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Two Gram-stain-positive bacterial strains, EXRC-4A-4T and RC-2-3T, were isolated from soil samples collected at Union Glacier, Antarctica. Based on 16S rRNA gene sequence similarity, strain EXRC-4A-4T was identified as belonging to the genus Rhodococcus, and strain RC-2-3T to the genus Pseudarthrobacter. Further genomic analyses, including average nucleotide identity and digital DNA-DNA hybridization, suggested that these strains represent new species. Strain EXRC-4A-4T exhibited growth at temperatures ranging from 4 to 28 °C (optimum between 20 and 28 °C), at pH 5.0-9.0 (optimum, pH 6.0), and in the presence of 0-5.0% NaCl (optimum between 0 and 1% NaCl). Strain RC-2-3T grew at 4-28 °C (optimum growth at 28 °C), pH 6.0-10 (optimum, pH 7.0) and in the presence of 0-5.0% NaCl (optimum, 1% NaCl). The fatty acid profile of EXRC-4A-4T was dominated by C17:1 ω-7, while that of RC-2-3T was dominated by anteiso-C15 : 0. The draft genome sequences revealed a DNA G+C content of 64.6 mol% for EXRC-4A-4T and 65.8 mol% for RC-2-3T. Based on this polyphasic study, EXRC-4A-4T and RC-2-3T represent two novel species within the genera Rhodococcus and Pseudarthrobacter, respectively. We propose the names Rhodococcus navarretei sp. nov. and Pseudarthrobacter quantipunctorum sp. nov. The type strains are Rhodococcus navarretei EXRC-4A-4T and Pseudarthrobacter quantipunctorum RC-2-3T. These strains have been deposited deposited in the CChRGM and BCCM/LMG culture collections with entry numbers RGM 3539/LMG 33621 and RGM 3538/LMG 33620, respectively.

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The CMNR group comprises bacteria of the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus and share cell wall and DNA content characteristics. Many pathogenic CMNR bacteria cause diseases such as mastitis, lymphadenitis, and pneumonia in farmed animals, which cause economic losses for breeders and represent a threat to public health. Traditional diagnosis in CMNR involves isolating target bacteria on general or selective media and conducting metabolic analyses with the assistance of laboratory biochemical identification systems. Advanced mass spectrometry may also support diagnosing these bacteria in the clinic's daily routine despite some challenges, such as the need for isolated bacteria. In difficult identification among some CMNR members, molecular methods using polymerase chain reaction (PCR) emerge as reliable options for correct specification that is sometimes achieved directly from clinical samples such as tracheobronchial aspirates and feces. On the other hand, immunological diagnostics such as the skin test or Enzyme-Linked Immunosorbent Assay (ELISA) for Mycobacterium tuberculosis yield promising results in subclinical infections with no bacterial growth involved. In this review, we present the methods most commonly used to diagnose pathogenic CMNR bacteria and discuss their advantages and limitations, as well as challenges and perspectives on adopting new technologies in diagnostics.

