The mechanism of anthracene degradation by tryptophan -2,3-dioxygenase (T23D) in Comamonas testosteroni.

It is well known that anthracene is a persistent organic pollutant. Among the four natural polycyclic aromatic hydrocarbons (PAHs) degrading strains, Comamonas testosterone (CT1) was selected as the strain with the highest degradation efficiency. In the present study, prokaryotic transcriptome analysis of CT1 revealed an increase in a gene that encodes tryptophane-2,3-dioxygenase (T23D) in the anthracene and erythromycin groups compared to CK. Compared to the wild-type CT1 strain, anthracene degradation by the CtT23D knockout mutant (CT-M1) was significantly reduced. Compared to Escherichia coli (DH5α), CtT23D transformed DH5α (EC-M1) had a higher degradation efficiency for anthracene. The recombinant protein rT23D oxidized tryptophan at pH 7.0 and 37 °C with an enzyme activity of 2.42 ± 0.06 µmol min-1·mg-1 protein. In addition, gas chromatography-mass (GC-MS) analysis of anthracene degradation by EC-M1 and the purified rT23D revealed that 2-methyl-1-benzofuran-3-carbaldehyde is an anthracene metabolite, suggesting that it is a new pathway.

Critical Role of Monooxygenase in Biodegradation of 2,4,6-Trinitrotoluene by Buttiauxella sp. S19-1.

2,4,6-Trinitrotoluene (TNT) is an aromatic pollutant that is difficult to be degraded in the natural environment. The screening of efficient degrading bacteria for bioremediation of TNT has received much attention from scholars. In this paper, transcriptome analysis of the efficient degrading bacterium Buttiauxella sp. S19-1 revealed that the monooxygenase gene (BuMO) was significantly up-regulated during TNT degradation. S-ΔMO (absence of BuMO gene in S19-1 mutant) degraded TNT 1.66-fold less efficiently than strain S19-1 (from 71.2% to 42.9%), and E-MO mutant (Escherichia coli BuMO-expressing strain) increased the efficiency of TNT degradation 1.33-fold (from 52.1% to 69.5%) for 9 h at 180 rpm at 27 °C in LB medium with 1.4 µg·mL-1 TNT. We predicted the structure of BuMO and purified recombinant BuMO (rBuMO). Its specific activity was 1.81 µmol·min-1·mg-1 protein at pH 7.5 and 35 °C. The results of gas chromatography mass spectrometry (GC-MS) analysis indicated that 4-amino-2,6-dinitrotoluene (ADNT) is a metabolite of TNT biodegradation. We speculate that MO is involved in catalysis in the bacterial degradation pathway of TNT in TNT-polluted environment.

Cloning and identification of a new repressor of 3,17ß-Hydroxysteroid dehydrogenase of Comamonas testosteroni.

BACKGROUND: 3,17ß-hydroxysteroid dehydrogenase (3,17ß-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17ß-HSD. Previous reports showed that three tetR genes are located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first tetR gene encoded a potential repressor of 3,17ß-HSD by sensing environmental signals. However, whether the other proposed tetR genes act as repressors of 3,17ß-HSD are still unknown. METHODS AND RESULTS: In the present study, we cloned the second tetR gene and analyzed the regulatory mechanism of the protein on 3,17ß-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second tetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17ß-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17ß-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological genetic stability, and the expression of 3,17ß-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction. CONCLUSIONS: The second tetR gene acts as a negative regulator in 3,17ß-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment.

Degradation of 2,4,6-Trinitrotoluene (TNT): Involvement of Protocatechuate 3,4-Dioxygenase (P34O) in Buttiauxella sp. S19-1.

Extensive use and disposal of 2,4,6-trinitrotoluene (TNT), a primary constituent of explosives, pollutes the environment and causes severe damage to human health. Complete mineralization of TNT via bacterial degradation has recently gained research interest as an effective method for the restoration of contaminated sites. Here, screening for TNT degradation by six selected bacteria revealed that Buttiauxella sp. S19-1, possesses the strongest degrading ability. Moreover, BuP34O (a gene encoding for protocatechuate 3,4-dioxygenase-P34O, a key enzyme in the ß-ketoadipate pathway) was upregulated during TNT degradation. A knockout of BuP34O in S19-1 to generate S-M1 mutant strain caused a marked reduction in TNT degradation efficiency compared to S19-1. Additionally, the EM1 mutant strain (Escherichia coli DH5α transfected with BuP34O) showed higher degradation efficiency than DH5α. Gas chromatography mass spectrometry (GC-MS) analysis of TNT degradation by S19-1 revealed 4-amino-2,6-dinitrotolune (ADNT) as the intermediate metabolite of TNT. Furthermore, the recombinant protein P34O (rP34O) expressed the activity of 2.46 µmol/min·mg. Our findings present the first report on the involvement of P34O in bacterial degradation of TNT and its metabolites, suggesting that P34O could catalyze downstream reactions in the TNT degradation pathway. In addition, the TNT-degrading ability of S19-1, a Gram-negative marine-derived bacterium, presents enormous potential for restoration of TNT-contaminated seas.

