Direct ammonia oxidation (Dirammox) is favored over cell growth in Alcaligenes ammonioxydans HO-1 to deal with the toxicity of ammonium.

Bacteria capable of direct ammonia oxidation (Dirammox) play important roles in global nitrogen cycling and nutrient removal from wastewater. Dirammox process, NH3 → NH2 OH → N2 , first defined in Alcaligenes ammonioxydans HO-1 and encoded by dnf gene cluster, has been found to widely exist in aquatic environments. However, because of multidrug resistance in Alcaligenes species, the key genes involved in the Dirammox pathway and the interaction between Dirammox process and the physiological state of Alcaligenes species remain unclear. In this work, ammonia removal via the redistribution of nitrogen between Dirammox and microbial growth in A. ammonioxydans HO-1, a model organism of Alcaligenes species, was investigated. The dnfA, dnfB, dnfC, and dnfR genes were found to play important roles in the Dirammox process in A. ammonioxydans HO-1, while dnfH, dnfG, and dnfD were not essential genes. Furthermore, an unexpected redistribution phenomenon for nitrogen between Dirammox and cell growth for ammonia removal in HO-1 was revealed. After the disruption of the Dirammox in HO-1, more consumed NH4 + was recovered as biomass-N via rapid metabolic response and upregulated expression of genes associated with ammonia transport and assimilation, tricarboxylic acid cycle, sulfur metabolism, ribosome synthesis, and other molecular functions. These findings deepen our understanding of the molecular mechanisms for Dirammox process in the genus Alcaligenes and provide useful information about the application of Alcaligenes species for ammonia-rich wastewater treatment.

Optimized Antibiotic Resistance Genes Monitoring Scenarios Promote Sustainability of Urban Water Cycle.

Dissemination of antibiotic resistance genes (ARGs) in urban water bodies has become a significant environmental and health concern. Many approaches based on real-time quantitative PCR (qPCR) have been developed to offer rapid and highly specific detection of ARGs in water environments, but the complicated and time-consuming procedures have hindered their widespread use. Herein, we developed a facile one-step approach for rapid detection of ARGs by leveraging the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA). This efficient method matches the sensitivity and specificity of qPCR and requires no complex equipment. The results show a strong correlation between the prevalence of four ARG markers (ARGs: sul1, qnrA-1, mcr-1, and class 1 integrons: intl1) in tap water, human urine, farm wastewater, hospital wastewater, municipal wastewater treatment plants (WWTPs), and proximate natural aquatic ecosystems, indicating the circulation of ARGs within the urban water cycle. Through monitoring the ARG markers in 18 WWTPs in 9 cities across China during both peak and declining stages of the COVID epidemic, we found an increased detection frequency of mcr-1 and qnrA-1 in wastewater during peak periods. The ARG detection method developed in this work may offer a useful tool for promoting a sustainable urban water cycle.

Radiofrequency radiation reshapes tumor immune microenvironment into antitumor phenotype in pulmonary metastatic melanoma by inducing active transformation of tumor-infiltrating CD8+ T and NK cells.

Immunosuppression by the tumor microenvironment is a pivotal factor contributing to tumor progression and immunotherapy resistance. Priming the tumor immune microenvironment (TIME) has emerged as a promising strategy for improving the efficacy of cancer immunotherapy. In this study we investigated the effects of noninvasive radiofrequency radiation (RFR) exposure on tumor progression and TIME phenotype, as well as the antitumor potential of PD-1 blockage in a model of pulmonary metastatic melanoma (PMM). Mouse model of PMM was established by tail vein injection of B16F10 cells. From day 3 after injection, the mice were exposed to RFR at an average specific absorption rate of 9.7 W/kg for 1 h per day for 14 days. After RFR exposure, lung tissues were harvested and RNAs were extracted for transcriptome sequencing; PMM-infiltrating immune cells were isolated for single-cell RNA-seq analysis. We showed that RFR exposure significantly impeded PMM progression accompanied by remodeled TIME of PMM via altering the proportion and transcription profile of tumor-infiltrating immune cells. RFR exposure increased the activation and cytotoxicity signatures of tumor-infiltrating CD8+ T cells, particularly in the early activation subset with upregulated genes associated with T cell cytotoxicity. The PD-1 checkpoint pathway was upregulated by RFR exposure in CD8+ T cells. RFR exposure also augmented NK cell subsets with increased cytotoxic characteristics in PMM. RFR exposure enhanced the effector function of tumor-infiltrating CD8+ T cells and NK cells, evidenced by increased expression of cytotoxic molecules. RFR-induced inhibition of PMM growth was mediated by RFR-activated CD8+ T cells and NK cells. We conclude that noninvasive RFR exposure induces antitumor remodeling of the TIME, leading to inhibition of tumor progression, which provides a promising novel strategy for TIME priming and potential combination with cancer immunotherapy.