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BACKGROUND: Quantum Dots (QDs) are fluorescent nanoparticles with exceptional optical and optoelectronic properties, finding widespread utility in diverse industrial applications. Presently, chemically synthesized QDs are employed in solar cells, bioimaging, and various technological domains. However, many applications demand QDs with prolonged lifespans under conditions of high-energy radiation. Over the past decade, microbial biosynthesis of nanomaterials has emerged as a sustainable and cost-effective process. In this context, the utilization of extremophile microorganisms for synthesizing QDs with unique properties has recently been reported. RESULTS: In this study, UV-resistant bacteria were isolated from one of the most extreme environments in Antarctica, Union Glacier at the Ellsworth Mountains. Bacterial isolates, identified through 16 S sequencing, belong to the genera Rhodococcus, Pseudarthrobacter, and Arthrobacter. Notably, Rhodococcus sp. (EXRC-4 A-4), Pseudarthrobacter sp. (RC-2-3), and Arthrobacter sp. (EH-1B-1) tolerate UV-C radiation doses ≥ 120 J/m². Isolated UV-resistant bacteria biosynthesized CdS QDs with fluorescence intensities 4 to 8 times higher than those biosynthesized by E. coli, a mesophilic organism tolerating low doses of UV radiation. Transmission electron microscopy (TEM) analysis determined QD sizes ranging from 6 to 23 nm, and Fourier-transform infrared (FTIR) analysis demonstrated the presence of biomolecules. QDs produced by UV-resistant Antarctic bacteria exhibit high photostability after exposure to UV-B radiation, particularly in comparison to those biosynthesized by E. coli. Interestingly, red fluorescence-emitting QDs biosynthesized by Rhodococcus sp. (EXRC-4 A-4) and Arthrobacter sp. (EH-1B-1) increased their fluorescence emission after irradiation. Analysis of methylene blue degradation after exposure to irradiated QDs biosynthesized by UV-resistant bacteria, indicates that the QDs transfer their electrons to O2 for the formation of reactive oxygen species (ROS) at different levels. CONCLUSIONS: UV-resistant Antarctic bacteria represent a novel alternative for the sustainable generation of nanostructures with increased radiation tolerance-two characteristics favoring their potential application in technologies requiring continuous exposure to high-energy radiation.

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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.

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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.

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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.

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Rhodococcus is a genus of actinomycetes that has been explored by the scientific community for different purposes, especially for bioremediation uses. However, the mechanisms governing Rhodococcus-mediated bioremediation processes are far from being fully elucidated. In this sense, this work aimed to compile the recent advances in the use of Rhodococcus for the bioremediation of organic and inorganic contaminants present in different environmental compartments. We reviewed the bioremediation capacity and mechanisms of Rhodococcus spp. in the treatment of polycyclic aromatic hydrocarbons, phenolic substances, emerging contaminants, heavy metals, and dyes given their human health risks and environmental concern. Different bioremediation techniques were discussed, including experimental conditions, treatment efficiencies, mechanisms, and degradation pathways. The use of Rhodococcus strains in the bioremediation of several compounds is a promising approach due to their features, primarily the presence of appropriate enzyme systems, which result in high decontamination efficiencies; but that vary according to experimental conditions. Besides, the genus Rhodococcus contains a small number of opportunistic species and pathogens, representing an advantage from the point of view of safety. Advances in analytical detection techniques and Molecular Biology have been collaborating to improve the understanding of the mechanisms and pathways involved in bioremediation processes. In the context of using Rhodococcus spp. as bioremediation agents, there is a need for more studies that 1) evaluate the role of these actinomycetes on a pilot and field scale; 2) use genetic engineering tools and consortia with other microorganisms to improve the bioremediation efficiency; and 3) isolate new Rhodococcus strains from environments with extreme and/or contaminated conditions aiming to explore their adaptive capabilities for bioremediation purposes.

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Production of transgenic plants with desired agronomic and horticultural traits has gained great importance to fulfill demands of the growing population. Genetic transformation is also a fundamental step to study basics of plant sciences. Different transformation protocols have been developed and used which are reliable and efficient. These protocols used antibiotic or herbicide resistance genes incorporated along with gene of interest to identify transformed plants from non-transformed ones. These marker genes may pose a threat to human and environment. Use of visual markers enables direct and easier observation of transformed plants with more precision. In current study a gene cassette with 'pigment production hydroxylase (PPH) gene under fiber specific promoter (GhSCFP) and downstream Nos-terminator was designed. After checking the structural and functional efficiency of codon optimized gene using bioinformatics tools, the cassette was sent for chemical synthesis from commercial source. The pigment gene cassette (PPH_CEMB), cloned in pCAMBIA-1301, was transformed into Agrobacterium through electroporation. Agrobacterium-mediated floral dip method was used to transform Camelina sativa inflorescence. After seed setting a total of 600 seed were observed for change in color and out of these, 19 seeds developed a reddish-brown coloration, while the remaining 581 seeds remained yellow. The transformation efficiency calculated on basis of color change was 1.0%. PCR analysis of leaves obtained after sowing reddish seeds confirmed the transformation of pigment production gene, while no PCR amplification was observed in leaves of plants from wild type seeds. From the results it is evident that Agrobacterium-mediated transformation of C. sativa inflorescence is very efficient and environment friendly technique not only for detection of transformed plants but also to study basic cellular processes.

