Identification of an important function of CYP123: Role in the monooxygenase activity in a novel estradiol degradation pathway in bacteria.

Nowadays, 17ß-estradiol (E2) biodegradation pathway has still not been identified in bacteria. To bridge this gap, we have described a novel E2 degradation pathway in Rhodococcus sp. P14 in this study, which showed that estradiol could be first transferred to estrone (E1) and thereby further converted into 16-hydroxyestrone, and then transformed into opened estrogen D ring. In order to identify the genes, which may be responsible for the pathway, transcriptome analysis was performed during E2 degradation in strain P14. The results showed that the expression of a short-chain dehydrogenase (SDR) gene and a CYP123 gene in the same gene cluster could be induced significantly by E2. Based on gene analysis, this gene cluster was found to play an important role in transforming E2 to 16-hydroxyestrone. The function of CYP123 was unknown before this study, and was found to harbor the activity of 16-estrone hydratase. Moreover, the global response to E2 in strain P14 was also analyzed by transcriptome analysis. It was observed that various genes involved in the metabolism processes, like the TCA cycle, lipid and amino acid metabolism, as well as glycolysis showed a significant increase in mRNA levels in response to strain P14 that can use E2 as the single carbon source. Overall, this study provides us an in depth understanding of the E2 degradation mechanisms in bacteria and also sheds light about the ability of strain P14 to effectively use E2 as the major carbon source for promoting its growth.

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.

Rhodococcus spongiicola sp. nov. and Rhodococcus xishaensis sp. nov., from marine sponges.

Two novel Rhodococcus strains, LHW50502T and LHW51113T, were isolated from marine sponges obtained on Xisha Island, Hainan Province, PR China. Rods and cocci, typical characteristics of the genus Rhodococcus, were observed. The strains contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall hydrolysates and galactose, arabinose, ribose and glucose as the whole-cell sugars. The major fatty acid identified was C16 : 0. MK-8(H4) was the predominat menaquinone of both strains. Stains LHW50502T and LHW51113T had almost identical (99.6 %) 16S rRNA gene sequences but shared relatively low similarities with previously characterized Rhodococcus species (well below 98.7 %). The results of phylogenetic analysis supported their closest relationship; however, the average nucleotide identity and digital DNA-DNA hybridization values between these two strains indicated that they belonged to distinct species. Taken together, the results of this study indicate that strains LHW50502T and LHW51113T represent two novel species of the genus Rhodococcus, for which the names Rhodococcus spongiicola sp. nov. (type strain LHW50502T=DSM 106291T=CCTCC AA 2018033T) and Rhodococcus xishaensis sp. nov. (type strain LHW51113T=DSM 106204T=CCTCC AA 2018034T) are proposed.

Rhodococcus spelaei sp. nov., isolated from a cave, and proposals that Rhodococcus biphenylivorans is a later synonym of Rhodococcus pyridinivorans, Rhodococcus qingshengii and Rhodococcus baikonurensis are later synonyms of Rhodococcus erythropolis, and Rhodococcus percolatus and Rhodococcus imtechensis are later synonyms of Rhodococcus opacus.

