or conformational state of the protein. RNAseq analysis indicated that MLDSR not only controls the expression of its own gene cluster but also of several genes involved in central, lipid, and redox metabolism, among others. We also identified putative MLDSR-binding sites on the upstream regions of genes coding for lipid catabolic enzymes and validated them by EMSA assays. Overexpression of mldsr gene under nitrogen-rich conditions promoted an increase of TAG accumulation, and further cell lysis with TAG release to the culture medium. Our results suggested that MLDSR is a fatty acid-responsive regulator that plays a dual role in cells by repression or activation of several metabolic genes in R. jostii RHA1. MLDSR seems to play an important role in the fine-tuning regulation of TAG accumulation, LD formation, and cellular lipid homeostasis, contributing to the oleaginous phenotype of R. jostii RHA1 and R. opacus PD630.

Lipid storage in high-altitude Andean Lakes extremophiles and its mobilization under stress conditions in Rhodococcus sp. A5, a UV-resistant actinobacterium.

The production of triacylglycerols (TAG) or wax esters (WS) seems to be a widespread feature among extremophile bacteria living in high-altitude Andean Lakes (HAAL), Argentina. Twelve out of twenty bacterial strains isolated from HAAL were able to produce TAG or WS (between 2 and 17 % of cellular dry weight) under nitrogen-limiting culture conditions. Among these strains, the extremophile Rhodococcus sp. A5 accumulated significant amounts of TAG during growth on glucose (17 %, CDW) and hexadecane (32 %, CDW) as sole carbon sources. The role of accumulated TAG in the response to carbon starvation, osmotic stress, UV-radiation and desiccation was investigated in Rhodococcus sp. A5 using an inhibitor of TAG degradation. Cells degraded TAG during these stresses in the absence of the inhibitor. The inhibition of TAG mobilization affected cell survival during osmotic stress only during the initial growth stage. Little or no surviving cells were observed after carbon starvation, UV-treatment and desiccation, when TAG mobilization was inhibited. These results suggested that TAG metabolism is relevant for the adaptation and survival of A5 cells under carbon starvation, osmotic stress and UV irradiation, and essential under desiccation conditions, which prevail in HAAL environments.

The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327-2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25-30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.