Identification and characterization of a strong constitutive promoter stnYp for activating biosynthetic genes and producing natural products in streptomyces.

BACKGROUND: Streptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest. RESULTS: A strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 - 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain. CONCLUSIONS: The promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species.

Bayesian Inference for Mixed Gaussian GARCH-Type Model by Hamiltonian Monte Carlo Algorithm.

MCMC algorithm is widely used in parameters' estimation of GARCH-type models. However, the existing algorithms are either not easy to implement or not fast to run. In this paper, Hamiltonian Monte Carlo (HMC) algorithm, which is easy to perform and also efficient to draw samples from posterior distributions, is firstly proposed to estimate for the Gaussian mixed GARCH-type models. And then, based on the estimation of HMC algorithm, the forecasting of volatility prediction is investigated. Through the simulation experiments, the HMC algorithm is more efficient and flexible than the Griddy-Gibbs sampler, and the credibility interval of forecasting for volatility prediction is also more accurate. A real application is given to support the usefulness of the proposed HMC algorithm well.

Characterization of an 17ß-estradiol-degrading bacterium Stenotrophomonas maltophilia SJTL3 tolerant to adverse environmental factors.

Bioremediation of environmental estrogens requires microorganisms with stable degradation efficiency and great stress tolerance in complex environments. In this work, Stenotrophomonas maltophilia SJTL3 isolated from wastewater was found to be able to degrade over 90% of 10 µg/mL 17ß-estradiol (E2) in a week and the degradation dynamic was fitted by the first-order kinetic equations. Estrone was the first and major intermediate of E2 biodegradation. Strain SJTL3 exhibited strong tolerance to several adverse conditions like extreme pH (3.0-11.0), high osmolality (2%), co-existing heavy metals (6.25 µg/mL of Cu2+) and surfactants (5 CMC of Tween 80), and retained normal cell vitality and stable E2-degradaing efficiency. In solid soil, strain SJTL3 could remove nearly 100% of 1 µg/mL of E2 after the bacteria inoculation and 8-day culture. As to the contamination of 10 µg/mL E2 in soil, the biodegradation efficiency was about 90%. The further obtainment of the whole genome of strain SJTL3 and genome analysis revealed that this strain contained not only the potential genes responsible for estrogen degradation, but also the genes encoding proteins involved in stress tolerance. This work could promote the estrogen-biodegrading mechanism study and provide insights into the bioremediation application.

Characterization of 17ß-hydroxysteroid dehydrogenase and regulators involved in estrogen degradation in Pseudomonas putida SJTE-1.

In bacteria, the enzyme catalyzing the transformation of 17ß-estradiol is considered the key enzyme for its metabolism, whose enzymatic activity and regulatory network influence the biodegradation efficiency of this typical estrogen. In this work, a novel 17ß-hydroxysteroid dehydrogenase (17ß-HSD) was characterized from the estrogen-degrading strain Pseudomonas putida SJTE-1, and two regulators were identified. This 17ß-HSD, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, could be induced by 17ß-estradiol and catalyzed the oxidization reaction at the C17 site of 17ß-estradiol efficiently. Its Km value was 0.068 mM, and its Vmax value was 56.26 µmol/min/mg; over 98% of 17ß-estradiol was oxidized into estrone in 5 min, indicating higher efficiency than other reported bacterial 17ß-HSDs. Furthermore, two genes (crgA and oxyR) adjacent to 17ß-hsd were studied which encoded the potential CrgA and OxyR regulators. Overexpression of crgA could enhance the transcription of 17ß-hsd, while that of oxyR resulted in the opposite effect. They could bind to the specific and different sites in the promoter region of 17ß-hsd gene directly, and binding of OxyR could be released by 17ß-estradiol. OxyR repressed the expression of 17ß-hsd by its specific binding to the conserved motif of GATA-N9-TATC, while CrgA activated the expression of this gene through its binding to the motif of T-N11-A. Therefore, this 17ß-HSD transformed 17ß-estradiol efficiently and the two regulators regulated its expression directly. This work could promote the study of the enzymatic mechanism and regulatory network of the estrogen biodegradation pathway in bacteria.

One 3-oxoacyl-(acyl-Carrier-protein) reductase functions as 17ß-hydroxysteroid dehydrogenase in the estrogen-degrading Pseudomonas putida SJTE-1.

