Degradation of gaseous hydrocarbons in aerated stirred bioreactors inoculated with Rhodococcus erythropolis: Effect of the carbon source and SIFT-MS method development.

The study of microbial hydrocarbons removal is of great importance for the development of future bioremediation strategies. In this study, we evaluated the removal of a gaseous mixture containing toluene, m-xylene, ethylbenzene, cyclohexane, butane, pentane, hexane and heptane in aerated stirred bioreactors inoculated with Rhodococcus erythropolis and operated under non-sterile conditions. For the real-time measurement of hydrocarbons, a novel systematic approach was implemented using Selected-Ion Flow Tube Mass Spectrometry (SIFT-MS). The effect of the carbon source (∼9.5 ppmv) on (i) the bioreactors' performance (BR1: dosed with only cyclohexane as a single hydrocarbon versus BR2: dosed with a mixture of the 8 hydrocarbons) and (ii) the evolution of microbial communities over time were investigated. The results showed that cyclohexane reached a maximum removal efficiency (RE) of 53% ± 4% in BR1. In BR2, almost complete removal of toluene, m-xylene and ethylbenzene, being the most water-soluble and easy-to-degrade carbon sources, was observed. REs below 32% were obtained for the remaining compounds. By exposing the microbial consortium to only the five most recalcitrant hydrocarbons, REs between 45% ± 5% and 98% ± 1% were reached. In addition, we observed that airborne microorganisms populated the bioreactors and that the type of carbon source influenced the microbial communities developed. The abundance of species belonging to the genus Rhodococcus was below 10% in all bioreactors at the end of the experiments. This work provides fundamental insights to understand the complex behavior of gaseous hydrocarbon mixtures in bioreactors, along with a systematic approach for the development of SIFT-MS methods.

Enhancing the stress resistance of nitrile hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and ß- subunits ligation.

BACKGROUND: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and ß-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. METHODS AND RESULTS: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of ß-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of ß-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. CONCLUSION: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.

Simultaneously degradation of various phthalate esters by Rhodococcus sp. AH-ZY2: Strain, omics and enzymatic study.

Phthalate esters (PAEs) are widely used as plasticizers and cause serious complex pollution problem in environment. Thus, strains with efficient ability to simultaneously degrade various PAEs are required. In this study, a newly isolated strain Rhodococcus sp. AH-ZY2 can degrade 500 mg/L Di-n-octyl phthalate completely within 16 h and other 500 mg/L PAEs almost completely within 48 h at 37 °C, 180 rpm, and 2 % (v/v) inoculum size of cultures with a OD600 of 0.8. OD600 = 0.8, 2 % (v/v). Twenty genes in its genome were annotated as potential esterase and four of them (3963, 4547, 5294 and 5359) were heterogeneously expressed and characterized. Esterase 3963 and 4547 is a type I PAEs esterase that hydrolyzes PAEs to phthalate monoesters. Esterase 5294 is a type II PAEs esterase that hydrolyzes phthalate monoesters to phthalate acid (PA). Esterase 5359 is a type III PAEs esterase that simultaneously degrades various PAEs to PA. Molecular docking results of 5359 suggested that the size and indiscriminate binding feature of spacious substrate binding pocket may contribute to its substrate versatility. AH-ZY2 is a potential strain for efficient remediation of PAEs complex pollution in environment. It is first to report an esterase that can efficiently degrade mixed various PAEs.

Judicial Opinion 130.

Opinion 130 deals with a Request for an Opinion asking the Judicial Commission to clarify whether the genus name Rhodococcus Zopf 1891 (Approved Lists 1980) is illegitimate. The Request is approved and an answer is given. The name Rhodococcus Zopf 1891 (Approved Lists 1980) is illegitimate because it is a later homonym of the validly published cyanobacterial name Rhodococcus Hansgirg 1884. The Judicial Commission also clarifies that it has the means to resolve such cases by conserving a name over an earlier homonym. It is concluded that the name Rhodococcus Zopf 1891 (Approved Lists 1980) is significantly more important than the name Rhodococcus Hansgirg 1884 and therefore the former is conserved over the latter. This makes the name Rhodococcus Zopf 1891 (Approved Lists 1980) legitimate.

