A new bacterium, Rhodococcus sp. S2-17, which could completely degrade an emerging organic pollutant, benzophenone-3 (BP-3), was isolated from contaminated sediment through an enrichment procedure, and its BP-3 catabolic pathway and genes were identified through metabolic intermediate and transcriptomic analyses and biochemical and genetic studies. Metabolic intermediate analysis suggested that strain S2-17 may degrade BP-3 using a catabolic pathway progressing via the intermediates BP-1, 2,4,5-trihydroxy-benzophenone, 3-hydroxy-4-benzoyl-2,4-hexadienedioic acid, 4-benzoyl-3-oxoadipic acid, 3-oxoadipic acid, and benzoic acid. A putative BP-3 catabolic gene cluster including cytochrome P450, flavin-dependent oxidoreductase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, and α/β hydrolase genes was identified through genomic and transcriptomic analyses. Genes encoding the cytochrome P450 complex that demethylates BP-3 to BP-1 were functionally verified through protein expression, and the functions of the other genes were also verified through knockout mutant construction and intermediate analysis. This study suggested that strain S2-17 might have acquired the ability to catabolize BP-3 by recruiting the cytochrome P450 complex and α/β hydrolase, which hydrolyzes 4-benzoyl-3-oxoadipic acid to benzoic acid and 3-oxoadipic acid, genes, providing insights into the recruitment of genes of for the catabolism of emerging organic pollutants.

Despite publication of numerous of papers, the effects of fluoxetine on fish behaviour remains mired in controversy and contradiction. One reason for this controversy is that fluoxetine displays distinct and opposing acute and chronic effects. A second reason is that most studies have been limited to two or at the most three concentrations. To address these deficiencies we exposed adult zebrafish, both single females and shoals consisting of one male and two females, to seven fluoxetine concentrations, ranging from 5 ng/L to 5 μg/L and measured their swimming behaviour, and response to a conspecific alarm substance (CAS) at seven, 14 and 28 days. We also measured the light startle response of unexposed F1 larvae at days seven and 28 post-hatch and the response to CAS at day 28. On day 7 fluoxetine decreased swimming speed at concentrations ≥500 ng/L. After addition of CAS fish exposed to 5, 500 and 1000 ng/L decreased swimming, while fish exposed to 10, 500 and 1000 ng/L significantly increased time motionless. On day 14 only fish exposed to 50 ng/L were significantly slower than controls before addition of CAS, but afterwards fish exposed to 5, 50, 1000 and 5000 ng/L showed significant differences from controls. On day 28 fish exposed to 50 and 5000 ng/L had slower average swimming speeds than controls before addition of CAS. After addition all fish except controls and those exposed to 500 ng/L showed decreased average speed. At seven days post-hatch, F1 larvae whose parents were exposed to 100 ng/L showed significantly higher activity than controls and those exposed to 500 ng/L fluoxetine showed lower activity in the light startle response. This study shows that the effects of fluoxetine vary with time and also in a non-monotonic manner. We suggest that the complex nature of the serotonergic system with multilateral effects at the genomic, biochemical and physiological levels interacting with environmental stimuli result in non-linear dose-response behavioural patterns.

Numerous recent studies have underlined the crucial players of noncoding RNAs (ncRNAs), i.e., microRNAs(miRNAs), long noncoding RNAs(lncRNAs) and circle RNAs(circRNAs) participating in genotoxic responses induced by a wide variety of environmental genotoxicants consistently. Genotoxic-derived ncRNAs provide us a new epigenetic molecular–ecological network (MEN) insights into the underlying mechanisms regarding genotoxicant exposure and genotoxic effects, which can modify ncRNAs to render them “genotoxic” and inheritable, thus potentially leading to disease risk via epigenetic changes. In fact, the spatial structures of ncRNAs, particularly of secondary and three-dimensional structures, diverse environmental genotoxicants as well as RNA splicing and editing forma dynamic pool of ncRNAs, which constructs a MEN in cells together with their enormous targets and interactions, making biological functions more complicated. We nonetheless suggest that ncRNAs have both beneficial(positive) and harmful(negative) effects, i.e., are “double-edged” in regulating genotoxicant toxic responses. Understanding the “double-edged” effects of ncRNAs is of crucial importance for our further comprehension of the pathogenesis of human diseases induced by environmental toxicants and for the construction of novel prevention and therapy targets. Furthermore, the MEN formed by ncRNAs and their interactions each other as well as downstream targets in the cells is important for considering the active relationships between external agents (environmental toxicants) and inherent genomic ncRNAs, in terms of suppression or promotion (down- or upregulation), and engineered ncRNA therapies can suppress or promote the expression of inherent genomic ncRNAs that are targets of environmental toxicants. Moreover, the MEN would be expected to be would be applied to the mechanistic explanation and risk assessment at whole scene level in environmental genotoxicant exposure. As molecular biology evolves rapidly, the proposed MEN perspective will provide a clearer or more comprehensive holistic view.