Pseudomonas putida SJTE-1 can utilize 17ß-estradiol (E2) as its carbon source, while the enzymes for E2 transformation in this strain is still unclear. 17ß-hydroxysteroid dehydrogenases (17ß-HSD) can catalyze the reduction/oxidation at C17 site of steroid hormone specifically, critical for steroid transformation. Here a novel 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (ANI02794.1) was identified as it could bß-estradiol, and was proved to be capable of functioning as 17ß-HSD. Sequences alignment showed it contained the two consensus regions and the conserved residues of short-chain dehydrogenase/reductase (SDR). Its encoding gene was cloned and over-expressed in Escherichia coli BL21(DE3) strain, and the recombinant protein was purified by the metal-ion affinity chromatography with the yield of 18 mg/L culture. HPLC (High Performance Liquid Chromatography) detection showed this enzyme could convert 17ß-estradiol into estrone using NAD+ as cofactor. Its Km value was 0.082 mM and its Vmax value was 0.81 mM/s; its transformation efficiency of 17ß-estradiol into estrone was over 96.6% in five minutes. Its optimal temperature was 37 °C and optimal was pH 9.0; the divalent ions had different effects on the enzymatic activity. In conclusion, this 3-oxoacyl-ACP reductase functioned as 17ß-HSD in P. putida SJTE-1 and played important role in its estrogen metabolism.

RT-qPCR with chimeric dU stem-loop primer is efficient for the detection of bacterial small RNAs.

Small non-coding RNAs are considered be involved in the regulation of multiple cellular processes. Quantitative reverse transcription PCR (RT-qPCR) is widely used in the detection of eukaryotic microRNA, and the stem-loop primers can improve the specificity and efficiency of reverse transcription. However, the loop structure of primers probably influence the next quantitative amplification due to the base stacking and steric hindrance. Here, we designed a chimeric stem-loop primer with a deoxyuracil (dU) base located near the RNA matching part. After the reverse transcription, uracil-DNA glycosylase (UDG) treatment was used to remove the dU base and destroy the stem-loop structure of RT product. Enzymatic assay confirmed that the recombinant UDG could efficiently eliminate the dU base in the oligonucleotide. Transcriptions of two small RNAs (TFF and ryeA) in Escherichia coli were detected by RT-qPCR with different primers. Results showed that the use of the chimeric dU stem-loop primer and UDG treatment could enhance the detection specificity and sensitivity about 1.1- to 3.4-fold, compared to those with traditional stem-loop primer and linear primer. Total RNA of 1-10 pg was enough for efficient detection with the chimeric stem-loop primers. In a word, this strategy could promote the RT-qPCR detection efficiency on the transcription of bacterial small RNAs even in trace samples and can facilitate the detection of exiguous change in cellular metabolism.

Isolation and characterization of Pseudomonas aeruginosa strain SJTD-2 for degrading long-chain n-alkanes and crude oil.

OBJECTIVE: Oil pollution poses a severe threat to ecosystems, and bioremediation is considered as a safe and efficient alternative to physicochemical. METHODS: for eliminating this contaminant. In this study, a gram-negative bacteria strain SJTD-2 isolated from oil-contaminated soil was found capable of utilizing n-alkanes and crude oil as sole energy sources. The efficiency of this strain in degrading these pollutants was analyzed. METHODS: Strain SJTD-2 was identified on the basis of its phenotype, its physiological features, and a comparative genetic analysis using 16S rRNA sequence. Growth of strain SJTD-2 with different carbon sources (n-alkanes of different lengths and crude oil) was assessed, and the gas chromatography-mass spectrometry method was used to analyze the degradation efficiency of strain SJTD-2 for n-alkanes and petroleum by detecting the residual n-alkane concentrations. RESULTS: Strain SJTD-2 was identified as Pseudomonas aeruginosa based on the phenotype, physiological features, and 16S rRNA sequence analysis. This strain can efficiently decompose medium-chain and long-chain n-alkanes (C10-C26), and petroleum as its sole carbon sources. It preferred the long-chain n-alkanes (C18-C22), and n-docosane was considered as the best carbon source for its growth. In 48 h, 500 mg/L n-docosane could be degraded completely, and 2 g/L n-docosane was decomposed to undetectable levels within 72 h. Moreover, strain SJTD-2 could utilize about 88% of 2 g/L crude oil in 7days. Compared with other alkane-utilizing strains, strain SJTD-2 showed outstanding degradation efficiency for long-chain n-alkanes and high tolerance to petroleum at elevated concentrations. CONCLUSION: The isolation and characterization of strain SJTD-2 would help researchers study the mechanisms underlying the biodegradation of n-alkanes, and this strain could be used as a potential strain for environmental governance and soil bioremediation.

