Common and unique testosterone and 17 beta-estradiol degradation mechanisms in Comamonas testosteroni JLU460ET by transcriptome analysis.

Strain C. testosteroni JLU460ET was isolated for testosterone and 17 beta-estradiol degradation by our group. In this study, strain C. testosteroni JLU460ET was induced by testosterone and 17 beta-estradiol and then subjected to transcriptome analysis. There were 2,047 upregulated genes after 3 h of testosterone induction, 2,040 upregulated genes after 13 h of testosterone induction, 2,078 upregulated genes after 3 h of 17 beta-estradiol induction, and 2,095 upregulated genes after 13 h of 17 beta-estradiol induction. Significantly upregulated genes were mainly involved in steroid and aromatic compound degradation. A 100 kb steroid-degrading gene cluster was found by transcriptome analysis, which included 92 annotated genes and 58 novel genes. Among them, MucB/RseB and Fiu are secretory proteins for sensing substrates in the environment. MFS-1 and TonB are transporters of testosterone and 17 beta-estradiol. Ring-cleavage enzymes and beta-oxidation enzymes are important for degradation. The genes upregulated by both substrates were almost the same, but the degree of induction by testosterone was higher than that by 17 beta-estradiol. Nine upregulated genes were selected for verification by quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR results were consistent with the transcriptome sequencing results. In this study, the common and unique metabolic mechanisms of testosterone and 17 beta-estradiol were compared by transcriptome analysis in C. testosteroni JLU460ET for the first time.

Genomic analysis of Gordonia polyisoprenivorans strain R9, a highly effective 17 beta-estradiol- and steroid-degrading bacterium.

The increasing levels of estrogens and pollution by other steroids pose considerable challenges to the environment. In this study, the genome of Gordonia polyisoprenivorans strain R9, one of the most effective 17 beta-estradiol- and steroid-degrading bacteria, was sequenced and annotated. The circular chromosome of G. polyisoprenivorans R9 was 6,033,879 bp in size, with an average GC content of 66.91%. More so, 5213 putative protein-coding sequences, 9 rRNA, 49 tRNA, and 3 sRNA genes were predicted. The core-pan gene evolutionary tree for the genus Gordonia showed that G. polyisoprenivorans R9 is clustered with G. polyisoprenivorans VH2 and G. polyisoprenivorans C, with 93.75% and 93.8% similarity to these two strains, respectively. Altogether, the three G. polyisoprenivorans strains contained 3890 core gene clusters. Strain R9 contained 785 specific gene clusters, while 501 and 474 specific gene clusters were identified in strains VH2 and C, respectively. Furthermore, whole genome analysis revealed the existence of the steroids and estrogens degradation pathway in the core genome of all three G. polyisoprenivorans strains, although the G. polyisoprenivorans R9 genome contained more specific estrogen and steroid degradation genes. In strain R9, 207 ABC transporters, 95 short-chain dehydrogenases (SDRs), 26 monooxygenases, 21 dioxygenases, 7 aromatic ring-hydroxylating dioxygenases, and 3 CoA esters were identified, and these are very important for estrogen and steroid transport, and degradation. The results of this study could enhance our understanding of the role of G. polyisoprenivorans R9 in estradiol and steroid degradation as well as evolution within the G. polyisoprenivorans species.

Identification and genome analysis of Comamonas testosteroni strain JLU460ET, a novel steroid-degrading bacterium.