Dibenzofuran (DBF) derivatives have caused serious environmental problems, especially those produced by paper pulp bleaching and incineration processes. Prominent for its resilient mutagenicity and toxicity, DBF poses a major challenge to human health. In the present study, a new strain of Pseudomonas aeruginosa, FA-HZ1, with high DBF-degrading activity was isolated and identified. The determined optimum conditions for cell growth of strain FA-HZ1 were a temperature of 30 °C, pH 5.0, rotation rate of 200 rpm and 0.1 mM DBF as a carbon source. The biochemical and physiological features as well as usage of different carbon sources by FA-HZ1 were studied. The new strain was positive for arginine double hydrolase, gelatinase and citric acid, while it was negative for urease and lysine decarboxylase. It could utilize citric acid as its sole carbon source, but was negative for indole and H2S production. Intermediates of DBF 1,2-dihydroxy-1,2-dihydrodibenzofuran, 1,2-dihydroxydibenzofuran, 2-hydroxy-4-(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid, 2,3-dihydroxybenzofuran, 2-oxo-2-(2′-hydrophenyl)lactic acid, and 2-hydroxy-2-(2′-hydroxyphenyl)acetic acid were detected and identified through liquid chromatography-mass analyses. FA-HZ1 metabolizes DBF by both the angular and lateral dioxygenation pathways. The genomic study identified 158 genes that were involved in the catabolism of aromatic compounds. To identify the key genes responsible for DBF degradation, a proteomic study was performed. A total of 1459 proteins were identified in strain FA-HZ1, of which 100 were up-regulated and 104 were down-regulated. A novel enzyme “HZ6359 dioxygenase”, was amplified and expressed in pET-28a in E. coli BL21(DE3). The recombinant plasmid was successfully constructed, and was used for further experiments to verify its function. In addition, the strain FA-HZ1 can also degrade halogenated analogues such as 2, 8-dibromo dibenzofuran and 4-(4-bromophenyl) dibenzofuran. Undoubtedly, the isolation and characterization of new strain and the designed pathways is significant, as it could lead to the development of cost-effective and alternative remediation strategies. The degradation pathway of DBF by P. aeruginosa FA-HZ1 is a promising tool of biotechnological and environmental significance.

Exposure to pesticides results in DNA damage and genomic instability. We previously predicted that endosulfan might be associated with leukemia, but the role of endosulfan in leukemia cells has been unexplored. The aim of this study is to elucidate molecular mechanism of endosulfan-induced DNA damage response in human leukemia cells. We performed endosulfan exposure experiments in K562 cells with varying concentrations of endosulfan for 48 h and found that endosulfan lowered cell viability in a dose-dependent manner. We observed the dramatic DNA damage using comet assay and the increase of micronucleus in 75 μM endosulfan-exposed cells. Endosulfan at 75 μM caused the expression alterations of ATM and DNA repair genes such as FANCD2, and BRCA1/2 at different exposure time points (12, 24, 48 h), which was reversed by ATM inhibitor KU-55933. Endosulfan significantly increased the mRNA expression levels of p53 and GADD45A, and decreased PCNA and XRCC2 at 48 h after exposure. Flow cytometric analysis showed that endosulfan at 50 and 75 μM induced cell cycle G1 arrest, a response attributed to down-regulation of CDK6 and up-regulation of p21. We also observed that endosulfan at 50 and 75 μM induced a considerable percentage of cells to undergo apoptosis, as detected by Annexin-V binding assays. Endosulfan resulted in the activation of caspase-3, and elevated the expression levels of PUMA and the ratio of BAX/Bcl-2. These findings suggest that endosulfan caused DNA damage response throughATM-p53 signaling pathway, implicating the potential correlation between endosulfan and leukemia.