Selective pressure leads to an improved synthetic consortium fit for dye degradation.

Microorganisms have great potential for bioremediation as they have powerful enzymes and machineries that can transform xenobiotics. The use of a microbial consortium provides more advantages in application point of view than pure cultures due to cross-feeding, adaptations, functional redundancies, and positive interactions among the organisms. In this study, we screened about 107 isolates for their ability to degrade dyes in aerobic conditions and without additional carbon source. From our screening results, we finally limited our synthetic consortium to Gordonia and Rhodococcus isolates. The synthetic consortium was trained and optimized for azo dye degradation using sequential treatment of small aromatic compounds such as phenols that act as selective pressure agents. After four rounds of optimization with different aims for each round, the consortium was able to decolorize and degrade various dyes after 48 h (80%-100% for brilliant black bn, methyl orange, and chromotrop 2b; 50-70% for orange II and reactive orange 16; 15-30% for chlorazol black e, reactive red 120, and allura red ac). Through rational approaches, we can show that treatment with phenolic compounds at micromolar dosages can significantly improve the degradation of bulky dyes and increase its substrate scope. Moreover, our selective pressure approach led to the production of various dye-degrading enzymes as azoreductase, laccase-like, and peroxidase-like activities were detected from the phenol-treated consortium. Evidence of degradation was also shown as metabolites arising from the degradation of methyl red and brilliant black bn were detected using HPLC and LC-MS analysis. Therefore, this study establishes the importance of rational and systematic screening and optimization of a consortium. Not only can this approach be applied to dye degradation, but this study also offers insights into how we can fully maximize microbial consortium activity for other applications, especially in biodegradation and biotransformation.

Physiology and comparative genomics of the haloalkalitolerant and hydrocarbonoclastic marine strain Rhodococcus ruber MSA14.

Marine hydrocarbonoclastic bacteria can use polycyclic aromatic hydrocarbons as carbon and energy sources, that makes these bacteria highly attractive for bioremediation in oil-polluted waters. However, genomic and metabolic differences between species are still the subject of study to understand the evolution and strategies to degrade PAHs. This study presents Rhodococcus ruber MSA14, an isolated bacterium from marine sediments in Baja California, Mexico, which exhibits adaptability to saline environments, a high level of intrinsic pyrene tolerance (> 5 g L- 1), and efficient degradation of pyrene (0.2 g L- 1) by 30% in 27 days. Additionally, this strain demonstrates versatility by using naphthalene and phenanthrene as individual carbon sources. The genome sequencing of R. ruber MSA14 revealed a genome spanning 5.45 Mbp, a plasmid of 72 kbp, and three putative megaplasmids, lengths between 110 and 470 Kbp. The bioinformatics analysis of the R. ruber MSA14 genome revealed 56 genes that encode enzymes involved in the peripheral and central pathways of aromatic hydrocarbon catabolism, alkane, alkene, and polymer degradation. Within its genome, R. ruber MSA14 possesses genes responsible for salt tolerance and siderophore production. In addition, the genomic analysis of R. ruber MSA14 against 13 reference genomes revealed that all compared strains have at least one gene involved in the alkanes and catechol degradation pathway. Overall, physiological assays and genomic analysis suggest that R. ruber MSA14 is a new haloalkalitolerant and hydrocarbonoclastic strain toward a wide range of hydrocarbons, making it a promising candidate for in-depth characterization studies and bioremediation processes as part of a synthetic microbial consortium, as well as having a better understanding of the catabolic potential and functional diversity among the Rhodococci group.

Transcriptome Analysis Reveals the Biocontrol Mechanism of Endophytic Bacterium AM201, Rhodococcus sp., against Root Rot Disease of Atractylodes macrocephala.