Two novel actinobacterial strains, designated C9-5T and C3-43, were isolated from soil samples of a cave in Jeju Island, Republic of Korea, and subjected to taxonomic study by a polyphasic approach. The organisms exhibited a typical rod-coccus developmental cycle during growth and grew at 10-30 °C, pH 5-9 and 0-3 % (w/v) NaCl. In 92 single-copy core gene sequence analysis, strain C9-5T was loosely associated with Rhodococcus tukisamuensis, albeit sharing low 16S rRNA gene sequence similarity (97.4 %). A combination of morphological and chemotaxonomic characteristics supported assignment with the genus Rhodococcus. With respect to 16S rRNA gene sequence similarity, the novel isolates showed the highest identity to the type strain of Rhodococcus subtropicus (98.7 % sequence similarity), followed by Rhodococcus olei (98.5 %) and Rhodococcus pedocola (98.4 %).The average nucleotide identity and digital DNA-DNA hybridization values between strain C9-5T and members of the genus Rhodococcus were ≤81.5 and ≤37.1 %, respectively. A set of physiological and chemotaxonomic properties together with overall genomic relatedness differentiated the novel isolates from members of the genus Rhodococcus, for which the name Rhodococcus spelaei sp. nov. is proposed. The type strain is C9-5T (=KACC 19822T=DSM 107558T). Based on genome analysis performed here, it is also proposed that Rhodococcus biphenylivorans Su et al. 2015 is a later heterotypic synonym of Rhodococcus pyridinivorans Yoon et al. 2000, Rhodococcus qingshengii Xu et al. 2007 and Rhodococcus baikonurensis Li et al. 2004 are later heterotypic synonyms of Rhodococcus erythropolis (Gray and Thornton 1928) Goodfellow and Alderson 1979 (Approved Lists 1980), and Rhodococcus percolatus Briglia et al. 1996 and Rhodococcus imtechensis Ghosh et al. 2006 are later heterotypic synonyms of Rhodococcus opacus Klatte et al. 1995.

Catabolic enzyme activities during biodegradation of three-ring PAHs by novel DTU-1Y and DTU-7P strains isolated from petroleum-contaminated soil.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants having health hazards. PAH-utilizing bacterial strains were isolated from petroleum-contaminated soil from siding area, Bijwasan supply location of BPCL, Delhi, India. Bacterial strains with different morphology were isolated and acclimatized to a mixture of low molecular weight PAH compounds in the concentration range of 50-10,000 mg/L. Two bacterial strains surviving at 10,000 mg/L PAH concentration were identified as Kocuria flava and Rhodococcus pyridinivorans, based on 16S rRNA gene sequencing and phylogenetic analysis over MEGA X, are reported for the first time for PAH degradation. The strain K. flava could degrade phenanthrene, anthracene, and fluorene with efficiency of 55.13%, 59.01%, and 63.46%, whereas R. pyridinivorans exhibited 62.03%, 64.99%, and 66.79% degradation for respective PAHs at initial PAH concentration of 10 mg/L. Slightly lower degradation of phenanthrene could be attributed to its more stable chemical structure. The consortium of both the strains degraded 61.32%, 64.72%, and 66.64%, of 10 mg/L of phenanthrene, anthracene, and fluorene, respectively, in 15 days of incubation period indicating no synergistic or antagonistic effect towards degradation. Catechol 2,3-dioxygenase (C23O), dehydrogenase and peroxidase enzyme activities during PAH degradation coincided with degradation of PAHs, thus highlighting the role of these enzymes in catabolising three-ring PAHs. This is the first investigation confirming the participation of C23O, dehydrogenase and peroxidases enzyme profiles throughout the period of degradation. The study concludes that these strains can play significant role in microbial remediation of PAH-contaminated environment.

A bifunctional enzyme belonging to cytochrome P450 family involved in the O-dealkylation and N-dealkoxymethylation toward chloroacetanilide herbicides in Rhodococcus sp. B2.

BACKGROUND: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown. RESULTS: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1 °C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCDB2), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthRB2), a ferredoxin reductase (EthAB2), a cytochrome P450 monooxygenase (EthBB2), a ferredoxin (EthCB2) and a 10-kDa protein of unknown function (EthDB2). Complementation with EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCDB2 was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthDB2 acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. CONCLUSIONS: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.

Biodegradation of phenol by a highly tolerant strain Rhodococcus ruber C1: Biochemical characterization and comparative genome analysis.