Identification of an efficient phenanthrene-degrading Pseudarthrobacter sp. L1SW and characterization of its metabolites and catabolic pathway.

Phenanthrene, a typical chemical of polycyclic aromatic hydrocarbons (PAHs) pollutants, severely threatens health of wild life and human being. Microbial degradation is effective and environment-friendly for PAH removal, while the phenanthrene-degrading mechanism in Gram-positive bacteria is unclear. In this work, one Gram-positive strain of plant growth-promoting rhizobacteria (PGPR), Pseudarthrobacter sp. L1SW, was isolated and identified with high phenanthrene-degrading efficiency and great stress tolerance. It degraded 96.3% of 500 mg/L phenanthrene in 72 h and kept stable degradation performance with heavy metals (65 mg/L of Zn2+, 5.56 mg/L of Ni2+, and 5.20 mg/L of Cr3+) and surfactant (10 CMC of Tween 80). Strain L1SW degraded phenanthrene mainly through phthalic acid pathway, generating intermediate metabolites including cis-3,4-dihydrophenanthrene-3,4-diol, 1-hydroxy-2-naphthoic acid, and phthalic acid. A novel metabolite (m/z 419.0939) was successfully separated and identified as an end-product of phenanthrene, suggesting a unique metabolic pathway. With the whole genome sequence alignment and comparative genomic analysis, 19 putative genes associated with phenanthrene metabolism in strain L1SW were identified to be distributed in three gene clusters and induced by phenanthrene and its metabolites. These findings advance the phenanthrene-degrading study in Gram-positive bacteria and promote the practical use of PGPR strains in the bioremediation of PAH-contaminated environments.

Cytochrome P450 Mining for Bufadienolide Diversification.

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1ß-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

A three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D.

Modulation of protein function through allosteric regulation is central in biology, but biomacromolecular systems involving multiple subunits and ligands may exhibit complex regulatory mechanisms at different levels, which remain poorly understood. Here, we discover an aldo-keto reductase termed AKRtyl and present its three-level regulatory mechanism. Specifically, by combining steady-state and transient kinetics, X-ray crystallography and molecular dynamics simulation, we demonstrate that AKRtyl exhibits a positive synergy mediated by an unusual Monod-Wyman-Changeux (MWC) paradigm of allosteric regulation at low concentrations of the cofactor NADPH, but an inhibitory effect at high concentrations is observed. While the substrate tylosin binds at a remote allosteric site with positive cooperativity. We further reveal that these regulatory mechanisms are conserved in AKR12D subfamily, and that substrate cooperativity is common in AKRs across three kingdoms of life. This work provides an intriguing example for understanding complex allosteric regulatory networks.

RNase HIII from Chlamydophila pneumoniae can efficiently cleave double-stranded DNA carrying a chimeric ribonucleotide in the presence of manganese.

Two ribonuclease Hs (RNase Hs) have been found in Chlamydophila pneumoniae, CpRNase HII and CpRNase HIII. This work is the first report that CpRNase HIII can efficiently cleave DNA-rN(1) -DNA/DNA (rN(1) , monoribonucleotide) in vitro in the presence of Mn(2+) , whereas the enzymatic activity of CpRNase HII on the same substrate was inhibited by Mn(2+) and dependent on Mg(2+) . However, the ability of both CpRNase Hs to cleave other alternative substrates (RNA/DNA hybrids and Okazaki-like substrates), was insensitive to the divalent ions changes, suggesting that high concentrations of Mn(2+) specifically repressed the ability of CpRNase HII to cleave DNA-rN(1) -DNA/DNA but activated this function in CpRNase HIII. Further in vivo experiments showed that the CpRNase HII complementation of Escherichia coli rnh(-) mutations in an Mg(2+) environment was suppressed by Mn(2+) . In contrast, Mn(2+) was indispensable for CpRNase HIII to complement the same mutations. Further, the cell growth inhibition and the genomic DNA sensitivity to alkali in the bacterial strain lacking RNase HII activity could be relieved by functional CpRNase HII or HIII with its compatible ion. Therefore, CpRNase HIII can execute cleavage activity on DNA-rN(1) -DNA/DNA under a Mn(2+) -rich environment and may function as a substitute for CpRNase HII under special physiological states.

