Microplastics (MPs) impair wastewater treatment by lowering pollutant removal and accumulating in systems, yet their effects on microbial metabolic interactions remain largely unexplored. This study investigated the effects of polystyrene MPs (PS-MPs) on granular sludge and elucidated the effects of quorum sensing (QS) across two modes of low-dose chronic addition and high-dose acute addition. Using sequencing batch reactors, changes in phosphorus removal, extracellular polymeric substances (EPS), QS molecules, and microbial community, and key genes were monitored. Results showed that, under continuous exposure to 10 mg/L and acute exposure to 100 mg/L PS-MPs, anaerobic phosphorus release decreased from 16.91 mg/L to 7.72 mg/L and from 10.92 mg/L to 5.54 mg/L, respectively. Concurrently, phosphorus removal efficiency dropped from 87.52% to 53.83% and from 88.17% to 58.14%, respectively. Notably, continuous dosing with 10 mg/L PS-MPs led to more persistent inhibition of phosphorus metabolism. EPS of granular sludge increased steadily from 85.18 to 145.59 mg/g SS in the 10 mg/L group, whereas acute dosing with 100 mg/L PS-MPs has no significant effect on EPS. Microbial community indicated that chronic exposure decreased the abundance of the dominant community polyphosphate-accumulating organisms (PAOs) and increased the species diversity, confirming that PS-MPs affect community structure related to phosphorus removal and EPS. A strong positive correlation was observed between QS molecules (C8-HSL and 3OC8-HSL) and EPS components, particularly polysaccharides (PS) and proteins (PN) (P < 0.05). Metatranscriptomic analysis further revealed that continuous exposure to 10 mg/L PS-MPs can increase EPS secretion and inhibit carbon-phosphorus metabolisms of dominant community PAOs through phbB, ppk and ppx. These findings offer the novel insights for sustaining phosphorus removal and enhancing resilience against the different exposure mode of MPs.

Competition between glycogen-accumulating organisms (GAOs) and polyphosphate-accumulating organisms (PAOs) may significantly contribute to the deterioration of enhanced biological phosphorus removal (EBPR). However, the metabolic shift of GAOs sludge from glycogen accumulating metabolism (GAM) to polyphosphate accumulating metabolism (PAM) through operational mode conversion still remains unclear. Here, the model of metabolic shift was constructed by altering the operational mode, and the metabolic characteristic was investigated. Our findings indicated a gradual increase in intracellular poly-P from 0.0125 to 0.0300 g/g SS during mode conversion. Phosphorus release during the anaerobic period surged from 1.739 to 30 mg/L. The system demonstrated the typical characteristics of biological phosphorus removal for 41 days, with 99 % phosphorus consumption occurring within the initial 2 h of the aerobic period. Batch experiments and stoichiometric ratios further validated the phosphorus removal performance, confirming the successful transition of GAOs sludge metabolism from GAM to PAM. Community structure analysis revealed a gradual increase in the highest abundance of Candidatus Accumulibacter to 4.73 %, which was consistently lower than that of Candidatus Contendobacter. The analysis of key genes revealed high expression of ppk, ppa, and hppA genes associated with phosphorus metabolism in Ca. Contendobacter, indicating its involvement in the PAM. Additionally, seven key genes, including phbB, phaJ, ccr, ecm, mcd, mch, and mcl were identified to regulate the synthesis of intracellular storage substances, polyhydroxybutyrate (PHB), and polyhydroxyvalerate (PHV) in Ca. Contendobacter. This study can offer novel insights into the metabolic behavior of Ca. Contendobacter and the optimization of EBPR deterioration.