In this work, C. testosteroni JLU460ET isolated from animal waste was confirmed to have great degradation capability for 17ß-estradiol and testosterone. This bacterium could degrade nearly 90% of 17ß-estradiol (5 mg L-1) in 4 days and transform it into estrone for further degradation. One hundred percent testosterone (144 mg L-1) could be completely degraded after 9 h of incubation. This is the first report of C. testosteroni strains with the ability to degrade both estrogens and testosterone. The whole genome sequence of C. testosteroni JLU460ET was obtained and annotated, containing one chromosome (5,497,097 bp) with 61.37% GC content. A total of 4805 protein-coding genes and 134 RNA genes (including 29 rRNA genes, 102 tRNA genes and three ncRNA genes) were identified. Furthermore, the complete genome sequence of C. testosteroni JLU460ET was compared with four other C. testosteroni strains. Altogether, these five C. testosteroni strains contain 3508 core genes and 7616 pan genes. A steroid degradation pathway including 11 steroid degradation genes exists in core genes of five C. testosteroni strains. Twenty-two steroid degradation genes were found in the C. testosteroni JLU460ET genome, which has the most reported steroid degradation genes among the five C. testosteroni genomes. Further functional genomic analysis identified a gene cluster responsible for testosterone degradation in C. testosteroni JLU460ET, as well as a gene encoding 17ß-HSD, the key enzyme for transforming 17ß-estradiol into estrone. This work could enrich the genome sources of steroid-degrading strains and promote the study of steroid-degradation mechanism in bacteria.

Isolation and characterization of a new highly effective 17ß-estradiol-degrading Gordonia sp. strain R9.

In this report, Gordonia sp. strain R9 isolated from an enrichment culture of chicken leachate was confirmed to degrade 17ß-estradiol (E2), which can also use other estrogens (estrone, estriol, and 17α-ethynylestradiol) and testosterone as sole carbon and energy sources. Optimization of growth conditions showed that Gordonia sp. strain R9 can tolerate a very wide range of temperature (4-40 °C) and pH (1.0-11.0), and is sensitive to antibiotics including kanamycin, ampicillin, chloramphenicol, and carbenicillin. Optimal culture conditions for E2 degradation were 30 °C and pH 7.0 with almost 100% degradation of E2 concentrations ranging from 50 µg/L to 5 mg/L within 24 h. The E2 intermediates so generated included estrone (E1), estratriol (E3), (3Z)-3-(3-hydroxy-3a-methyl-7-oxododecahydro-6H-cyclopenta[a]naphthalen-6-ylidene) propanoic acid and 3-hydroxy-3a-methyl-7-oxododecahydro-1H-cyclopenta[a]naphthalene-6-carboxylic acid. These results indicate that the highly effective E2-degradative ability of Gordonia sp. strain R9 merits further investigation as a candidate for large-scale estrogen biodegradation.

Simultaneous determination of four trace estrogens in feces, leachate, tap and groundwater using solid-liquid extraction/auto solid-phase extraction and high-performance liquid chromatography with fluorescence detection.

A simple and selective high-performance liquid chromatography method coupled with fluorescence detection was developed for the simultaneous measurement of trace levels of four estrogens (estrone, estradiol, estriol and 17α-ethynyl estradiol) in environmental matrices. For feces samples, solid-liquid extraction was applied with a 1:1 v/v mixture of acetonitrile and ethyl acetate as the extraction solvent. For liquid samples (e.g., leachate and groundwater), hydrophobic/lipophilic balanced automated solid-phase extraction disks were selected due to their high recoveries compared to conventional C18 disks. Chromatographic separations were performed on a reversed-phase C18 column gradient-eluted with a 45:55 v/v mixture of acetonitrile and water. The detection limits were down to 1.1 × 10(-2) (estrone), 4.11 × 10(-4) (estradiol), 5.2 × 10(-3) (estriol) and 7.18 × 10(-3) µg/L (17α-ethynyl estradiol) at excitation/emission wavelengths of 288/310 nm, with recoveries in the range of 96.9 ± 3.2-105.4 ± 3.2% (n = 3). The method was successfully applied to determine estrogens in feces and water samples collected at livestock farms and a major river in Northeast China. We observed relatively high abundance and widespread distribution of all four estrogens in our sample collections, implying the urgency for a comprehensive and intricate investigation of estrogenic fate and contamination in our researched area.

Analysis and characterization of eight estradiol inducible genes and a strong promoter from the steroid degrading marine bacterial strain S19-1.