Bioaugmentation removal and microbiome analysis of the synthetic estrogen 17α-ethynylestradiol from hostile conditions and environmental samples by Pseudomonas citronellolis SJTE-3.

Synthetic estrogens are emerging environmental contaminants with great estrogenic activities and stable structures that are widespread in various ecological systems and significantly threaten the health of organisms. Pseudomonas citronellolis SJTE-3 is reported to degrade the synthetic estrogen 17α-ethynylestradiol (EE2) efficiently in laboratory conditions. In this work, the environmental adaptability, the EE2-degrading properties, and the ecological effects of P. citronellolis SJTE-3 under different hostile conditions (heavy metals and surfactants) and various natural environment samples (solid soil, lake water, and pig manure) were studied. Strain SJTE-3 can tolerate high concentrations of Zn2+ and Cr3+, but is relatively sensitive to Cu2+. Tween 80 of low concentration can significantly promote EE2 degradation by strain SJTE-3, different from the repressing effect of Triton X-100. High concentration of Tween 80 prolonged the lagging phase of EE2-degrading process, while the final EE2 removal efficiency was improved. More importantly, strain SJTE-3 can grow normally and degrade estrogen stably in various environmental samples. Inoculation of strain SJTE-3 removed the intrinsic synthetic and natural estrogens (EE2 and estrone) in lake water samples in 4 days, and eliminated over 90% of the amended 1 mg/L EE2 in 2 days. Bioaugmentation of strain SJTE-3 in EE2-supplied solid soil and pig manure samples achieved a removal rate of over 55% and 70% of 1 mg/kg EE2 within 2 weeks. Notably, the bioaugmentation of extrinsic strain SJTE-3 had a slight influence on indigenous bacterial community in pig manure samples, and its relative abundance decreased significantly after EE2 removal. Amendment of EE2 or strain SJTE-3 in manure samples enhanced the abundance of Proteobacteria and Actinobacteria, implying their potential in utilizing EE2 or its metabolites. These findings not only shed a light on the environment adaptability and degradation efficiency of strain SJTE-3, but also provide insights for bioremediation application in complex and synthetic estrogen polluted environments.

Identification of a 17ß-estradiol-degrading Microbacterium hominis SJTG1 with high adaptability and characterization of the genes for estrogen degradation.

Environmental estrogen contamination poses severe threat to wildlife and human. Biodegradation is an efficient strategy to remove the wide-spread natural estrogen, while strains suitable for hostile environments and fit for practical application are rare. In this work, Microbacterium hominis SJTG1 was isolated and identified with high degrading efficiency for 17ß-estradiol (E2) and great environment fitness. It could degrade nearly 100% of 10 mg/L E2 in minimal medium in 6 days, and remove 93% of 1 mg/L E2 and 74% of 10 mg/L E2 in the simulated E2-polluted solid soil in 10 days. It maintained stable E2-degrading efficiency in various harsh conditions like non-neutral pH, high salinity, stress of heavy metals and surfactants. Genome mining and comparative genome analysis revealed that there are multiple genes potentially associated with steroid degradation in strain SJTG1. One 3ß/17ß-hydroxysteroid dehydrogenase HSD-G129 induced by E2 catalyzed the 3ß/17ß-dehydrogenation of E2 and other steroids efficiently. The transcription of hsd-G129 gene was negatively regulated by the adjacent LysR-type transcriptional regulator LysR-G128, through specific binding to the conserved site. E2 can release this binding and initiate the degradation process. This work provides an efficient and adaptive E2-degrading strain and promotes the biodegrading mechanism study and actual remediation application.

JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication.