Guidance Point Generation-Based Cooperative UGV Teleoperation in Unstructured Environment.

Teleoperation is widely used for unmanned ground vehicle (UGV) navigation in military and civilian fields. However, the human operator has to limit speed to ensure the handling stability because of the low resolution of video, limited field of view and time delay in the control loop. In this paper, we propose a novel guidance point generation method that is well suited for human-machine cooperative UGV teleoperation in unstructured environments without a predefined goal position. The key novelty of this method is that the guidance points used for navigation can be generated with only the local perception information of the UGV. Firstly, the locally occupied grid map (OGM) was generated utilizing a probabilistic grid state description method, and converted into binary image to constructed the convex hull of obstacle area. Secondly, we proposed an improved thinning algorithm to extract skeletons of navigable regions from binary images, and find out the target skeleton related to the position of the UGV utilizing the k-nearest neighbor (kNN) algorithm. The target skeleton was reconstructed at the midline position of the navigable region using the decreasing gradient algorithm in order to obtain the appropriate skeleton end points for use as candidate guidance points. For visually presenting the driving trend of the UGV and convenient touch screen operation, we transformed guidance point selection into trajectory selection by generating the predicted trajectory correlative to candidate guidance points based on the differential equation of motion. Experimental results show that the proposed method significantly increases the speed of teleoperated UGV.

Characterization of a LuxR repressor for 3,17ß-HSD in Comamonas testosteroni ATCC11996.

3,17ß-Hydroxysteroid dehydrogenase in Comamonas testosteroni (C. testosteroni) is a key enzyme involved in the degradation of steroid compounds. Recently, we found that LuxR is a negative regulator in the expression of the 3,17ß-HSD gene. In the present work, we cultured wild-type and LuxR knock-out mutants of C. testosteroni with inducers such as testosterone, estradiol, progesterone or estrone. HPLC analysis showed that the degradation activities towards testosterone, estradiol, progesterone, and estrone by C.T.-LuxR-KO1 were increased by 7.1%, 9.7%, 11.9% and 3.1%, respectively compared to the wild-type strain. Protein conformation of LuxR was predicted by Phyre 2 Server software, where the N-terminal 86(Ile), 116(Ile), 118(Met) and 149(Phe) residues form a testosterone binding hydrophobic pore, while the C-terminus forms the DNA binding site (HTH). Further, luxr point mutant plasmids were prepared by PCR and co-transformed with pUC3.2-4 into E. coli HB101. ELISA was used to determine 3,17ß-HSD expression after testosterone induction. Compared to wild-type luxr, 3,17ß-HSD expression in mutants of I86T, I116T, M118T and F149S were decreased. The result indicates that testosterone lost its capability to bind to LuxR after the four amino acid residues had been exchanged. No significant changes of 3,17ß-HSD expression were found in K354I and Y356 N mutants compared to wild-type luxr, which indicates that these two amino acid residues in LuxR might relate to DNA binding. Native LuxR protein was prepared from inclusion bodies using sodium lauroylsarcosinate. Molecular interaction experiments showed that LuxR protein binds to a nucleotide sequence which locates 87 bp upstream of the ßhsd promoter. Our results revealed that steroid induction of 3,17ß-HSD in C. testosteroni in fact appears to be a de-repression, where testosterone prevents the LuxR regulator protein binding to the 3,17ß-HSD promoter domain.

NarL, a Novel Repressor for CYP108j1 Expression during PAHs Degradation in Rhodococcus sp. P14.

Rhodococcus sp. P14 was isolated from crude-oil-contaminated sediments, and a wide range of polycyclic aromatic hydrocarbons (PAHs) could be used as the sole source of carbon and energy. A key CYP450 gene, designated as cyp108j1 and involved in the degradation of PAHs, was identified and was able to hydroxylate various PAHs. However, the regulatory mechanism of the expression of cyp108j1 remains unknown. In this study, we found that the expression of cyp108j1 is negatively regulated by a LuxR (helix-turn-helix transcription factors in acyl-homoserine lactones-mediated quorum sensing) family regulator, NarL (nitrate-dependent two-component regulatory factor), which is located upstream of cyp108j1. Further analysis revealed that NarL can directly bind to the promoter region of cyp108j1. Mutational experiments demonstrated that the binding site between NarL and the cyp108j1 promoter was the palindromic sequence GAAAGTTG-CAACTTTC. Together, the finding reveal that NarL is a novel repressor for the expression of cyp108j1 during PAHs degradation.