A novel phenol-degrading strain was isolated and identified as Rhodococcus ruber C1. The degradation analysis shows that 1806 mg/L of phenol can be completely degraded by strain C1 within 38 h, and the maximum specific growth rate (µmax=1.527 h-1) and maximum specific phenol degradation rate (qmax=3.674 h-1) indicate its excellent phenol metabolism capability. More importantly, phenol can be degraded by strain C1 in the temperature range of 20-45 °C within 72 h, and with longer degradation time, phenol can be completely degraded even at 10, 15 and 50 °C. The whole genome of strain C1 was sequenced, and a comparative genome analysis of strain C1 with 36 other genomes of Rhodococcus was performed. A remarkable gene family expansion occurred during the evolution of Rhodococcus, and a comprehensive evolutionary picture of Rhodococcus at genomic level was presented. Moreover, the copy number of genes involved in phenol metabolism was compared among genus Rhodococcus, and the results demonstrate high phenol degradation capability of strain C1 at genomic level. These findings suggest that Rhodococcus ruber C1 is a bacterium capable of degrading phenol efficiently in the temperature range of 10-50 °C.

Sequence analysis of 16S rDNA, gyrB and alkB genes of plant-associated Rhodococcus species from Tunisia.

The genus Rhodococcus contains several species with agricultural, biotechnological and ecological importance. Within this genus, many phyllosphere, rhizosphere and endosphere strains are plant growth promoting bacteria, whereas strains designated as R. fascians are plant pathogens. In this study, we isolated 47 Rhodococcus strains from a range of herbaceous and woody plant species. Phylogenetic analysis based on 16S rDNA, gyrB and alkB genes was used to compare our strains with type strains of Rhodococcus. For most of our strains, sequence similarity of the 16S rDNA, gyrB and alkB regions to type strains ranged from 98-100 %. Results of the concatenated gene sequence comparisons identified 18 strains of R. fascians and three strains of R. kroppenstedtii. The remaining strains were unclassified, and may represent novel species of Rhodococcus. Phylogenetic analysis based on gyrB sequences provided a more precise classification of our strains to species level than 16S rDNA sequences, whereas analysis of alkB sequences was unable to identify strains with orange-coloured colonies to species level.

Rhodococcus cavernicola sp. nov., isolated from a cave, and Rhodococcus degradans is a later heterosynonym of Rhodococcus qingshengii.

A Gram-reaction-positive, strictly aerobic, catalase-positive, oxidase-negative, non-motile actinobacterium, designated C1-24T, was isolated from a soil sample collected inside a natural cave. The organism exhibited a rod-coccus developmental cycle during its growth phase. Results of 16S rRNA gene-based phylogenetic analysis showed that the novel strain belonged to the genus Rhodococcus and formed a distinct sublineage at the base of the radiation including a Rhodococcus enclensis-Rhodococcus kroppenstedtii-Rhodococcus corynebacterioides-Rhodococcus trifoli cluster. In the results of phylogenomic analysis, the novel strain was loosely associated to Rhodococcus corynebacterioides. The closest relatives were Rhodococcus qingshengii (98.01 % 16S rRNA gene sequence similarity) and Rhodococcus degradans (98.01 %). The genome size was 5.66 Mbp and the DNA G+C content was 64.30 mol%. Whole-cell hydrolysates contained meso-diaminopimelic acid, arabinose and galactose as the diagnostic diamino acid and sugars. MK-8(H2) was the predominant menaquinone. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, an unidentified glycolipid and three unidentified phospholipids. Mycolic acids were present. The major fatty acids were C16 : 0, C18 : 1 ω9c, C16 : 1 ω7c and/or C16 : 1 ω6c and 10-methyl C18 : 0. Digital DNA-DNA hybridization and average nucleotide identity values revealed that the novel strain should be assigned to a different species. Based on the combined data obtained here, strain C1-24T (=KACC 19964T=DSM 109484T) represents a new species of the genus Rhodococcus, for which Rhodococcus cavernicola sp. nov. is proposed. Also, it is proposed that R. degradans is a later heterosynonym of R. qingshengii based on analyses of 16S rRNA gene and whole-genome sequences.

Rhodococcus oryzae sp. nov., a novel actinobacterium isolated from rhizosphere soil of rice (Oryza sativa L.).