Atractylodes macrocephala Koidz (AMK) is a perennial herb from the plant family Asteraceae (formerly Compositae). This herb is mainly distributed in mountainous wetlands in Zhejiang, Sichuan, Yunnan, and Hunan provinces of China. Its medicinal production and quality, however, are severely impacted by root rot disease. In our previous study, endophytic bacterium designated AM201 exerted a high biocontrol effect on the root rot disease of AMK. However, the molecular mechanisms underlying this effect remain unclear. In this study, the identity of strain AM201 as Rhodococcus sp. was determined through analysis of its morphology, physiological and biochemical characteristics, as well as 16S rDNA sequencing. Subsequently, we performed transcriptome sequencing and bioinformatics analysis to compare and analyze the transcriptome profiles of root tissues from two groups: AM201 (AMK seedlings inoculated with Fusarium solani [FS] and AM201) and FS (AMK seedlings inoculated with FS alone). We also conducted morphological, physiological, biochemical, and molecular identification analyses for the AM201 strain. We obtained 1,560 differentially expressed genes, including 187 upregulated genes and 1,373 downregulated genes. We screened six key genes (GOLS2, CIPK25, ABI2, egID, PG1, and pgxB) involved in the resistance of AM201 against AMK root rot disease. These genes play a critical role in reactive oxygen species (ROS) clearance, Ca2+ signal transduction, abscisic acid signal inhibition, plant root growth, and plant cell wall defense. The strain AM201 was identified as Rhodococcus sp. based on its morphological characteristics, physiological and biochemical properties, and 16S rDNA sequencing results. The findings of this study could enable to prevent and control root rot disease in AMK and could offer theoretical guidance for the agricultural production of other medicinal herbs.

The metabolic landscape of the bacteria Rhodococcus erythropolis used in the biodesulfurization of petroleum products: an emphasis on 2-hydroxybiphenyl.

Microorganisms produce diverse classes of metabolites under various physiological conditions. Many bacterial strains have been reported to carry out the process of desulfurization in a cost-effective manner by converting dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) and then using the 2-HBP as a carbon source for growth and development. Key rate-limiting factors and an increased concentration of 2HBP (400 µM) affect the biodesulfurization activity of bacteria through the produced metabolites. Thus, this study was designed to explore the nature of the metabolites produced by Rhodococcus erythropolis in the presence of DBT and 2HBP supplemented with a culture medium. A total of 330 metabolites were detected, and the key metabolites identified were 11Z-eicosaenoyl-EA, 1-carboxyethylisoleucine, 1(3)-glyceryl-PGF2alpha, taurine, 2-hydroxynicotinic acid, 4,4-dimethyl-14alpha-hydroxymethyl-5alpha-cholest-8-en-3beta-ol, and 10-nitrooleic acid. The supplementation of DBT and DBT-2HBP resulted in the differential regulation of these metabolites, either through downregulation or overexpression. Furthermore, at high concentrations of 2-HBP, 1-carboxyethylisoleucine, taurine, 2-hydroxynicotinic acid, and nicotinic acid were upregulated. This work proposes that the identified metabolites may play a role in bacteria-mediated desulphurization and could be beneficial in developing a cost-effective method of desulphurization for refining petroleum.

Taxonomic identification, phenol biodegradation and soil remediation of the strain Rhodococcus sacchari sp. nov. Z13T.