Repurposing CRISPR RNA-guided integrases system for one-step, efficient genomic integration of ultra-long DNA sequences.

Genomic integration techniques offer opportunities for generation of engineered microorganisms with improved or even entirely new functions but are currently limited by inability for efficient insertion of long genetic payloads due to multiplexing. Herein, using Shewanella oneidensis MR-1 as a model, we developed an optimized CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST system), which enables programmable, RNA-guided transposition of ultra-long DNA sequences (30 kb) onto bacterial chromosomes at ∼100% efficiency in a single orientation. In this system, a crRNA (CRISPR RNA) was used to target multicopy loci like insertion-sequence elements or combining I-SceI endonuclease, thereby allowing efficient single-step multiplexed or iterative DNA insertions. The engineered strain exhibited drastically improved substrate diversity and extracellular electron transfer ability, verifying the success of this system. Our work greatly expands the application range and flexibility of genetic engineering techniques and may be readily extended to other bacteria for better controlling various microbial processes.

Efficient and precise control of gene expression in Geobacter sulfurreducens through new genetic elements and tools for pollutant conversion.

Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well-characterized genetic elements and gene expression tools constrains the effective and precise fine-tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene-aroK and morphogenic genes-ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO3 ), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications.

Multiple roles of released c-type cytochromes in tuning electron transport and physiological status of Geobacter sulfurreducens.

Dissimilatory metal-reducing bacteria (DMRB) can transfer electrons to extracellular insoluble electron acceptors and play important roles in geochemical cycling, biocorrosion, environmental remediation, and bioenergy generation. c-type cytochromes (c-Cyts) are synthesized by DMRB and usually transported to the cell surface to form modularized electron transport conduits through protein assembly, while some of them are released as extracellularly free-moving electron carriers in growth to promote electron transport. However, the type of these released c-Cyts, the timing of their release, and the functions they perform have not been unrevealed yet. In this work, after characterizing the types of c-Cyts released by Geobacter sulfurreducens under a variety of cultivation conditions, we found that these c-Cyts accumulated up to micromolar concentrations in the surrounding medium and conserved their chemical activities. Further studies demonstrated that the presence of c-Cyts accelerated the process of microbial extracellular electron transfer and mediated long-distance electron transfer. In particular, the presence of c-Cyts promoted the microbial respiration and affected the physiological state of the microbial community. In addition, c-Cyts were observed to be adsorbed on the surface of insoluble electron acceptors and modify electron acceptors. These results reveal the overlooked multiple roles of the released c-Cyts in acting as public goods, delivering electrons, modifying electron acceptors, and even regulating bacterial community structure in natural and artificial environments.

Oxidation of Sb(III) by Shewanella species with the assistance of extracellular organic matter.

Antimony (Sb) is a toxic substance that poses a serious ecological threat when released into the environment. The species and redox state of Sb determine its environmental toxicity and fate. Understanding the redox transformations and biogeochemical cycling of Sb is crucial for analyzing and predicting its environmental behavior. Dissolved organic matter (DOM) in the environment greatly affects the fate of Sb. Microbially produced DOM is a vital component of environmental DOM; however, its specific role in Sb(III) oxidation has not been experimentally confirmed. In this work, the oxidation capacity of several Shewanella strains and their derived DOM to Sb(III) was confirmed. The oxidation rate of Sb(III) shows a positive correlation with DOM concentration, with higher rates observed under neutral and weak alkaline conditions, regardless of the presence of light. Incubation experiments indicated that extracellular enzymes and common reactive oxygen species were not involved in the oxidation of Sb(III). Characteristics of DOM suggests that microbial humic acid-like and fulvic acid-like substances are the potential contributors to Sb(III) oxidation. These findings not only experimentally validate the role of bacterial-derived DOM in Sb(III) oxidation but also reveal the significance of Shewanella and biogenic DOM in the biogeochemical cycling of Sb.