Background & Aims: HBV infection is a global health burden. Covalently closed circular DNA (cccDNA) transcriptional regulation is a major cause of poor cure rates of chronic hepatitis B (CHB) infection. Herein, we evaluated whether targeting host factors to achieve functional silencing of cccDNA may represent a novel strategy for the treatment of HBV infection. Methods: To evaluate the effects of Jumonji C domain-containing (JMJD2) protein subfamily JMJD2A-2D proteins on HBV replication, we used lentivirus-based RNA interference to suppress the expression of isoforms JMJD2A-2D in HBV-infected cells. JMJD2D-knockout mice were generated to obtain an HBV-injected model for in vivo experiments. Co-immunoprecipitation and ubiquitylation assays were used to detect JMJD2D-HBx interactions and HBx stability modulated by JMJD2D. Chromatin immunoprecipitation assays were performed to investigate JMJD2D-cccDNA and HBx-cccDNA interactions. Results: Among the JMJD2 family members, JMJD2D was significantly upregulated in mouse livers and human hepatoma cells. Downregulation of JMJD2D inhibited cccDNA transcription and HBV replication. Molecularly, JMJD2D sustained HBx stability by suppressing the TRIM14-mediated ubiquitin-proteasome degradation pathway and acted as a key co-activator of HBx to augment HBV replication. The JMJD2D-targeting inhibitor, 5C-8-HQ, suppressed cccDNA transcription and HBV replication. Conclusion: Our study clarified the mechanism by which JMJD2D regulates HBV transcription and replication and identified JMJD2D as a potential diagnostic biomarker and promising drug target against CHB, and HBV-associated hepatocarcinoma. Impact and implications: HBV cccDNA is central to persistent infection and is a major obstacle to healing CHB. In this study, using cellular and animal HBV models, JMJD2D was found to stabilise and cooperate with HBx to augment HBV transcription and replication. This study reveals a potential novel translational target for intervention in the treatment of chronic hepatitis B infection.

Genome sequence of Pseudomonas putida strain SJTE-1, a bacterium capable of degrading estrogens and persistent organic pollutants.

Pseudomonas putida strain SJTE-1 can utilize 17ß-estradiol and other environmental estrogens/toxicants, such as estrone, and naphthalene as sole carbon sources. We report the draft genome sequence of strain SJTE-1 (5,551,505 bp, with a GC content of 62.25%) and major findings from its annotation, which could provide insights into its biodegradation mechanisms.

Genome sequence of Pseudomonas aeruginosa strain SJTD-1, a bacterium capable of degrading long-chain alkanes and crude oil.

Pseudomonas aeruginosa strain SJTD-1 can utilize long-chain alkanes, diesel oil, and crude oil as sole carbon sources. We report the draft genome sequence of strain SJTD-1 (6,074,058 bp, with a GC content of 66.83%) and major findings from its annotation, which could provide insights into its petroleum biodegradation mechanism.

The 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase HSD-X1 of Pseudomonas Citronellolis SJTE-3 Catalyzes the Conversion of 17ß-estradiol to Estrone.

BACKGROUND: Pseudomonas citronellolis SJTE-3 can efficiently degrade 17ß-estradiol (E2) and other estrogenic chemicals. However, the enzyme responsible for E2 metabolism within strain SJTE-3 has remained unidentified. OBJECTIVE: Here, a novel 3-oxoacyl-(acyl-carrier protein) (ACP) reductase, HSD-X1 (WP_ 009617962.1), was identified in SJTE-3 and its enzymatic characteristics for the transformation of E2 were investigated. METHODS: Multiple sequence alignment and homology modelling were used to predict the protein structure of HSD-X1. The concentrations of different steroids in the culture of recombinant strains expressing HSD-X1 were determined by high performance liquid chromatography. Additionally, the transcription of hsd-x1 gene was investigated using reverse transcription and quantitative PCR analysis. Heterologous expression and affinity purification were used to obtain recombinant HSD- X1. RESULTS: The transcription of hsd-x1 gene in P. citronellolis SJTE-3 was induced by E2. Multiple sequence alignment (MSA) indicated that HSD-X1 contained the two consensus regions and conserved residues of short-chain dehydrogenase/reductases (SDRs) and 17ß-hydroxysteroid dehydrogenases (17ß-HSDs). Over-expression of hsd-x1 gene allowed the recombinant strain to degrade E2. Recombinant HSD-X1 was purified with a yield of 22.15 mg/L and used NAD+ as its cofactor to catalyze the oxidization of E2 into estrone (E1) while exhibiting a Km value of 0.025 ± 0.044 mM and a Vmax value of 4.92 ± 0.31 mM/min/mg. HSD-X1 could tolerate a wide range of temperature and pH, while the presence of divalent ions exerted little influence on its activity. Further, the transformation efficiency of E2 into E1 was over 98.03% across 15 min. CONCLUSION: Protein HSD-X1 efficiently catalyzed the oxidization of E2 and participated in estrogen degradation by P. citronellolis SJTE-3.