The overuse of tetracycline (TC) a novel reverse ELISA assay integrating quantum dots (QDs) with an allosteric transcription factor (TetR) was developed for rapid tetracycline (TC) detection. Specifically, biotin-modified double-stranded DNA (dsDNA) was immobilized on a streptavidin-coated 96-well plate, after which QD-TetR conjugates were added. In the presence of TC, the QD-TetR conjugate binds TC and undergoes allosteric changes that result in its dissociation from the dsDNA. The developed QD-TetR-based reverse ELISA achieved quantitative TC detection within a linear dynamic range of 0.05–100 μM in just 5 min, with a method detection limit of 0.02 μM. The recoveries ranged from 93.43

Antibiotics are extensively utilized in agriculture and livestock farming; however, their environmental residues may foster the proliferation of drug-resistant bacteria and disrupt ecosystems, underscoring the necessity for sensitive detection technologies. Allosteric transcription factors (aTFs) represent a class of proteins that can specifically recognize small molecules and regulate gene expression. Owing to their high specificity and sensitivity, aTFs have been widely adopted in the development of biosensors. This study utilized the properties of allosteric transcription factors to alter their DNA affinity upon ligand binding. By measuring the changes in affinity of TetR and MphR before and after DNA binding using SPR, the study quantitatively analyzed the concentrations of tetracycline and erythromycin. Through SPR technology, quantitative detection of these affinity changes was achieved. Under optimized conditions regarding sample incubation time and system injection time, the affinity values exhibited changes of two orders of magnitude. The detection limits for tetracycline and erythromycin using this technique were determined to be 0.8 nM and 1 nM, respectively. These results indicate that the developed SPR detection method demonstrates high sensitivity and accuracy for detecting tetracycline and erythromycin, making it a promising tool for various applications, including environmental monitoring and quality control.

Antibiotic resistance genes present a major public health challenge and have potential implications for global biogeochemical cycles. However, their impacts on biological nitrogen removal systems remain poorly understood. In the ammonia-oxidizing bacteria Nitrosomonas europaea ATCC 19718 harboring the multidrug-resistant plasmid RP4, a significant decrease in ammonia oxidation efficiency was observed, accompanied by markedly elevated levels of cyclic di-guanylate (c-di-GMP) and acyl-homoserine lactones (AHLs), compared to plasmid-free controls. The results demonstrated that c-di-GMP facilitates the secretion of AHLs, while elevated levels of AHLs inhibit the ammonia oxidation efficiency of Nitrosomonas europaea ATCC 19718. These results revealed that RP4 plasmid significantly impaired ammonia oxidation efficiency through the c-di-GMP and AHLs pathways. Our findings indicate that the multidrug-resistant plasmid RP4 adversely affects the nitrogen metabolism of ammonia-oxidizing bacteria, potentially disrupting the nitrogen biogeochemical cycle and posing substantial ecological and environmental risks.

The quartz crystal microbalance (QCM) is a powerful analytical instrument that enables the quantification of substance mass by precisely measuring frequency changes. To meet the requirements of trace detection limits, traditional QCM sensors typically need to use nanomaterials to connect to target during the detection process to gradually increase the mass of the target, thereby enhancing frequency signal changes and improving the detection limit. However, this increases the processing time for the target. To address these limitations, we have developed a QCM biosensor based on allosteric transcription factors (aTFs). Through the implementation of a signal amplification synergy effect strategy-specifically, increasing the number of aTFs and employing quantum dots for mass signal amplification-the detection sensitivity for Pb2+ has been enhanced by 20-fold, achieving an ultra-low limit of detection (LOD) of 0.05 pM. Meanwhile, the dynamic range has been extended by two orders of magnitude, showing a linear response from 0.05 pM to 200 nM. It is worth noting that leveraging the rapid and specific recognition of Pb2+ by aTFs, the incubation time has been shortened to 10 min. In addition, the QCM biosensor can be regenerated up to five times. These results indicate that the QCM biosensor based on aTFs effectively overcomes the limitations of traditional QCM in the quantitative analysis of low-mass substances. It also has certain economic applicability, providing a new strategy for the development of QCM biosensors suitable for various analytes.