A Gram-stain-positive, non-motile, creamy-white actinobacterium, which has an elementary branching rod-coccus life cycle was isolated from the rhizosphere soil of rice (Oryza sativa L.) collected from Northeast Agricultural University in Harbin, Heilongjiang province, north-east PR China, and its taxonomic status was examined by using a polyphasic approach. Results from the 16S rRNA gene sequence study showed that the isolate, designated strain NEAU-CX67T, belonged to the genus Rhodococcus and formed a cluster with Rhodococcus maanshanensis DSM 44675T, Rhodococcus kronopolitis NEAU-ML12T and Rhodococcus tukisamuensis JCM 11308T (98.3, 98.1 and 97.7% gene sequence similarity, respectively). The major fatty acids were C16 : 0, 10-methyl C18 : 0, C18 : 1 ω9c and C16 : 1 ω7c. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The major isoprenoid quinone was MK-8(H2). Whole-cell hydrolysates contained meso-diaminopimelic acid. Arabinose, galactose and ribose were detected as diagnostic sugars from whole-cell hydrolysates. Mycolic acids were detected. The genomic DNA G+C content of strain NEAU-CX67T was 64.6 mol%. Strain NEAU-CX67T exhibited low average nucleotide identity and digital DNA-DNA hybridization values with R. maanshanensis DSM 44675T (92.1 and 45.4 %) and R. tukisamuensis JCM 11308T (81.9 and 24.4 %). On the basis of results of phylogenetic, genotypic, physiological and chemotaxonomic analysis, strain NEAU-CX67T is considered to represent a novel species of the genus Rhodococcus for which the name Rhodococcus oryzae sp. nov. is proposed. The type strain is NEAU-CX67T (=DSM 107701T=CCTCC AB 2018233T).

Exploring the abundance of oleate hydratases in the genus Rhodococcus-discovery of novel enzymes with complementary substrate scope.

Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD+-dependent oxidation of 10-hydroxystearic acid.

Analysis of the biodegradative and adaptive potential of the novel polychlorinated biphenyl degrader Rhodococcus sp. WAY2 revealed by its complete genome sequence.

The complete genome sequence of Rhodococcus sp. WAY2 (WAY2) consists of a circular chromosome, three linear replicons and a small circular plasmid. The linear replicons contain typical actinobacterial invertron-type telomeres with the central CGTXCGC motif. Comparative phylogenetic analysis of the 16S rRNA gene along with phylogenomic analysis based on the genome-to-genome blast distance phylogeny (GBDP) algorithm and digital DNA-DNA hybridization (dDDH) with other Rhodococcus type strains resulted in a clear differentiation of WAY2, which is likely a new species. The genome of WAY2 contains five distinct clusters of bph, etb and nah genes, putatively involved in the degradation of several aromatic compounds. These clusters are distributed throughout the linear plasmids. The high sequence homology of the ring-hydroxylating subunits of these systems with other known enzymes has allowed us to model the range of aromatic substrates they could degrade. Further functional characterization revealed that WAY2 was able to grow with biphenyl, naphthalene and xylene as sole carbon and energy sources, and could oxidize multiple aromatic compounds, including ethylbenzene, phenanthrene, dibenzofuran and toluene. In addition, WAY2 was able to co-metabolize 23 polychlorinated biphenyl congeners, consistent with the five different ring-hydroxylating systems encoded by its genome. WAY2 could also use n-alkanes of various chain-lengths as a sole carbon source, probably due to the presence of alkB and ladA gene copies, which are only found in its chromosome. These results show that WAY2 has a potential to be used for the biodegradation of multiple organic compounds.

A new Rhodococcus aetherivorans strain isolated from lubricant-contaminated soil as a prospective phenol-biodegrading agent.