Buttiauxella strain S19-1 is a new marine bacterium, isolated from the Baltic Sea, which can degrade steroids. In this report, a meta-genomic approach was used to isolate estradiol inducible genes from S19-1. SalI-fragments from the chromosomal DNA of S19-1 were ligated into plasmid pKEGFP2 bearing an EGFP gene as the reporter system. All resulting plasmids harboring SalI-fragments were transformed into Escherichia coli HB101 to measure the relative fluorescent units (RFU). E. coli cells showing higher RFU after estradiol induction than those without estradiol induction, were selected and the respective plasmids were sequenced. Sequences of 8 positive plasmids were analyzed and aligned by BLAST. Among the predicted genes we found similarities to the major facilitator superfamily, glycerol dehydratase activator, formate acetyltransferase activating enzyme, histidinol-phosphate/aromatic aminotransferase, ABC-transporter, transcriptional regulator nadR, lipoate-protein ligase A, and alcohol phosphatidyl-transferase. Interestingly, one of the E. coli cell clones (containing plasmid p302) showed up in green color by normal light microscopy, which indicated that a strong promoter was present in this plasmid. Sequencing and deletion-mutagenesis revealed that the putative promoter comprises a 108 bp DNA fragment within p302, from which the putative -10 and -35 regions are TTTGAT and TTGGTT, respectively. The promoter might be used to construct S19-1 mutants in which steroid degradation occurs at high levels.

Characterization of the steroid degrading bacterium S19-1 from the Baltic Sea at Kiel, Germany.

Steroid contamination of sea water is an ever growing problem and impacts population dynamics of all kinds of sea animals. We have long experience with the soil bacterium Comamonas testosteroni ATCC11996 which is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons, and which might be used in the bioremediation of contaminated places. In C. testosteroni about 20 enzymes which are involved in steroid degradation could be induced by 0.5 mM testosterone. In this work, we isolated a marine bacterial strain, S19-1, from the Baltic Sea at Kiel (Germany) which degrades steroids and is able to use steroids as carbon source. S19-1 was characterized as being Gram negative, and 16S rRNA analysis showed that it belongs to the genus Buttiauxella of the Enterobacteriaceae family. Strain S19-1 could be best grown in SIN medium supplemented with 0.6-4.1% NaCl at 20°C. As a carbon source, testosterone, estradiol or cholesterol in minimal medium could be used by S19-1. Moreover, S19-1 could grow up in medium with 50 µg/ml erythromycin. Plasmids pK18 and other plasmids were successfully transformed into S19-1 by chemical transformation (CaCl(2)) and shown to replicate in the cells. Currently, work is ongoing to find the steroid inducible genes.

The effects of telocinobufagin isolated from Chan Su on the activation and cytokine secretion of immunocytes in vitro.

Many traditional Chinese medicines have been used as immunomodulators that act as either immunosuppressants or immunostimulators. Recently, our lab successfully isolated a monomer telocinobufagin (TCB) from the chloroform extract of Chan Su (Venenum Bufonis). In the present paper, we evaluated the immunomodulatory effects of this compound in vitro. We found that TCB significantly stimulates splenocyte proliferation when administered alone or in combination with polyclonal T-cell mitogens concanavalin A (Con A) and lipopolysaccharide. Telocinobufagin markedly enhances natural killer cell and peritoneal macrophage activation. Telocinobufagin increases the percentage of CD4, CD8 positive cells within a population of splenocytes. Moreover, we found that the level of several Th1 cytokines, including interleukin-2 (IL-2), interleukin-12 (IL-12), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), are significantly increased after TCB treatment, while the level of the Th2 cytokine interleukin-4 (IL-4) is significantly decreased. As a result, the ratio of Th1/Th2 is significantly increased. Taken together, these results indicate that TCB has potential immune system regulatory effects and suggest that this compound could be developed as a novel immunotherapeutic agent to treat cancer and other immune-mediated diseases, and it may become a new immunomodulatory agent in many regions.