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BACKGOUND: Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy. METHODS: We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2. RESULTS: All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules. CONCLUSIONS: Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications. GENERAL SIGNIFICANCE: Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.

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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.

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Abstract An increasing production of natural rubber (NR) products has led to major challenges in waste management. In this study, the degradation of rubber latex gloves in a mineral salt medium (MSM) using a bacterial consortium, a mixed culture of the selected bacteria and a pure culture were studied. The highest 18% weight loss of the rubber gloves were detected after incubated with the mixed culture. The increased viable cell counts over incubation time indicated that cells used rubber gloves as sole carbon source leading to the degradation of the polymer. The growth behavior of NR-degrading bacteria on the latex gloves surface was investigated using the scanning electron microscope (SEM). The occurrence of the aldehyde groups in the degradation products was observed by Fourier Transform Infrared Spectroscopy analysis. Rhodococcus pyridinivorans strain F5 gave the highest weight loss of rubber gloves among the isolated strain and posses latex clearing protein encoded by lcp gene. The mixed culture of the selected strains showed the potential in degrading rubber within 30 days and is considered to be used efficiently for rubber product degradation. This is the first report to demonstrate a strong ability to degrade rubber by Rhodococcus pyridinivorans.

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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.

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An increasing production of natural rubber (NR) products has led to major challenges in waste management. In this study, the degradation of rubber latex gloves in a mineral salt medium (MSM) using a bacterial consortium, a mixed culture of the selected bacteria and a pure culture were studied. The highest 18% weight loss of the rubber gloves were detected after incubated with the mixed culture. The increased viable cell counts over incubation time indicated that cells used rubber gloves as sole carbon source leading to the degradation of the polymer. The growth behavior of NR-degrading bacteria on the latex gloves surface was investigated using the scanning electron microscope (SEM). The occurrence of the aldehyde groups in the degradation products was observed by Fourier Transform Infrared Spectroscopy analysis. Rhodococcus pyridinivorans strain F5 gave the highest weight loss of rubber gloves among the isolated strain and posses latex clearing protein encoded by lcp gene. The mixed culture of the selected strains showed the potential in degrading rubber within 30 days and is considered to be used efficiently for rubber product degradation. This is the first report to demonstrate a strong ability to degrade rubber by Rhodococcus pyridinivorans.

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A novel actinobacterium, designated strain CMAA 1533T, was isolated from the rhizosphere of Deschampsia antarctica collected at King George Island, Antarctic Peninsula. Strain CMAA 1533T was found to grow over a wide range of temperatures (4-28 °C) and pH (4-10). Macroscopically, the colonies were observed to be circular shaped, smooth, brittle and opaque-cream on most of the culture media tested. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CMAA 1533T belongs to the family Nocardiaceae and forms a distinct phyletic line within the genus Rhodococcus. Sequence similarity calculations indicated that the novel strain is closely related to Rhodococcus degradans CCM 4446T, Rhodococcus erythropolis NBRC 15567T and Rhodococcus triatomae DSM 44892T (≤ 96.9%). The organism was found to contain meso-diaminopimelic acid, galactose and arabinose in whole cell hydrolysates. Its predominant isoprenologue was identified as MK-8(H2) and the polar lipids as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The major fatty acids were identified as Summed feature (C16:1 ω6c and/or C16:1 ω7c), C16:0, C18:1 ω9c and 10-methyl C18:0. The G+C content of genomic DNA was determined to be 65.5 mol%. Unlike the closely related type strains, CMAA 1533T can grow at 4 °C but not at 37 °C and was able to utilise adonitol and galactose as sole carbon sources. Based on phylogenetic, chemotaxonomic and physiological data, it is concluded that strain CMAA 1533T (= NRRL B-65465T = DSM 104532T) represents a new species of the genus Rhodococcus, for which the name Rhodococcus psychrotolerans sp. nov. is proposed.