Microbe-based decontamination of phenol-polluted environments has significant advantages over physical and chemical approaches by being relatively cheaper and ensuring complete phenol degradation. There is a need to search for commercially prospective bacterial strains that are resistant to phenol and other co-pollutants, e.g. oil hydrocarbons, in contaminated environments, and able to carry out efficient phenol biodegradation at a variable range of concentrations. This research characterizes the phenol-biodegrading ability of a new actinobacteria strain isolated from a lubricant-contaminated soil environment. Phenotypic and phylogenetic analyses showed that the novel strain UCM Ac-603 belonged to the species Rhodococcus aetherivorans, and phenol degrading ability was quantitatively characterized for the first time. R. aetherivorans UCM Ac-603 tolerated and assimilated phenol (100% of supplied concentration) and various hydrocarbons (56.2-94.4%) as sole carbon sources. Additional nutrient supplementation was not required for degradation and this organism could grow at a phenol concentration of 500 mg L-1 without inhibition. Complete phenol assimilation occurred after 4 days at an initial concentration of 1750 mg L-1 for freely-suspended cells and at 2000 mg L-1 for vermiculite-immobilized cells: 99.9% assimilation of phenol was possible from a total concentration of 3000 mg L-1 supplied at daily fractional phenol additions of 750 mg L-1 over 4 days. In terms of phenol degradation rates, R. aetherivorans UCM Ac-602 showed efficient phenol degradation over a wide range of initial concentrations with the rates (e.g. 35.7 mg L-1 h-1 at 500 mg L-1 phenol, and 18.2 mg L-1 h-1 at 1750 mg L-1 phenol) significantly exceeding (1.2-5 times) reported data for almost all other phenol-assimilating bacteria. Such efficient phenol degradation ability compared to currently known strains and other beneficial characteristics of R. aetherivorans UCM Ac-602 suggest it is a promising candidate for bioremediation of phenol-contaminated environments.

Microaerobic conditions caused the overwhelming dominance of Acinetobacter spp. and the marginalization of Rhodococcus spp. in diesel fuel/crude oil mixture-amended enrichment cultures.

The aim of the present study was to reveal how different microbial communities evolve in diesel fuel/crude oil-contaminated environments under aerobic and microaerobic conditions. To investigate this question, aerobic and microaerobic bacterial enrichments amended with a diesel fuel/crude oil mixture were established and analysed. The representative aerobic enrichment community was dominated by Gammaproteobacteria (64.5%) with high an abundance of Betaproteobacteriales (36.5%), followed by Alphaproteobacteria (8.7%), Actinobacteria (5.6%), and Candidatus Saccharibacteria (4.5%). The most abundant alkane monooxygenase (alkB) genotypes in this enrichment could be linked to members of the genus Rhodococcus and to a novel Gammaproteobacterium, for which we generated a high-quality draft genome using genome-resolved metagenomics of the enrichment culture. Contrarily, in the microaerobic enrichment, Gammaproteobacteria (99%) overwhelmingly dominated the microbial community with a high abundance of the genera Acinetobacter (66.3%), Pseudomonas (11%) and Acidovorax (11%). Under microaerobic conditions, the vast majority of alkB gene sequences could be linked to Pseudomonas veronii. Consequently, results shed light on the fact that the excellent aliphatic hydrocarbon degrading Rhodococcus species favour clear aerobic conditions, while oxygen-limited conditions can facilitate the high abundance of Acinetobacter species in aliphatic hydrocarbon-contaminated subsurface environments.

Rhodococcus subtropicus sp. nov., a new actinobacterium isolated from a cave.

