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.

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.

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.

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.

Isolation and characterization of a marine testosterone-degrading bacterium: Vibrio sp. N3.

Steroids, including testosterone, estrone, 17ß-estradiol, estriol and 17ß-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 17ß-estradiol (E2), 17ß-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 48h.

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.

Toward a More Complete, Flexible, and Safer Speed Planning for Autonomous Driving via Convex Optimization.

In this paper, we present a complete, flexible and safe convex-optimization-based method to solve speed planning problems over a fixed path for autonomous driving in both static and dynamic environments. Our contributions are five fold. First, we summarize the most common constraints raised in various autonomous driving scenarios as the requirements for speed planner developments and metrics to measure the capacity of existing speed planners roughly for autonomous driving. Second, we introduce a more general, flexible and complete speed planning mathematical model including all the summarized constraints compared to the state-of-the-art speed planners, which addresses limitations of existing methods and is able to provide smooth, safety-guaranteed, dynamic-feasible, and time-efficient speed profiles. Third, we emphasize comfort while guaranteeing fundamental motion safety without sacrificing the mobility of cars by treating the comfort box constraint as a semi-hard constraint in optimization via slack variables and penalty functions, which distinguishes our method from existing ones. Fourth, we demonstrate that our problem preserves convexity with the added constraints, thus global optimality of solutions is guaranteed. Fifth, we showcase how our formulation can be used in various autonomous driving scenarios by providing several challenging case studies in both static and dynamic environments. A range of numerical experiments and challenging realistic speed planning case studies have depicted that the proposed method outperforms existing speed planners for autonomous driving in terms of constraint type covered, optimality, safety, mobility and flexibility.

A novel 17ß-hydroxysteroid dehydrogenase in Rhodococcus sp. P14 for transforming 17ß-estradiol to estrone.

17ß-hydroxysteroid dehydrogenases (17ß-HSD) are a group of oxidoreductase enzymes that exhibit high specificity for 17C reduction/oxidation. However, the mechanism of 17ß-HSD in oxidizing steroid hormone 17ß-estradiol to estrone in bacterium is still unclear. In this work, a functional bacterium Rhodococcus sp. P14 was identified having rapid ability to oxidize estradiol into estrone in mineral salt medium (MSM) within 6 h. The functional genes encoding NADH-dependent oxidoreductase were successfully detected with the help of bioinformatics, and it was identified that it contained two consensus regions affiliated to the short-chain dehydrogenase/reductase (SDR) superfamily. Expression of 17ß-HSD could be induced by estradiol in strain P14. The 17ß-HSD gene from Rhodococcus sp. P14 was expressed in Escherichia coli strain BL21. Furthermore, recombinant 17ß-HSD-expressing BL21 cells showed a high transformation rate, they are capable of transforming estradiol to estrone up to 94%. The purified His-17ß-HSD protein also exhibited high catalyzing efficiency. In conclusion, this study provides the first evidence that a novel 17ß-HSD in Rhodococcus sp. P14 can catalyze the oxidation of estradiol.

Quantitative determination of testosterone levels with biolayer interferometry.

Natural and synthetic steroid hormones are widely spread in the environment and are considered as pollutants due to their endocrine activities, even at low concentrations, which are harmful to human health. To detect steroid hormones in the environment, a novel biosensor system was developed based on the principle of biolayer interferometry. Detection is based on changes in the interference pattern of white light reflected from the surface of an optical fiber with bound biomolecules. Monitoring interactions between molecules does not require radioactive, enzymatic, or fluorescent labels. Here, 2 double-stranded DNA fragments of operator 1 (OP1) and OP2 containing 10-bp palindromic sequences in chromosomal Comamonas testosteroni DNA (ATCC11996) were surface-immobilized to streptavidin sensors. Interference changes were detected when repressor protein RepA bound the DNA sequences. DNA-protein interactions were characterized and kinetic parameters were obtained. The dissociation constants between the OP1 and OP2 DNA sequences and RepA were 9.865 × 10-9 M and 2.750 × 10-8 M, respectively. The reactions showed high specifically and affinity. Because binding of the 10-bp palindromic sequence and RepA was affected by RepA-testosterone binding, the steroid could be quantitatively determined rapidly using the biosensor system. The mechanism of the binding assay was as follows. RepA could bind both OP1 and testosterone. RepA binding to testosterone changed the protein conformation, which influenced the binding between RepA and OP1. The percentage of the signal detected negative correlation with the testosterone concentration. A standard curve was obtained, and the correlation coefficient value was approximately 0.97. We could quantitatively determine testosterone levels between 2.13 and 136.63 ng/ml. Each sample could be quantitatively detected in 17 min. These results suggested that the specific interaction between double-stranded OP1 DNA and the RepA protein could be used to rapidly and quantitatively determine environmental testosterone levels by the biolayer interferometry technique.