Characterization of the Tellurite-Resistance Properties and Identification of the Core Function Genes for Tellurite Resistance in Pseudomonas citronellolis SJTE-3.

Tellurite is highly toxic to bacteria and commonly used in the clinical screening for pathogens; it is speculated that there is a potential relationship between tellurite resistance and bacterial pathogenicity. Until now, the core function genes of tellurite resistance and their characteristics are still obscure. Pseudomonas citronellolis SJTE-3 was found able to resist high concentrations of tellurite (250 µg/mL) and formed vacuole-like tellurium nanostructures. The terZABCDE gene cluster located in the large plasmid pRBL16 endowed strain SJTE-3 with the tellurite resistance of high levels. Although the terC and terD genes were identified as the core function genes for tellurite reduction and resistance, the inhibition of cell growth was observed when they were used solely. Interestingly, co-expression of the terA gene or terZ gene could relieve the burden caused by the expression of the terCD genes and recover normal cell growth. TerC and TerD proteins commonly shared the conserved sequences and are widely distributed in many pathogenic bacteria, highly associated with the pathogenicity factors.

Metabolism analysis of 17α-ethynylestradiol by Pseudomonas citronellolis SJTE-3 and identification of the functional genes.

Synthetic estrogens are the most hazardous and persistent environmental estrogenic contaminants, with few reports on their biodegradation. Pseudomonas citronellolis SJTE-3 degraded natural steroids efficiently and metabolized 17α-ethynylestradiol (EE2) with the addition of different easily used energy sources (glucose, peptone, ethanol, yeast extract, fulvic acid and ammonia). Over 92% of EE2 (1 mg/L) and 55% of EE2 (10 mg/L) in culture were removed in seven days with the addition of 0.1% ethanol, and the EE2-biotransforming efficiency increased with the increasing ethanol concentrations. Two novel intermediate metabolites of EE2 (C22H22O and C18H34O2) were identified with high-performance liquid chromatography (HPLC) and GC-Orbitrap/MS. Comparative analysis and genome mining revealed strain SJTE-3 contained a unique genetic basis for EE2 metabolism, and the putative EE2-degrading genes exhibited dispersed distribution. The EE2 metabolism of strain SJTE-3 was inducible and the transcription of eight genes were significantly induced by EE2. Three genes (sdr3, yjcH and cyp2) encoding a short-chain dehydrogenase, a membrane transporter and a cytochrome P450 hydroxylase, respectively, were vital for EE2 metabolism in strain SJTE-3; their over-expression accelerated EE2 metabolic processes and advanced the generation of intermediate metabolites. This work could promote the study of bacterial EE2 metabolism mechanisms and facilitate efficient bioremediation for EE2 pollution.

Construction of a bacterial assay for estrogen detection based on an estrogen-sensitive intein.

Escherichia coli strain DIER was constructed for estrogen detection by inserting an estrogen-sensitive intein (VMA(ER) intein) into the specific site of the constitutively expressed chromosomal lacZ gene. This VMA(ER) intein was generated by replacing the endonuclease region of the Saccharomyces cerevisiae VMA intein with the estrogen binding region of the human estrogen receptor α (hERα). When there were estrogens or analogs, the splicing of the VMA(ER) intein was induced to produce the mature LacZ protein, which was detected through a ß-galactosidase colorimetric assay. Eight typical chemicals (17-ß-estradiol, bisphenol A, chrysene, 6-OH-chrysene, benz[a]anthracene, pyrene, progesterone, and testosterone) were detected using this DIER strain, and the whole detection procedure was accomplished in 2 h. Their 50% effective concentrations (EC(50)), relative estrogenic activities, and estradiol equivalency factors were calculated and were quite consistent with those detected with the yeast estrogen screening (YES) system. Furthermore, the estrogenic activities of the synthetic musk samples extracted from the wastewater and waste sludge of a sewage treatment plant of Shanghai (China) were detected, and their results were comparable to those obtained from the YES system and gas chromatography-mass spectrometry (GC-MS). In conclusion, the DIER bioassay could fill a niche for the efficient, rapid, high-throughput screening of estrogenic compounds and has potential for the remote, near-real-time monitoring of environmental estrogens.