A novel Gram-stain-positive actinobacterial strain, designated C9-28T, was isolated from soil sampled in a natural cave on Jeju Island, Republic of Korea. Strain C9-28T morphologically exhibited a rod-coccus life cycle and grew at 10-37 °C (optimum, 30 °C), pH 6-9 (optimum, pH 7) and 0-3 % (optimum, absence of NaCl). In the maximum-likelihood tree based on 16S rRNA gene sequences, strain C9-28T formed a sublineage between a Rhodococcus equi-Rhodococcus soli-Rhodococcus agglutinans clade and the type strain of Rhodococcus defluvii. The closest relatives of strain C9-28T were the type strains of R. defluvii (98.88 % 16S rRNA gene sequence similarity), R. equi (98.88 %) and R. soli (98.60 %). The phylogenomic tree based on whole genome sequences supported the distinct position of the novel strain within the genus Rhodococcus. The following chemotaxonomic characteristics also supported the assignment to the genus: meso-diaminopimelic acid; arabinose and galactose in whole-cell hydrolysates; the predominant menaquinone of MK-8(H2); and polar lipids including diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, three unidentified glycolipids and two unidentified lipids. The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω9c and C14 : 0. Based on the values of average nucleotide identity and digital DNA-DNA hybridization from whole genome sequences, and in vitro DNA-DNA hybridization between the isolate and the closest relatives, strain C9-28T (=KACC 19823T=DSM 107559T) represents a novel species of the genus Rhodococcus, for which the name Rhodococcussubtropicus sp. nov. is proposed.

Characterization of trehalose lipids produced by a unique environmental isolate bacterium Rhodococcus qingshengii strain FF.

AIMS: The aim of this study was to elucidate the chemical properties and applications of trehalose lipids produced by Rhodococcus qingshengii strain FF and optimize its production yield. METHODS AND RESULTS: Strain FF was identified as R. qingshengii. It was observed to produce biosurfactants in the presence of n-hexadecane. The biosurfactants were identified as the mixture of trehalose triesters and trehalose tetraesters, mainly consisting of TrehC12 C3 C6 C12 :10, TrehC11 C8 C6 :6, TrehC11 C6 C4 :5 and TrehC6 C4 C6 :5 based on the analysis of thin layer chromatography, Fourier transform infrared and flight tandem mass spectrometry. The best carbon source and nitrogen source for producing trehalose lipids was the mixture of n-hexadecane and oleic acid (m : m = 1 : 1) and the organic nitrogen, urea. Under this condition, the production of trehalose lipids could reach 7·97 g l-1 . The crude trehalose lipids showed extremely high surface-active properties and were proven to promote the degradation of naphthalene. CONCLUSIONS: The trehalose lipids produced by R. qingshengii strain FF exhibited high surfactant activity under various conditions and were proven to promote the degradation of naphthalene. SIGNIFICANCE AND IMPACT OF THE STUDY: Rhodococcus qingshengii strain FF is a potential candidate for bioremediation. The trehalose lipids might be used as unique biosurfactants in cosmetic industries, biological formulations and other applications.

Comparative Analysis of Draft Genome Sequence of Rhodococcus sp. Eu-32 with Other Rhodococcus Species for Its Taxonomic Status and Sulfur Metabolism Potential.

Rhodococcus sp. Eu-32 has shown an extended novel dibenzothiophene desulfurization sulfur-specific 4S pathway and could remove significant amounts of organic sulfur from coal. Here, we present the draft genome sequence of Eu-32 with a genome size of approximately 5.61 Mb, containing 5065 protein coding sequences with a G+C content of 65.1%. The Rhodococcus sp. Eu-32 showed ~ 99% identity at the 16S rRNA gene sequence level while < 34% digital DNA-DNA hybridization and < 81% average nucleotide identity values with the genome sequence of most closely related known Rhodococcus species, suggesting that it is taxonomically different from the already reported Rhodococcus species. Among the annotated genes, 90 are involved in the metabolism of sulfur. Comparative genome analysis suggests many commonalities in sulfur metabolism gene sets that may have evolved due to many factors including ecological pressures. Our study and the genome sequence data will be available for further research and will provide insights into potential biotechnological and industrial applications of this bacterium.

New insights into the genome of Rhodococcus ruber strain Chol-4.