Phenols are highly toxic chemicals that are extensively used in industry and produce large amounts of emissions. Notably, phenols released into the soil are highly persistent, causing long-term harm to human health and the environment. In this study, a gram-positive, aerobic, and rod-shaped bacterial strain, Z13T, with efficient phenol degradation ability, was isolated from the soil of sugarcane fields. Based on the physiological properties and genomic features, strain Z13T is considered as a novel species of the genus Rhodococcus, for which the name Rhodococcus sacchari sp. nov. is proposed. The type strain is Z13T (= CCTCC AB 2022327T = JCM 35797T). This strain can use phenol as its sole carbon source. Z13T was able to completely degrade 1200 mg/L phenol within 20 h; the maximum specific growth rate was µmax = 0.93174 h-1, and the maximum specific degradation rate was qmax = 0.47405 h-1. Based on whole-genome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, strain Z13T contains a series of phenol degradation genes, including dmpP, CatA, dmpB, pcaG, and pcaH, and can metabolize aromatic compounds. Moreover, the potential of strain Z13T for soil remediation was investigated by introducing Z13T into simulated phenol-contaminated soil, and the soil microbial diversity was analyzed. The results showed that 100% of the phenol in the soil was removed within 7.5 d. Furthermore, microbial diversity analysis revealed an increase in the relative species richness of Oceanobacillus, Chungangia, and Bacillus.

Synthesis of Isoamyl Fatty Acid Ester, a Flavor Compound, by Immobilized Rhodococcus Cutinase.

Isoamyl fatty acid esters (IAFEs) are widely used as fruity flavor compounds in the food industry. In this study, various IAFEs were synthesized from isoamyl alcohol and various fatty acids using a cutinase enzyme (Rcut) derived from Rhodococcus bacteria. Rcut was immobilized on methacrylate divinylbenzene beads and used to synthesize isoamyl acetate, butyrate, hexanoate, octanoate, and decanoate. Among them, Rcut synthesized isoamyl butyrate (IAB) most efficiently. Docking model studies showed that butyric acid was the most suitable substrate in terms of binding energy and distance from the active site serine (Ser114) γ-oxygen. Up to 250 mM of IAB was synthesized by adjusting reaction conditions such as substrate concentration, reaction temperature, and reaction time. When the enzyme reaction was performed by reusing the immobilized enzyme, the enzyme activity was maintained at least six times. These results demonstrate that the immobilized Rcut enzyme can be used in the food industry to synthesize a variety of fruity flavor compounds, including IAB.

Assessment of biodegradation and toxicity of alternative plasticizer di(2-ethylhexyl) terephthalate: Impacts on microbial biofilms, metabolism, and reactive oxygen species-mediated stress response.

Although di(2-ethylhexyl) terephthalate (DOTP) is being widely adopted as a non-phthalate plasticizer, existing research primarily focuses on human and rat toxicity. This leaves a significant gap in our understanding of their impact on microbial communities. This study assessed the biodegradation and toxicity of DOTP on microbes, focusing on its impact on biofilms and microbial metabolism using Rhodococcus ruber as a representative bacterial strain. DOTP is commonly found in mass fractions between 0.6 and 20% v/v in various soft plastic products. This study used polyvinyl chloride films (PVC) with varying DOTP concentrations (range 1-10% v/v) as a surface for analysis of biofilm growth. Cell viability and bacterial stress responses were tested using LIVE/DEAD™ BacLight™ Bacterial Viability Kit and by the detection of reactive oxygen species using CellROX™ Green Reagent, respectively. An increase in the volume of dead cells (in the plastisphere biofilm) was observed with increasing DOTP concentrations in experiments using PVC films, indicating the potential negative impact of DOTP on microbial communities. Even at a relatively low concentration of DOTP (1%), signs of stress in the microbes were noticed, while concentrations above 5% compromised their ability to survive. This research provides a new understanding of the environmental impacts of alternative plasticizers, prompting the need for additional research into their wider effects on both the environment and human health.

Biosynthesis of photostable CdS quantum dots by UV-resistant psychrotolerant bacteria isolated from Union Glacier, Antarctica.

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.

Degradation of petroleum hydrocarbon contaminants by Rhodococcus erythropolis KB1 synergistic with alfalfa (Medicago sativa L.).