Ligand-Assisted Formation of Soluble Mn(III) and Bixbyite-like Mn2O3 by Shewanella putrefaciens CN32.

Functional material synthesis through biomineralization is effective and environmentally friendly. Biomineralized manganese (Mn) oxides are important for remediation and energy storage. Manganese(II) biomineralization is achieved by a diverse group of bacteria. We show that in the presence of oxygen the dissimilatory manganese-reducing bacterium Shewanella putrefaciens CN32 can oxidize Mn(II). The Mn(II) oxidation was accelerated with the increase in the initial Mn(II) concentration from 0.5 to 3 mM. The reaction was mainly associated with a cell-free filtrate, rather than the direct enzymatic oxidation or indirect oxidation by reactive oxygen species or macrocyclic siderophores. Instead, indirect oxidization of Mn(II) into soluble Mn(III) and bixbyite-like Mn2O3 via microbially produced extracellular ligands (molecular weights of 1-3 kDa) was identified. This work broadens our view about microbial Mn(II) oxidation and unveils the important roles of Shewanella species in the geochemical cycling of manganese.

Detection and Quantification of Antimicrobial-Resistant Cells in Aquatic Environments by Bioorthogonal Noncanonical Amino Acid Tagging.

Aquatic environments are important reservoirs of antibiotic wastes, antibiotic resistance genes, and bacteria, enabling the persistence and proliferation of antibiotic resistance in different bacterial populations. To prevent the spread of antibiotic resistance, effective approaches to detect antimicrobial susceptibility in aquatic environments are highly desired. In this work, we adopt a metabolism-based bioorthogonal noncanonical amino acid tagging (BONCAT) method to detect, visualize, and quantify active antimicrobial-resistant bacteria in water samples by exploiting the differences in bacterial metabolic responses to antibiotics. The BONCAT approach can be applied to rapidly detect bacterial resistance to multiple antibiotics within 20 min of incubation, regardless of whether they act on proteins or DNA. In addition, the combination of BONCAT with the microscope enables the intuitive characterization of antibiotic-resistant bacteria in mixed systems at single-cell resolution. Furthermore, BONCAT coupled with flow cytometry exhibits good performance in determining bacterial resistance ratios to chloramphenicol and population heterogeneity in hospital wastewater samples. In addition, this approach is also effective in detecting antibiotic-resistant bacteria in natural water samples. Therefore, such a simple, fast, and efficient BONCAT-based approach will be valuable in monitoring the increase and spread of antibiotic resistance within natural and engineered aquatic environments.

Single Strain-Triggered Biogeochemical Cycle of Arsenic.

The microbial metabolism of arsenic plays a prominent role in governing the biogeochemical cycle of arsenic. Although diverse microbes are known to be involved in the redox transformation of inorganic arsenic, the underlying mechanisms about the arsenic redox cycle mediated by a single microbial strain remain unclear yet. Herein, we discover that Shewanella putrefaciens CN32, a well-known arsenate-respiring and dissimilatory metal-reducing bacterium, could mediate the reversible arsenic redox transformation under aerobic conditions. Genetic analysis shows that S. putrefaciens CN32 contains both ars and arr operon but lacks an As(III) oxidase encoding gene. Arsenic(V) reduction tests demonstrate that the ars operon is advantageous but not essential for As(V) respiration in S. putrefaciens CN32. The Arr complex encoded by the arr operon not only plays a crucial role in arsenate respiration under anaerobic conditions but also participates in the sequential process of As(V) reduction and As(III) oxidation under aerobic conditions. The Arr enzyme also contributes to the microbial As(III) resistance. The expression and catalysis directionality of Arr in S. putrefaciens CN32 are regulated by the carbon source types. Our results highlight the complexity of arsenic redox biotransformation in environments and provide new insights into the important contribution of Arr to the As biogeochemical cycle in nature.