Background: High-altitude hypoxia is known to adversely affect bone health, leading to accelerated bone loss and metabolic alterations. Recent studies suggest that factors such as bicarbonate and gut microbiota may play key roles in bone health. Mineral water, rich in bicarbonate, may influence bone health and the gut–bone axis under such conditions. Methods: Mice were exposed to hypoxia and treated with different concentrations of drinking water. Bone-related parameters were assessed using dual-energy X-ray absorptiometry (DXA) and Micro-CT. Bone health was assessed using the measurement of serum biomarkers. Additionally, Untargeted Metabolomics was employed to analyze differential metabolites between groups, while gut microbiota composition was analyzed using 16S rRNA sequencing. Results: BMW consumption increased bone mineral density (BMD) and helped alleviate the damage to the microstructure of bones caused by hypoxia and delayed the progression of osteoporosis. Additionally, BMW was shown to enhance probiotics such as Akkermansia and Dubosiella and regulate the longevity-regulating pathway as well as the PI3K/AKT/mTOR (PAM) signaling pathway. This study also discovered changes in metabolic products due to BMW intervention, predominantly in pathways such as the amino acid, prostaglandin, and purine metabolisms, with correlation analysis further exploring the relationships between gut microbiota and these differential metabolites. Conclusions: Long-term exposure to high-altitude hypoxic conditions affects the structure of gut microbiota and bone metabolism in mice. The consumption of BMW improves the structure of gut microbiota and regulates the metabolic pathways to maintain bone health under high-altitude hypoxia.

Antibiotic and heavy metal contamination in water poses a severe global public health risk, yet conventional detection methods remain constrained by reliance on complex laboratory instrumentation. Although cell-free biosensors utilizing allosteric transcription factors (aTFs) offer promising alternatives, their limited sensitivity impedes reliable detection of small-molecule contaminants. To address this limitation, we developed allosteric transcription factors (aTFs)-regulated rolling circle transcription assay (ARCTA) for ultrasensitive detection of water contaminants. The ARCTA system employs a circular DNA template comprising a double-stranded region with a T7 promoter and aTFs binding sequence, alongside a single-stranded domain transcribable into fluorescent RNA. Target-specific recognition by aTFs enables rapid, efficient transcription and amplification of fluorescent RNA, achieving an exceptionally low detection threshold. Applied to tetracycline, oxytetracycline, erythromycin, clarithromycin, Hg2+, Pb2+, Cd2+, and As3+ in water samples, ARCTA demonstrated detection limits of 1.25 nM, 0.93 nM, 0.28 nM, 0.45 nM, 0.034 nM, 0.006 nM, 0.13 nM, and 0.016 nM, respectively, and all had a broad dynamic ranges.Notably, ARCTA by designing distinct circular DNA templates to transcribe wavelength-specific fluorescent RNAs, enabling the simultaneous detection of multiple targets. In addition, the system also exhibited robust performance in real-world applications, underscoring its practicality. These results highlight ARCTA's potential as a transformative tool for ultrasensitive monitoring of diverse small-molecule contaminants.

Exposure to Pb2+ in the environment, especially in water, poses a significant threat to human health and urgently necessitates the development of highly sensitive Pb2+ detection methods. In this study, we have integrated the high sensitivity of electrochemical techniques with allosteric transcription factors (aTFs) to develop an innovative electrochemical biosensing platform. This biosensors leverage the specific binding and dissociation of DNA to the aTFs (PbrR) on electrode surfaces to detect Pb2+. Under the optimal conditions, the platform has a broad linear detection range from 1 pM to 10 nM and an exceptionally low detection threshold of 1 pM, coupled with excellent selectivity for Pb2+. Notably, the biosensor demonstrates regenerative capabilities, enabling up to five effective Pb2+ measurements. After one week of storage at 4 °C, effective lead ion detection was still possible, demonstrating the biosensor's excellent stability, this can effectively save the cost of detection. The biosensor also achieves a recovery rate of 93.3% to 106.6% in real water samples. The biosensor shows its potential as a robust tool for the ultrasensitive detection of Pb2+ in environmental monitoring. Moreover, this research provides new insights into the future applications of aTFs in electrochemical sensing.

Vibrio parahaemolyticus (V. parahaemolyticus) is a significant concern, as it can cause severe infections and hemolytic trauma. Given its prevalence in seawater and coastal seafood, it poses a substantial risk as a foodborne pathogen. Biosensor-based detection technology has been continuously evolving, and toehold switches have emerged as a promising area within it, especially in the detection of RNA viruses. Here, we have developed a cell-free toehold switch sensor for V. parahaemolyticus detection. Traditional toehold switch detection methods usually use green fluorescent protein (GFP) or enzyme LacZ as the output signal, with an incubation time as long as 2 h, and are also mainly applied to the detection of RNA viruses. In this study, we introduced a novel, artificially designed luciferase (LuxSit-i) as an output signal and constructed toehold switches with two different output signals (sfGFP, LuxSit-i), aimed at reducing the incubation time of toehold switches. Moreover, to further improve the detection process, we separately utilize recombinase polymerase amplification (RPA) and nucleic acid sequence-based amplification (NASBA) to amplify dead and live bacterial suspensions for detection and attempt to distinguish between dead and live bacteria. This study provided a convenient, rapid, and accurate method for the on-site detection of V. parahaemolyticus, especially beneficial for resource-limited settings. By eliminating the requirement for specialized facilities and personnel, this system has the potential to be a valuable tool in improving public health responses, especially in developing regions.