Petroleum hydrocarbons are a stubborn pollutant that is difficult to degrade globally, and plant-microbial degradation is the main way to solve this type of pollutant. In this study, the physiological and ecological responses of alfalfa to petroleum hydrocarbons in different concentrations of petroleum hydrocarbon-contaminated soil with KB1 (Rhodococcus erythropolis) were analyzed and determined by laboratory potting techniques. The growth of alfalfa (CK) and alfalfa with KB1 (JZ) in different concentrations of petroleum hydrocarbons contaminated soil was compared and analyzed. The results of the CK group showed that petroleum hydrocarbons could significantly affect the activity of alfalfa antioxidant enzyme system, inhibit the development of alfalfa roots and the normal growth of plants, especially in the high-concentration group. KB1 strain had the ability to produce IAA, form biofilm, fix nitrogen, produce betaine and ACC deaminase, and the addition of KB1 could improve the growth traits of alfalfa in the soil contaminated with different concentrations of petroleum hydrocarbons, the content of soluble sugars in roots, and the stress resistance and antioxidant enzyme activities of alfalfa. In addition, the degradation kinetics of the strain showed that the degradation rate of petroleum could reach 75.2% after soaking with KB1. Furthermore, KB1 can efficiently degrade petroleum hydrocarbons in advance and significantly alleviate the damage of high concentration of petroleum hydrocarbons to plant roots. The results showed that KB1 strains and alfalfa plants could effectively enhance the degradation of petroleum hydrocarbons, which provided new ideas for improving bioremediation strategies.

Cryo-EM structure of bacterial nitrilase reveals insight into oligomerization, substrate recognition, and catalysis.

Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.

Synergistic detoxification efficiency and mechanism of triclocarban degradation by a bacterial consortium in the liver-gut-microbiota axis of zebrafish (Danio rerio).

Triclocarban (TCC), an emerging organic contaminant, poses a potential threat to human health with long-term exposure. Here, Rhodococcus rhodochrous BX2 and Pseudomonas sp. LY-1 were utilized to degrade TCC at environmental related concentrations for enhancing TCC biodegradation and investigating whether the toxicity of intermediate metabolites is lower than that of the parent compound. The results demonstrated that the bacterial consortium could degrade TCC by 82.0% within 7 days. The calculated 96 h LC50 for TCC, as well as its main degradation product 3,4-Dichloroaniline (DCA) were 0.134 mg/L and 1.318 mg/L respectively. Biodegradation also alleviated histopathological lesions induced by TCC in zebrafish liver and gut tissues. Liver transcriptome analysis revealed that biodegradation weakened differential expression of genes involved in disrupted immune regulation and lipid metabolism caused by TCC, verified through RT-qPCR analysis and measurement of related enzyme activities and protein contents. 16 S rRNA sequencing indicated that exposure to TCC led to gut microbial dysbiosis, which was efficiently improved through TCC biodegradation, resulting in decreased relative abundances of major pathogens. Overall, this study evaluated potential environmental risks associated with biodegradation of TCC and explored possible biodetoxification mechanisms, providing a theoretical foundation for efficient and harmless bioremediation of environmental pollutants.

Metabolic cross-feeding between the competent degrader Rhodococcus sp. strain p52 and an incompetent partner during catabolism of dibenzofuran: Understanding the leading and supporting roles.

Microbial interactions, particularly metabolic cross-feeding, play important roles in removing recalcitrant environmental pollutants; however, the underlying mechanisms involved in this process remain unclear. Thus, this study aimed to elucidate the mechanism by which metabolic cross-feeding occurs during synergistic dibenzofuran degradation between a highly efficient degrader, Rhodococcus sp. strain p52, and a partner incapable of utilizing dibenzofuran. A bottom-up approach combined with pairwise coculturing was used to examine metabolic cross-feeding between strain p52 and Arthrobacter sp. W06 or Achromobacter sp. D10. Pairwise coculture not only promoted bacterial pair growth but also facilitated dibenzofuran degradation. Specifically, strain p52, acting as a donor, released dibenzofuran metabolic intermediates, including salicylic acid and gentisic acid, for utilization and growth, respectively, by the partner strains W06 and D10. Both salicylic acid and gentisic acid exhibited biotoxicity, and their accumulation inhibited dibenzofuran degradation. The transcriptional activity of the genes responsible for the catabolism of dibenzofuran and its metabolic intermediates was coordinately regulated in strain p52 and its cocultivated partners, thus achieving synergistic dibenzofuran degradation. This study provides insights into microbial metabolic cross-feeding during recalcitrant environmental pollutant removal.