Biotransformation strategies for steroid estrogen and androgen pollution.

The common steroid hormones are estrone (E1), 17ß-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), and testosterone (T). These steroids are reported to contaminate the environment through wastewater treatment plants. Steroid estrogens are widespread in the aquatic environment and therefore pose a potential risk, as exposure to these compounds has adverse impacts on vertebrates. Excessive exposure to steroid estrogens causes endocrine disruption in aquatic vertebrates, which affects the normal sexual life of these animals. Steroid pollutants also cause several health problems in humans and other animals. Microbial degradation is an efficient method for removing hormone pollutants from the environment by remediation. Over the last two decades, microbial metabolism of steroids has gained considerable attention due to its higher efficiency to reduce pollutants from the environment. The present review is focused on the major causes of steroid pollution, concentrations of these pollutants in surface water, groundwater, drinking water, and wastewater, their effect on humans and aquatic animals, as well as recent efforts by various research groups that seek better ways to degrade steroids by aerobic and anaerobic microbial systems. Detailed overview of aerobic and anaerobic microbial biotransformation of steroid estrogens and testosterone present in the environment along with the active enzyme systems involved in these biotransformation reactions is described in the review article, which helps readers to understand the biotransformation mechanism of steroids in depth. Other measures such as co-metabolic degradation, consortia degradation, algal, and fungal steroid biotransformation are also discussed in detail.

Aislamiento y caracterización de una bacteria marina degradadora de testosterona: Vibrio sp. N3 / Isolation and characterization of a marine testosterone-degrading bacterium: Vibrio sp. N3

Steroids, including testosterone, estrone, 17β-estradiol, estriol and 17β-ethinyl estradiol, are harmful not only to the population dynamics of aquatic life forms but also to public health. In this study, a marine testosterone-degrading bacterium (strain N3) was isolated from Nanao Island in the South China Sea. In addition, the strain could also use 17β-estradiol (E2), 17β-ethinyl estradiol (EE2), estriol (E3) or cholesterol as a sole carbon source. According to the 16S rRNA gene sequence analysis, strain N3 was identified as Vibrio sp. Further characterization showed that the strain is aerobic, gram-negative, and mobile and exhibits resistance to ampicillin, carbenicillin, penicillin and spectinomycin. For enhancing its capacity of testosterone degradation, the Plackett-Burman factorial design and the central composite design were used to optimize the culture condition. Under optimal conditions, 92% of testosterone was degraded by Vibrio sp. N3 in 48 h.

Los esferoides-que incluyen la testosterona, la estrona, el 17 β-estradiol, el estriol y el 17 p-etinilestradiol-son nocivos no solo para la población dinámica de las formas de vida acuática, sino también para la salud pública. En este estudio se aisló una bacteria marina degradadora de testosterona de la isla de Nanao, en el Mar del Sur de China, a la que se denominó cepa N3. Se determinó que esta cepa también podría usar 17 β-estradiol (E2), 17 p-etinilestradiol (EE2), estriol (E3) o colesterol como únicas fuentes de carbono. De acuerdo con el análisis de la secuencia del gen 16S rRNA, la cepa N3 se identificó como Vibrio sp. La caracterización adicional mostró que dicha bacteria es un organismo aerobio, gram negativo y móvil, y que presenta resistencia a ampicilina, carbenicilina, penicilina y espectinomicina. Para optimizar la condición de cultivo en relación con su capacidad de degradar la testosterona, se utilizaron el diseño factorial Plackett-Burman y el diseno compuesto central. En condiciones óptimas, el 92% de la testosterona fue degradada por Vibrio sp. N3 en 48 h.

A novel dehydrogenase 17ß-HSDx from Rhodococcus sp. P14 with potential application in bioremediation of steroids contaminated environment.

Steroids are endocrine disrupting compounds in human and are distributed in various environments. Our previous study showed that a marine bacterium Rhodococcus sp. P14 was able to efficiently degrade one typical steroid estradiol. In this study, we showed that P14 could also use other steroids, including estriol and testosterone, as sole carbon source for growth. Two dehydrogenation products, 16-hydroxestrone and androst-4-ene-3, 17-dione, were detected during estriol and testosterone degradation, respectively. By screening the genome, a short chain dehydrogenase gene was identified and named as 17ß-HSDx. Expression of 17ß-HSDx was induced in P14 when estriol, estradiol or testosterone was used as single carbon source. In addition, 17ß-HSDx was shown to have dehydrogenation ability of transforming estriol to 16-hydroxestrone, estradiol to estrone and testosterone to androst-4-ene-3, 17-dione. This is the first short chain dehydrogenase identified in bacteria with dehydrogenation ability on various steroids substrates. Overall, this study reveals that 17ß-HSDx has potential application in the bioremediation of steroids contaminated environment.