Cylindrospermopsin (CYN), a cyanobacterial toxin, has been detected in the global water environment. However, information concerning the potential environmental risk of CYN is limited, since the majority of previous studies have mainly focused on the adverse health effects of CYN through contaminated drinking water. The present study reported that CYN at environmentally relevant levels (0.1–100 μg/L) can significantly enhance the conjugative transfer of RP4 plasmid in Escherichia coli genera, wherein application of 10 μg/L of CYN led to maximum fold change of ∼6.5- fold at 16 h of exposure. Meanwhile, evaluation of underlying mechanisms revealed that environmental concentration of CYN exposure could increase oxidative stress in the bacterial cells, resulting in ROS overproduction. In turn, this led to an upregulation of antioxidant enzyme-related genes to avoid ROS attack. Further, inhibition of the synthesis of glutathione (GSH) was also detected, which led to the rapid depletion of GSH in cells and thus triggered the SOS response and promoted the conjugative transfer process. Increase in cell membrane permeability, upregulation of expression of genes related to pilus generation, ATP synthesis, and RP4 gene expression were also observed. These results highlight the potential impact on the spread of antimicrobial resistance in water environments.

Pentachlorophenol (PCP) was once used as a pesticide, germicide, and preservative due to its stable properties and resistance to degradation. This study aimed to design a biosensor for the quantitative and prompt detection of capable of PCP. A cell-free fluorescence biosensor was developed while employing NalC, an allosteric Transcription Factor responsive to PCP and In Vitro Transcription. By adding a DNA template and PCP and employing Electrophoretic Mobility Shift Assay while monitoring the dynamic fluorescence changes in RNA, this study offers evidence of NalC’s potential applicability in sensor systems developed for the specific detection of PCP. The biosensor showed the capability for the quantitative detection of PCP, with a Limit of Detection (LOD) of 0.21 μM. Following the addition of Nucleic Acid Sequence-Based Amplification, the fluorescence intensity of RNA revealed an excellent linear relationship with the concentration of PCP, showing a correlation coefficient (R2) of 0.9595. The final LOD was determined to be 0.002 μM. This study has successfully translated the determination of PCP into a fluorescent RNA output, thereby presenting a novel approach for detecting PCP within environmental settings.

The widespread use of disinfectants during the global response to the 2019 coronavirus pandemic has increased the co-occurrence of disinfection byproducts (DBPs) and antibiotic resistance genes (ARGs). Although DBPs pose major threats to public health globally, there is limited knowledge regarding their biological effects on ARGs. This study aimed to investigate the effects of two inorganic DBPs (chlorite and bromate) on the conjugative transfer of RP4 plasmid among Escherichia coli strains at environmentally relevant concentrations. Interestingly, the frequency of conjugative transfer was initially inhibited when the exposure time to chlorite or bromate was less than 24 h. However, this inhibition transformed into promotion when the exposure time was extended to 36 h. Short exposures to chlorite or bromate were shown to impede the electron transport chain, resulting in an ATP shortage and subsequently inhibiting conjugative transfer. Consequently, this stimulates the overproduction of reactive oxygen species (ROS) and activation of the SOS response. Upon prolonged exposure, the resurgent energy supply promoted conjugative transfer. These findings offer novel and valuable insights into the effects of environmentally relevant concentrations of inorganic DBPs on the conjugative transfer of ARGs, thereby providing a theoretical basis for the management of DBPs.