Study on the estradiol degradation gene expression and resistance mechanism of Rhodococcus R-001 under low-temperature stress.

Estradiol (E2), an endocrine disruptor, acts by mimicking or interfering with the normal physiological functions of natural hormones within organisms, leading to issues such as endocrine system disruption. Notably, seasonal fluctuations in environmental temperature may influence the degradation speed of estradiol (E2) in the natural environment, intensifying its potential health and ecological risks. Therefore, this study aims to explore how bacteria can degrade E2 under low-temperature conditions, unveiling their resistance mechanisms, with the goal of developing new strategies to mitigate the threat of E2 to health and ecological safety. In this paper, we found that Rhodococcus equi DSSKP-R-001 (R-001) can efficiently degrade E2 at 30 °C and 10 °C. Six genes in R-001 were shown to be involved in E2 degradation by heterologous expression at 30 °C. Among them, 17ß-HSD, KstD2, and KstD3, were also involved in E2 degradation at 10 °C; KstD was not previously known to degrade E2. RNA-seq was used to characterize differentially expressed genes (DEGs) to explore the stress response of R-001 to low-temperature environments to elucidate the strain's adaptation mechanism. At the low temperature, R-001 cells changed from a round spherical shape to a long rod or irregular shape with elevated unsaturated fatty acids and were consistent with the corresponding genetic changes. Many differentially expressed genes linked to the cold stress response were observed. R-001 was found to upregulate genes encoding cold shock proteins, fatty acid metabolism proteins, the ABC transport system, DNA damage repair, energy metabolism and transcriptional regulators. In this study, we demonstrated six E2 degradation genes in R-001 and found for the first time that E2 degradation genes have different expression characteristics at 30 °C and 10 °C. Linking R-001 to cold acclimation provides new insights and a mechanistic basis for the simultaneous degradation of E2 under cold stress in Rhodococcus adaptation.

Transcriptional dynamics during Rhodococcus erythropolis infection with phage WC1.

BACKGROUND: Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. RESULTS: By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production. CONCLUSIONS: The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

DszA Catalyzes C-S Bond Cleavage through N5-Hydroperoxyl Formation.

Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N5OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.

An effective strategy for biodegradation of high concentration phenol in soil via biochar-immobilized Rhodococcus pyridinivorans B403.

High concentration of phenol residues in soil are harmful to human health and ecological safety. However, limited information is available on the in-situ bioremediation of phenol-contaminated soil using biochar as a carrier for bacteria. In this study, bamboo -derived biochar was screened as a carrier to assemble microorganism-immobilized composite with Rhodococcus pyridinivorans B403. Then, SEM used to observe the micromorphology of composite and its bioactivity was detected in solution and soil. Finally, we investigated the effects of free B403 and biochar-immobilized B403 (BCJ) on phenol biodegradation in two types of soils and different initial phenol concentrations. Findings showed that bacterial cells were intensively distributed in/onto the carriers, showing high survival. Immobilisation increased the phenol degradation rate of strain B403 by 1.45 times (37.7 mg/(L·h)). The phenol removed by BCJ in soil was 81% higher than free B403 on the first day. Moreover, the removal of BCJ remained above 51% even at phenol concentration of 1,500 mg/kg, while it was only 15% for free B403. Compared with the other treatment groups, BCJ showed the best phenol removal effect in both tested soils. Our results indicate that the biochar-B403 composite has great potential in the remediation of high phenol-contaminated soil.