Controlling pathogenic risks of water treatment biotechnologies at the source by genetic editing means.

Antimicrobial-resistant pathogens in the environment and wastewater treatment systems, many of which are also important pollutant degraders and are difficult to control by traditional disinfection approaches, have become an unprecedented treat to ecological security and human health. Here, we propose the adoption of genetic editing techniques as a highly targeted, efficient and simple tool to control the risks of environmental pathogens at the source. An 'all-in-one' plasmid system was constructed in Aeromonas hydrophila to accurately identify and selectively inactivate multiple key virulence factor genes and antibiotic resistance genes via base editing, enabling significantly suppressed bacterial virulence and resistance without impairing their normal phenotype and pollutant-degradation functions. Its safe application for bioaugmented treatment of synthetic textile wastewater was also demonstrated. This genetic-editing technique may offer a promising solution to control the health risks of environmental microorganisms via targeted gene inactivation, thereby facilitating safer application of water treatment biotechnologies.

Extracellular electron transfer via multiple electron shuttles in waterborne Aeromonas hydrophila for bioreduction of pollutants.

Members of the genus Aeromonas prevail in aquatic habitats and have a great potential in biological wastewater treatment because of their unique extracellular electron transfer (EET) capabilities. However, the mediated EET mechanisms of Aeromonas have not been fully understood yet, hindering their applications in biological wastewater treatment processes. In this study, the electron shuttles in Aeromonas hydrophila, a model and widespread strain in aquatic environments and wastewater treatment plants, were explored. A. hydrophila was found to produce both flavins and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as electron shuttles and utilize them to accelerate its EET for the bioreduction of various pollutants. The Mtr-like respiratory pathway was essential for the reduction of flavins, but not involved in the ACNQ reduction. The electron shuttle activity of ACNQ for pollutant bioreduction involved the redox reactions that occurred inside the cell. These findings deepen our understanding about the underlying EET mechanisms in dissimilatory metal reducing bacteria and provide new insights into the roles of the genus Aeromonas in biological wastewater treatment.

Enhanced Bioreduction of Radionuclides by Driving Microbial Extracellular Electron Pumping with an Engineered CRISPR Platform.

Dissimilatory metal-reducing bacteria (DMRB) with extracellular electron transfer (EET) capability show great potential in bioremediating the subsurface environments contaminated by uranium through bioreduction and precipitation of hexavalent uranium [U(VI)]. However, the low EET efficiency of DMRB remains a bottleneck for their applications. Herein, we develop an engineered CRISPR platform to drive the extracellular electron pumping of Shewanella oneidensis, a representative DMRB species widely present in aquatic environments. The CRISPR platform allows for highly efficient and multiplex genome editing and rapid platform elimination post-editing in S. oneidensis. Enabled by such a platform, a genomic promoter engineering strategy (GPS) for genome-widely engineering the EET-encoding gene network was established. The production of electron conductive Mtr complex, synthesis of electron shuttle flavin, and generation of NADH as intracellular electron carrier are globally optimized and promoted, leading to a significantly enhanced EET ability. Applied to U(VI) bioreduction, the edited strains achieve up to 3.62-fold higher reduction capacity over the control. Our work endows DMRB with an enhanced ability to remediate the radionuclides-contaminated environments and provides a gene editing approach to handle the growing environmental challenges of radionuclide contaminations.

Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation.

Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.

Promoting bidirectional extracellular electron transfer of Shewanella oneidensis MR-1 for hexavalent chromium reduction via elevating intracellular cAMP level.

The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3',5'-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP-cyclic adenosine 3',5'-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP-CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.

CRISPRi System as an Efficient, Simple Platform for Rapid Identification of Genes Involved in Pollutant Transformation by Aeromonas hydrophila.