Increased disinfection of wastewater to preserve its microbiological quality during the coronavirus infectious disease-2019 (COVID-19) pandemic have inevitably led to increased production of toxic disinfection by-products (DBPs). However, there is limited information on such DBPs (i.e., trihalomethanes, haloacetic acids, nitrosamines, and haloacetonitriles). This review focused on the upsurge of chlorine-based disinfectants (such as chlorine, chloramine and chlorine dioxide) in wastewater treatment plants (WWTPs) in the global response to COVID-19. The formation and distribution of DBPs in wastewater were then analyzed to understand the impacts of these large-scale usage of disinfectants in WWTPs. In addition, potential ecological risks associated with DBPs derived from wastewater disinfection and its receiving water bodies were summarized. Finally, various approaches for mitigating DBP levels in wastewater and suggestions for further research into the environmental risks of increased wastewater disinfection were provided. Overall, this study presented a comprehensive overview of the formation, distribution, potential ecological risks, and mitigating approaches of DBPs derived from wastewater disinfection that will facilitate appropriate wastewater disinfection techniques selection, potential ecological risk assessment, and removal approaches and regulations consideration.

In wastewater treatment plants (WWTPs), ammonia oxidation is primarily carried out by three types of ammonia oxidation microorganisms (AOMs): ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and comammox (CMX). Antibiotic resistance genes (ARGs), which pose an important public health concern, have been identified at every stage of wastewater treatment. However, few studies have focused on the impact of ARGs on ammonia removal performance. Therefore, our study sought to investigate the effect of the representative multidrug-resistant plasmid RP4 on the functional microorganisms involved in ammonia oxidation. Using an inhibitor-based method, we first evaluated the contributions of AOA, AOB, and CMX to ammonia oxidation in activated sludge, which were determined to be 13.7%, 41.1%, and 39.1%, respectively. The inhibitory effects of C2H2, C8H14, and 3,4-dimethylpyrazole phosphate (DMPP) were then validated by qPCR. After adding donor strains to the sludge, fluorescence in situ hybridization (FISH) imaging analysis demonstrated the co-localization of RP4 plasmids and all three AOMs, thus confirming the horizontal gene transfer (HGT) of the RP4 plasmid among these microorganisms. Significant inhibitory effects of the RP4 plasmid on the ammonia nitrogen consumption of AOA, AOB, and CMX were also observed, with inhibition rates of 39.7%, 36.2%, and 49.7%, respectively. Moreover, amoA expression in AOB and CMX was variably inhibited by the RP4 plasmid, whereas AOA amoA expression was not inhibited. These results demonstrate the adverse environmental effects of the RP4 plasmid and provide indirect evidence supporting plasmid-mediated conjugation transfer from bacteria to archaea.

The enrichment and spread of antibiotic resistance genes (ARGs) induced by environmental chemical pollution further exacerbated the threat to human health and ecological safety. Several compounds are known to induce R plasmid-mediated conjugation through inducing reactive oxygen species (ROS), increasing cell membrane permeability, enhancing regulatory genes expression, and so forth. Up to now, there has been no substantial breakthrough in the studies of models and related mechanisms. Here, we established a new conjugation model using pheromone-responsive plasmid pCF10 and confirmed that five kinds of bisphenols (BPs) at environmentally relevant concentrations could significantly promote the conjugation of ARGs mediated by plasmid pCF10 in E. faecalis by up to 4.5-fold compared with untreated cells. Using qPCR, gene knockout and UHPLC, we explored the mechanisms behind this phenomenon using bisphenol A (BPA) as a model of BPs and demonstrated that BPA could upregulate the expression of pheromone, promote bacterial aggregation, and even directly activate conjugation as a pheromone instead of producing ROS and enhancing cell membrane permeability. Interestingly, the result of mathematical analysis showed that the pheromone effect of most BPs is more potent than that of synthetic pheromone cCF10. These findings provide new insight into the environmental behavior and biological effect of BPs and provided new method and theory to study on enrichment and spread of ARGs induced by environmental chemical pollution.