Functional analysis of a novel repressor LuxR in Comamonas testosteroni.

Comamonas testosteroni (C. testosteroni) ATCC11996 is a gram negative bacterium which can use steroid as a carbon and energy source. 3,17ß-hydroxysteroid dehydrogenase (3,17ß-HSD) is a key enzyme for the degradation of steroid hormones in C. testosteroni. The LuxR regulation family is a group of regulatory proteins which play important role in gram negative bacterium. The luxr gene is located on 58 kb upstream of 3,17ß-HSD gene with the opposite transcription orientation in the chromosomal DNA of C. testosteroni. An open reading frame of this putative luxr gene consists of 1125 bp and is translated into a protein containing 374 amino acids. The luxr gene was cloned into plasmid pK18 and plasmid pK-LuxR1 was obtained. E. coli HB101 was co-transformed by pK-LuxR1 and pUC912-10, pUC1128-5 or pUC3.2-4 (which contain ßhsd gene and different length promoter, repeat sequences). The result of ELISA showed that LuxR protein is a negative regulator for 3,17ß-HSD expression. The luxr gene in C. testosteroni was knock-out by homologous integration. 3,17ß-HSD expression was increased in the mutant (C.T.-L-KO1) comparing to that in wild-type C. testosteroni (C.T.) after 0.5 mM testosterone induction. The mutant C.T.-L-KO1 and wild-type C. testosteroni were cultured at 27 °C and 37 °C. The result of growth curve proved that LuxR has also effect on the bacterial growth.

Regulation of alkane degradation pathway by a TetR family repressor via an autoregulation positive feedback mechanism in a Gram-positive Dietzia bacterium.

n-Alkanes are ubiquitous in nature and serve as important carbon sources for both Gram-positive and Gram-negative bacteria. Hydroxylation of n-alkanes by alkane monooxygenases is the first and most critical step in n-alkane metabolism. However, regulation of alkane degradation genes in Gram-positive bacteria remains poorly characterized. We therefore explored the transcriptional regulation of an alkB-type alkane hydroxylase-rubredoxin fusion gene, alkW1, from Dietzia sp. DQ12-45-1b. The alkW1 promoter was characterized and so was the putative TetR family regulator, AlkX, located downstream of alkW1 gene. We further identified an unusually long 48 bp inverted repeat upstream of alkW1 and demonstrated the binding of AlkX to this operator. Analytical ultracentrifugation and microcalorimetric results indicated that AlkX formed stable dimers in solution and two dimers bound to one operator in a positive cooperative fashion characterized by a Hill coefficient of 1.64 (± 0.03) [k(D) = 1.06 (± 0.16) µM, k(D) ' = 0.05 (± 0.01) µM]. However, the DNA-binding affinity was disrupted in the presence of long-chain fatty acids (C10-C24), suggesting that AlkX can sense the concentrations of n-alkane degradation metabolites. A model was therefore proposed where AlkX controls alkW1 expression in a metabolite-dependent manner. Bioinformatic analysis revealed that the alkane hydroxylase gene regulation mechanism may be common among Actinobacteria.

Isolation and identification of a repressor TetR for 3,17ß-HSD expressional regulation in Comamonas testosteroni.

Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17ß-Hydroxysteroid dehydrogenase (3,17ß-HSD) from C. testosteroni is a key enzyme in steroid degradation. Understanding the mechanism of 3,17ß-HSD gene (ßhsd) induction may help us to elucidate its complete molecular regulation. Sequencing the C. testosteroni ATCC11996 genome lead us to identify the tetR (522 bp) downstream of ßhsd. Two repeat sequences (RS; 13 bp), that are separated to each other by 1661 bp, were found upstream of ßhsd. A bioinformatic analysis revealed that TetR family proteins act as transcriptional repressors which are sensitive to environmental signals. Since, C. testosteroni responds to environmental steroid induction and upregulates steroid catabolic genes, we hypothesized that TetR might act in C. testosteroni as repressor for ßhsd expression. The tetR was cloned into different plasmids, including an EGFP reporter system, for functional characterization and/or overexpression. The data indicate that, indeed, TetR acts as a repressor for 3,17ß-HSD expression. Testosterone in turn, which is known to induce ßhsd expression, could not resolve TetR repression. To further substantiate TetR as repressor for ßhsd expression, a tetR gene knock-out mutant of C. testosteroni was generated. TetR gene knock-out mutants showed the same basal low level of ßhsd expression as the C. testosteroni wild type cells. Interestingly, testosterone induction leads to a strong increase in ßhsd expression, especially in the tetR gene knock-out mutants. The result with the knock-out mutant, in principle, supports our hypothesis that TetR is a repressor for ßhsd expression, but the exact role of testosterone in this context remains unknown. Finally, it turned out that TetR is obviously also involved in the regulation of the hsdA gene.