Soil microorganisms represent one of the largest biodiversity reservoirs. However, most low-abundance, slow-growing or dormant microorganisms in soils are difficult to capture with traditional enrichment culture methods. These types of microorganisms represent a valuable “microbial seed bank”. To better exploit and utilize this “microbial dark matter”, we developed a novel strategy that integrates single-cell-level isolation with microfluidics technology and culture with resuscitation-promoting factor (Rpf) to isolate biphenyl-degrading bacteria from four typical soils (paddy soil, red soil, alluvial soil and black soil) in eastern China. Multitudinous bacteria were successfully isolated and cultured; some of the identified clades have not been previously linked to biphenyl biodegradation, such as Actinotalea, Curtobacterium and Rothia. Soil microcosmic experiments validated that some bacteria are responsible for biphenyl degradation in soil. In addition, genomic sequencing and Illumina MiSeq sequencing of 16S rRNA genes indicated that exogenous Rpf mainly promotes the recovery and growth of bacteria containing endogenous Rpf-encoding genes. In summary, this study provides a novel strategy for capturing target functional microorganisms in soils, indicates potential bioresources for the bioremediation of contaminated soils, and enhances our current understanding of the mechanisms involved in the response to exogenous Rpf.

Little is known about the association between ambient temperature and DNA methylation, which is a potential biological process through which ambient temperature affects health. This study aimed to evaluate the association between ambient temperature and DNA methylation across human genome. We included 479 Australian women, including 132 twin pairs and 215 sisters of these twins. Blood-derived DNA methylation was measured using the HumanMethylation450 BeadChip array. Data on average ambient temperature during eight different exposure windows [lag0d (the blood draw day), lag0-7d (the current day and previous seven days prior to blood draw), lag0-14d, lag0-21d, lag0-28d, lag0-90d, lag0-180d, and lag0-365d)] was linked to each participant's home address. For each cytosine-guanine dinucleotide (CpG), we evaluated the association between its methylation level and temperature using generalized estimating equations (GEE), adjusting for important covariates. We used comb-p and DMRcate to identify differentially methylated regions (DMRs). We identified 31 CpGs at which blood DNA methylation were significantly associated with ambient temperature with false discovery rate [FDR] < 0.05. There were 82 significant DMRs identified by both comb-p (Sidak p-value < 0.01) and DMRcate (FDR < 0.01). Most of these CpGs and DMRs only showed association with temperature during one specific exposure window. These CpGs and DMRs were mapped to 85 genes. These related genes have been related to many human chronic diseases or phenotypes (e.g., diabetes, arthritis, breast cancer, depression, asthma, body height) in previous studies. The signals of short-term windows (lag0d and lag0-21d) showed enrichment in biological processes related to cell adhesion. In conclusion, short-, medium-, and long-term exposures to ambient temperature were all associated with blood DNA methylation, but the target genomic loci varied by exposure window. These differential methylation signals may serve as potential biomarkers to understand the health impacts of temperature.

Mobile genetic elements (MGEs) such as plasmids or integrative conjugative elements (ICEs) are widely involved in the horizontal transfer of antibiotic resistant genes (ARGs), but their environmental host-range and reservoirs remain poorly known, as mainly assessed through the analysis of culturable and clinical bacterial isolates. In this study, we used a gradual approach for determining the environmental abundance and host-range of ICEs belonging to the SXT/R391 family, otherwise well known to bring ARGs in Vibrio spp. epidemic clones and other pathogens. First, by screening a set of aquatic bacteria libraries covering 1794 strains, we found that almost 1% of the isolates hosted an SXT/R391 element, all belonging to a narrow group of non-O1/non-O139 Vibrio cholerae. However, when SXT/R391 ICEs were then quantified in various aquatic communities, they appeared to be ubiquitous and relatively abundant, from 10⁻⁶ to 10⁻³ ICE copies per 16 S rDNA. Finally, the molecular exploration of the SXT/R391 host-range in two river ecosystems impacted by anthropogenic activities, using the single-cell genomic approach epicPCR, revealed several new SXT/R391 hosts mostly in the Proteobacteria phylum. Some, such as the pathogen Arcobacter cryaerophilus (Campylobacteraceae), have only been encountered in discharged treated wastewaters and downstream river waters, thus revealing a likely anthropogenic origin. Others, such as the non-pathogenic bacterium Neptunomonas acidivorans (Oceanospirillaceae), were solely identified in rivers waters upstream and downstream the treated wastewaters discharge points and may intrinsically belong to the SXT/R391 environmental reservoir. This work points out that not only the ICEs of the SXT/R391 family are more abundant in the environment than anticipated, but also that a variety of unsuspected hosts may well represent a missing link in the environmental dissemination of MGEs from and to bacteria of anthropogenic origin.