Chemical agents (CAs) and their analogues, as representative persistent organic pollutants, are of serious global concerns and can have devastating impacts on environments and human beings. Enzymes used for decontamination of such chemical pollutions often lack high efficacy and robustness against environmental pressures, thereby limiting their practical application in bioremediation. Here, we report living materials based on genetically engineered biofilms that exhibit remarkable enzymatic activities, superior environmental tolerance, self-regeneration, recyclable usage, and tunable functionality. We show that the designer living materials can degrade CAs and pesticides with high efficacy in an eco-friendly manner, and our systems enable actual elimination of CAs pollution in water and soil. The degradation capacity of the designer living materials can achieve more than 95% for 5 mg/ml HD, 5 mg/ml GD and 5 mg/ml VX within 60 min under laboratory conditions, and the turnover numbers of the designer living materials for HD, GD, and VX have increased by more than 1.3 times compared with free enzymes. In addition, the relative activities of the designer living materials remained almost unchanged after 5 recycles, retaining over 95% of their initial catalytic activities even after the final cycle. Combining the adhesive engineered biofilm living materials with electropositive granule media to form a bifunctional composite material, we further demonstrate coinstantaneous removal of biological and chemical pollutants in environmental water. Our work thus establishes a general approach to improve the robustness of enzymes against environmental pressures and provides a sustainable method for decontamination of chemical and biological pollutions.

Picornavirus hepatitis A virus (HAV) is a common cause of hepatitis worldwide. It is spread primarily through contaminated food and water or person-to-person contact. HAV I has been identified as the most common type of human HAV infection. Here, we have developed a cell-free toehold switch sensor for HAV I detection. We screened 10 suitable toehold switch sequences using NUPACK software, and the VP1 gene was used as the target gene. The optimal toehold switch sequence was selected by in vivo expression. The best toehold switch concentration was further found to be 20 nM in a cell-free system. 5 nM trigger RNA activated the toehold switch to generate visible green fluorescence. The minimum detection concentration decreased to 1 pM once combined with NASBA. HAV I trigger RNA could be detected accurately with excellent specificity. In addition, the cell-free toehold switch sensor was verified in HAV I entities. The successful construction of the cell-free toehold switch sensor provided a convenient, rapid, and accurate method for HAV I on-site detection, especially in developing countries, without the involvement of expensive facilities and additional professional operators.

Antibiotic resistance genes (ARGs) have recently become an important public health problem and therefore several studies have characterized ARG composition and distribution. However, few studies have assessed their impact on important functional microorganisms in the environment. Therefore, our study sought to investigate the mechanisms through which multidrug-resistant plasmid RP4 affected the ammonia oxidation capacity of ammonia-oxidizing bacteria, which play a key role in the nitrogen cycle. The ammonia oxidation capacity of N. europaea ATCC25978 (RP4) was significantly inhibited, and NO and N2O were produced instead of nitrite. Our findings demonstrated that the decrease in electrons from NH2OH decreased the ammonia monooxygenase (AMO) activity, leading to a decrease in ammonia consumption. In the ammonia oxidation process, N. europaea ATCC25978 (RP4) exhibited ATP and NADH accumulation. The corresponding mechanism was the overactivation of Complex Ⅰ, ATPase, and the TCA cycle by the RP4 plasmid. The genes encoding TCA cycle enzymes related to energy generation, including gltA, icd, sucD, and NE0773, were upregulated in N. europaea ATCC25978 (RP4). These results demonstrate the ecological risks of ARGs, including the inhibition of the ammonia oxidation process and an increased production of greenhouse gases such as NO and N2O.

Water quality monitoring requires a reliable and practical on-site detection method for heavy metal ions. Combining an in vitro transcription (IVT) technology with allosteric transcription factors (aTFs), we developed a cell-free paper-based biosensor for on-site detection of Hg2+ and Pb2+ in water. Suitable aTFs screened using surface plasmon resonance (SPR) were employed for building biosensors. ATFs could disassociate from DNA due to their specific affinity to metal ions, and fluorescent RNA was transcribed as a signal. The developed biosensor could quantitatively detect Hg2+ in a linear dynamic range of 0.5-500 nM and Pb2+ in a 1-250 nM range in a 1 h period. The LOD of the biosensor was 0.5 nM for Hg2+ and 0.1 nM for Pb2+. The recoveries ranged from 91.09% to 123.24% for actual water samples detection. Furthermore, freeze-drying was used to create a paper-based biosensor that could detect Hg2+ and Pb2+ simultaneously on-site. This research presents a useful technique for various heavy metal ion detections.