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Rhodococcus opacus PD630 accumulates significant amounts of triacylglycerols (TAG), but is not able to de novo synthesize wax esters (WE) from structural unrelated carbon sources, such as gluconate. In this study, strain PD630 was engineered to produce WE by heterologous expression of maqu_2220 gene, which encodes a fatty acyl-CoA reductase for the production of fatty alcohols in Marinobacter hydrocarbonoclasticus. Recombinant cells produced ca. 46% of WE and 54% of TAG (of total WE+TAG) from gluconate compared with the wild type, which produced 100% of TAG. Cell growth was not affected by the heterologous expression of MAQU_2220. Several saturated and monounsaturated WE species were produced by cells, with C18:C16, C16:C16 and C16:C18 as main species. The fatty acid composition of WE fraction in PD630maqu_2220 was enriched with C16:0, C18:0, whereas C16:0, C18:0 and C18:1 predominated in the TAG fraction. Significant amounts of WE and TAG were accumulated by PD630maqu_2220 from whey, an inexpensive waste material from dairy industries, without affecting cell biomass production. This is the first report on WE synthesis by R. opacus from gluconate, which demonstrates that lipid metabolism of this bacterium is flexible enough to assimilate heterologous components for the production of new lipid derivatives with industrial interest.

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BACKGROUND: Glycosylation is one of the most abundant posttranslational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. There is growing evidence about the importance of these modifications in host bacteria interactions in tuberculosis. It is known, that the sugars present in some Mycobacterium tuberculosis glycoproteins play an important role in both humoral and cellular immune response against the pathogen. Since this modification is lost in the recombinant proteins expressed in Escherichia coli, it is fundamental to search for host bacteria with the capacity to modify the foreign proteins. Amongst the bacteria that are likely to have this possibility are some members of Rhodococcus genus which are Gram-positive bacteria, with high GC-content and genetically very close related to M. tuberculosis. RESULTS: In this work, apa, pstS1 and lprG genes that coding for M. tuberculosis glycoproteins were cloned and expressed in Rhodococcus erythropolis. All recombinant proteins were mannosylated as demonstrated by their interaction with mannose binding lectin Concanavalin A. In addition, as native proteins recombinants Apa and PstS1 were secreted to the culture medium in contrast with LprG that was retained in the cell wall. CONCLUSIONS: Together these results, point out R. erythropolis, as a new host for expression of M. tuberculosis glycoproteins.

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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.

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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.

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Recently, there has been a lot of interest in the utilization of rhodococci in the bioremediation of petroleum contaminated environments. This study investigates the response of Rhodococcus erythropolis IBBPo1 cells to 1% organic solvents (alkanes, aromatics). A combination of microbiology, biochemical, and molecular approaches were used to examine cell adaptation mechanisms likely to be pursued by this strain after 1% organic solvent exposure. R. erythropolis IBBPo1 was found to utilize 1% alkanes (cyclohexane, n-hexane, n-decane) and aromatics (toluene, styrene, ethylbenzene) as the sole carbon source. Modifications in cell viability, cell morphology, membrane permeability, lipid profile, carotenoid pigments profile and 16S rRNA gene were revealed in R. erythropolis IBBPo1 cells grown 1 and 24 h on minimal medium in the presence of 1% alkanes (cyclohexane, n-hexane, n-decane) and aromatics (toluene, styrene, ethylbenzene). Due to its environmental origin and its metabolic potential, R. erythropolis IBBPo1 is an excellent candidate for the bioremediation of soils contaminated with crude oils and other toxic compounds. Moreover, the carotenoid pigments produced by this nonpathogenic Gram-positive bacterium have a variety of other potential applications.

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