Antibiotic resistance genes (ARGs) are now viewed as emerging contaminants posing a potential worldwide human health risk. The degree to which ARGs are transferred to other bacteria via mobile genetic elements (MGEs), including insertion sequences (ISs), plasmids, and phages, has a strong association with their likelihood to function as resistance transfer determinants. Consequently, understanding the structure and function of MGEs is paramount to assessing future health risks associated with ARGs in an environment subjected to strong antibiotic pressure. In this study we used whole genome sequencing, done using MinION and HiSeq platforms, to examine antibiotic resistance determinants among four multidrug resistant bacteria isolated from fish farm effluent in Jeju, South Korea. The combined data was used to ascertain the association between ARGs and MGEs. Hybrid assembly using HiSeq and MinION reads revealed the presence of IncFIB(K) and pVPH2 plasmids, whose sizes were verified using pulsed field gel electrophoresis. Twenty four ARGs and 95 MGEs were identified among the 955 coding sequences annotated on these plasmids. More importantly, 22 of 24 ARGs conferring resistance to various antibiotics were found to be located near MGEs, whereas about a half of the ARGs (11 out of 21) were so in chromosomes. Our results also suggest that the total phenotypic resistance exhibited by the isolates was mainly contributed by these putatively mobilizable ARGs. The study gives genomic insights into the origins of putatively mobilizable ARGs in bacteria subjected to selection pressure.

Chronic exposure to pyrethroid insecticides can result in strong selective pressures on non-target species in aquatic systems and drive the evolution of resistance and population-level changes. Characterizing the underlying mechanisms of resistance is essential to better understanding the potential consequences of contaminant-driven microevolution. The current study found that multiple mechanisms enhance the overall tolerance of Hyalella azteca to the pyrethroid permethrin. In H. azteca containing mutations in the voltage-gated sodium channel (VGSC), both adaptation and acclimation played a role in mitigating the adverse effects of pyrethroid exposures. Pyrethroid resistance is primarily attributed to the heritable mutation at a single locus of the VGSC, resulting in reduced target-site sensitivity. However, additional pyrethroid tolerance was conferred through enhanced enzyme-mediated detoxification. Cytochrome P450 monooxygenases (CYP450) and general esterases (GE) significantly contributed to the detoxification of permethrin in H. azteca. Over time, VGSC mutated H. azteca retained most of their pyrethroid resistance, though there was some increased sensitivity from parent to offspring when reared in the absence of pyrethroid exposure. Permethrin median lethal concentrations (LC50s) declined from 1809 ng/L in parent (P₀) individuals to 1123 ng/L in the first filial (F₁) generation, and this reduction in tolerance was likely related to alterations in acclimation mechanisms, rather than changes to target-site sensitivity. Enzyme bioassays indicated decreased CYP450 and GE activity from P₀ to F₁, whereas the VGSC mutation was retained. The permethrin LC50s in resistant H. azteca were still two orders-of-magnitude higher than non-resistant populations indicating that the largest proportion of resistance was maintained through the inherited VGSC mutation. Thus, the noted variation in tolerance in H. azteca is likely associated with inducible traits controlling enzyme pathways. A better understanding of the mechanistic and genomic basis of acclimation is necessary to more accurately predict the ecological and evolutionary consequences of contaminant-driven change in H. azteca.