BACKGROUND: Rhodococcus ruber strain Chol-4, a strain isolated from a sewage sludge sample, is able to grow in minimal medium supplemented with several compounds, showing a broad catabolic capacity. We have previously determined its genome sequence but a more comprehensive study of their metabolic capacities was necessary to fully unravel its potential for biotechnological applications. RESULTS: In this work, the genome of R. ruber strain Chol-4 has been re-sequenced, revised, annotated and compared to other bacterial genomes in order to investigate the metabolic capabilities of this microorganism. The analysis of the data suggests that R. ruber Chol-4 contains several putative metabolic clusters of biotechnological interest, particularly those involved on steroid and aromatic compounds catabolism. To demonstrate some of its putative metabolic abilities, R. ruber has been cultured in minimal media containing compounds belonging to several of the predicted metabolic pathways. Moreover, mutants were built to test the naphtalen and protocatechuate predicted catabolic gene clusters. CONCLUSIONS: The genomic analysis and experimental data presented in this work confirm the metabolic potential of R. ruber strain Chol-4. This strain is an interesting model bacterium due to its biodegradation capabilities. The results obtained in this work will facilitate the application of this strain as a biotechnological tool.

Ecological impact assessment of a bioaugmentation site on remediation of chlorinated ethylenes by multi-omics analysis.

Bioremediation may affect the ecological system around bioremediation sites. However, little is known about how microbial community structures change over time after the initial injection of degraders. In this study, we have assessed the ecological impact of bioaugmentation using metagenomic and metatranscriptomic approaches to remove trichlorinated ethylene/cis-dichloroethylene (TCE/cDCE) by Rhodococcus jostii strain RHA1 as an aerobic chemical compound degrader. Metagenomic analysis showed that the number of organisms belonging to the genus Rhodococcus, including strain RHA1, increased from 0.1% to 76.6% of the total microbial community on day 0 at the injection site. Subsequently, the populations of strain RHA1 and other TCE/cDCE-degrading bacteria gradually decreased over time, whereas the populations of the anaerobic dechlorinators Geobacter and Dehalococcoides increased at later stages. Metatranscriptomic analysis revealed a high expression of aromatic compound-degrading genes (bphA1-A4) in strain RHA1 after RHA1 injection. From these results, we concluded that the key dechlorinators of TCE/cDCE were mainly aerobic bacteria, such as RHA1, until day 1, after which the key dechlorinators changed to anaerobic bacteria, such as Geobacter and Dehalococcocides, after day 6 at the injection well. Based on the α-diversity, the richness levels of the microbial community were increased after injection of strain RHA1, and the microbial community composition had not been restored to that of the original composition during the 19 days after treatment. These results provide insights into the assessment of the ecological impact and bioaugmentation process of RHA1 at bioremediation sites.

Rhodococcus daqingensis sp. nov., isolated from petroleum-contaminated soil.

A novel Gram-stain positive, aerobic, non-motile bacterial strain, designated Z1T, was isolated from a sample of petroleum-contaminated soil collected in Daqing, Heilongjiang province, China and characterised with a series of taxonomic approaches. The morphological and chemotaxonomic properties of the isolate were typical of those of members of the genus Rhodococcus. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Z1T belongs to the genus Rhodococcus and clustered with Rhodococcus maanshanensis DSM 44675T (99.2%, sequence similarity) and Rhodococcus tukisamuensis JCM 11308T (97.9%), respectively. However, the DNA-DNA hybridizations between strain Z1T and R. maanshanensis DSM 44675T and R. tukisamuensis JCM 11308T were both less than 70%. The optimal growth temperature and pH for strain Z1T were found to be at 28 °C and at pH 7.0. The peptidoglycan was found to contain meso-diaminopimelic acid; arabinose, galactose and glucose were detected as diagnostic sugars. The main polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified lipid; MK-8(H2) was found as the major menaquinone. The major fatty acids were identified as C16:0, 10-methyl C18:0 and C18:1ω9c. Mycolic acids were found to be present. The G + C content of the genomic DNA was determined to be 66.7 mol%. Based on a comparative analysis of phenotypic and genotypic characteristics, in combination with DNA-DNA hybridization results, strain Z1T can be distinguished from the type strains of its two close neighbours as a novel species of the genus Rhodococcus, for which the name Rhodococcus daqingensis sp. nov. is proposed. The type strain is Z1T (= CGMCC 1.13630T = DSM 107227T).