Identification and isolation of a regulator protein for 3,17ß-HSD expressional regulation in Comamonas testosteroni.

Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17ß-Hydroxysteroid dehydrogenase (3,17ß-HSD) from C. testosteroni is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. It is an inducible and key enzyme in steroid degradation. Elucidating the mechanism of 3,17ß-HSD gene (ßhsd) regulation may help us to generate prospective C. testosteroni mutants for bioremediation. The genome of C. testosteroni ATCC11996 was sequenced in our previous work. Upon examining the genome with bioinformatics tools, a gene (brp) coding for a regulator protein (BRP) for 3,17ß-HSD expression was found upstream of the ßhsd gene. A Blast search revealed high identities to a nucleotide binding protein with unknown function in other bacteria. Two potential promoters and two repeat sequences (RS, 16 bp), spaced to each other by 1661 bp, were also found upstream of the ßhsd gene C. testosteroni. The brp gene was cloned into plasmid pK18 and pET-15b, expressed in Escherichia coli, and the recombinant BRP protein was purified on a Ni-column. In addition, a brp gene knock-out mutant of C. testosteroni was prepared. These knock-out mutants showed an enhanced expression of both the ßhsd gene and the hsdA gene (the latter coding for 3α-HSD/CR) in the presence of testosterone. To characterize the BRP functional DNA domain, different fragments of the ßhsd upstream regulatory region were tested in a cotransformation system. Our data reveal that the ßhsd gene undergoes complex regulation involving the two promoters, a loop structure via the two repeat sequences, and the steroid testosterone. Furthermore, a proximal repressor gene for ßhsd expression, phaR, had been identified in our previous investigations. The exact interplay between all these factors will be determined in future experiments.

Construction of a biosensor mutant of Comamonas testosteroni for testosterone determination by cloning the EGFP gene downstream to the regulatory region of the 3,17ß-HSD gene.

Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17ß-Hydroxysteroid dehydrogenase (3,17ß-HSD) from C. testosteroni is a testosterone-inducible protein and a key enzyme in steroid degradation. 3,17ß-HSD is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. We found that a 2.4 kb regulatory DNA fragment upstream of the 3,17ß-HSD gene (ßhsd) responds to steroids and triggers ßhsd gene induction. To exploit this cis-acting regulatory element for a steroid determination system, plasmids pK2.4-EGFP-4 and pBB2.4-EGFP-8 were constructed. Both plasmids contain the EGFP gene fused downstream to a 2.4 kb DNA fragment from C. testosteroni. However, whereas pK2.4-EGFP-4 could integrate into the chromosomal DNA of C. testosteroni and knock out the ßhsd gene promoter, pBB2.4-EGFP-8 could replicate in C. testosteroni cells as a free plasmid DNA. After integration of pK2.4-EGFP-4 into the ßhsd gene promoter, 3,17ß-HSD expression could not be induced such that EGFP expression in the mutant cells was at low levels. In contrast, in C. testosteroni cells transformed with pBB2.4-EGFP-8 the expression of EGFP was induced with testosterone. Our results showed that fluorescence counts (relative fluorescence units; RFU) increased in parallel with testosterone concentrations. Of note, estradiol and cholesterol could not induce the EGFP reporter gene. In summary, this new biosensor system might be used for the specific determination of testosterone.

Incorporating a wheeled vehicle model in a new monocular visual odometry algorithm for dynamic outdoor environments.

This paper presents a monocular visual odometry algorithm that incorporates a wheeled vehicle model for ground vehicles. The main innovation of this algorithm is to use the single-track bicycle model to interpret the relationship between the yaw rate and side slip angle, which are the two most important parameters that describe the motion of a wheeled vehicle. Additionally, the pitch angle is also considered since the planar-motion hypothesis often fails due to the dynamic characteristics of wheel suspensions and tires in real-world environments. Linearization is used to calculate a closed-form solution of the motion parameters that works as a hypothesis generator in a RAndom SAmple Consensus (RANSAC) scheme to reduce the complexity in solving equations involving trigonometric. All inliers found are used to refine the winner solution through minimizing the reprojection error. Finally, the algorithm is applied to real-time on-board visual localization applications. Its performance is evaluated by comparing against the state-of-the-art monocular visual odometry methods using both synthetic data and publicly available datasets over several kilometers in dynamic outdoor environments.