Aeromonas species are indigenous in diverse aquatic environments and play important roles in environmental remediation. However, the pollutant transformation mechanisms of these bacteria remain elusive, and their potential in pollution control is largely unexploited so far. In this work, we report an efficient and simple genome regulation tool to edit Aeromonas hydrophila and identify its biomolecular pathways for pollutant transformation. The genome regulation system, which is based on the type II clustered regularly interspaced short palindromic repeat interference (CRISPRi) system from Streptococcus pyogenes, can serve as a reversible and multiplexible platform for gene knockdown in A. hydrophila. A single-plasmid CRISPRi system harboring both dCas9 and the sgRNA was constructed in A. hydrophila and used to silence diverse genes with varied sizes and expression levels. With this system, up to 467-fold repression of gfp expression was achieved, and the function of the essential gene-ftsZ was identified quickly and accurately. Furthermore, simultaneous transcriptional repression of multiple targeted genes was realized. We discovered that the ars operon played an essential role in arsenic detoxification, and the extracellular electron transfer (EET) pathway was involved in methyl orange reduction, but not in vanadium reduction by A. hydrophila. Our method allows better insights and effective genetic manipulation of the pollutant transformation processes in Aeromonas, which might facilitate more efficient utilization of the Aeromonas species and other microbial species for environmental remediation applications.

Rediverting Electron Flux with an Engineered CRISPR-ddAsCpf1 System to Enhance the Pollutant Degradation Capacity of Shewanella oneidensis.

Pursuing efficient approaches to promote the extracellular electron transfer (EET) of extracellular respiratory bacteria is essential to their application in environmental remediation and waste treatment. Here, we report a new strategy of tuning electron flux by clustered regularly interspaced short palindromic repeat (CRISPR)-ddAsCpf1-based rediverting (namely STAR) to enhance the EET capacity of Shewanella oneidensis MR-1, a model extracellular respiratory bacterium widely present in the environment. The developed CRISPR-ddAsCpf1 system enabled approximately 100% gene repression with the green fluorescent protein (GFP) as a reporter. Using a WO3 probe, 10 representative genes encoding for putative competitive electron transfer proteins were screened, among which 7 genes were identified as valid targets for EET enhancement. Repressing the valid genes not only increased the transcription level of the l-lactate metabolism genes but also affected the genes involved in direct and indirect EET. Increased riboflavin production was also observed. The feasibility of this strategy to enhance the bioreduction of methyl orange, an organic pollutant, and chromium, a typical heavy metal, was demonstrated. This work implies a great potential of the STAR strategy with the CIRPSR-ddAsCpf1 system for enhancing bacterial EET to favor more efficient environmental remediation applications.

Sensing and Approaching Toxic Arsenate by Shewanella putrefaciens CN-32.

Although arsenic at a high concentration imposes strong selective pressure on microbes, various microbes have been found to grow in As-rich environments. So far, little is known about how microbes can sense and move toward arsenate in the environment, and the underlying molecular mechanisms have not been revealed. Here, we report the chemotaxis response toward arsenate (As(V)) by Shewanella putrefaciens CN-32, a model dissimilatory metal-reducing bacterium (DMRB), and elucidate the mechanisms. We find that S. putrefaciens CN-32 exhibits a chemotactic behavior toward As(V) and diverse electron acceptors. To sense As(V), S. putrefaciens CN-32 requires functional arsenate respiratory reductase but does not depend on its metal-reducing-like respiratory pathway. We observe that such a sense is governed by an energy taxis mechanism and mediated by several methyl-accepting chemotaxis proteins (MCPs), rather than a specific MCP. Moreover, we reveal that the chemotactic signal transduction pathway is conserved in Shewanella, and histidine kinase and flagella-mediated motility are essential for taxis toward As(V). This work reverses the conventional view about arsenic as a chemotactic inhibitor to microbes by revealing the positive chemotaxis of Shewanella to As(V).

Nicotinamide N-Methyltransferase Suppression Participates in Nickel-Induced Histone H3 Lysine9 Dimethylation in BEAS-2B Cells.

BACKGROUND: Nickel compounds are well-established human carcinogens with weak mutagenic activity. Histone methylation has been proposed to play an important role in nickel-induced carcinogenesis. Nicotinamide N-methyltransferase (NNMT) decreases histone methylation in several cancer cells by altering the cellular ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). However, the role of NNMT in nickel-induced histone methylation remains unclear. METHODS: BEAS-2B cells were exposed to different concentrations of nickel chloride (NiCl2) for 72 h or 200 µM NiCl2 for different time periods. Histone H3 on lysine 9 (H3K9) mono-, di-, and trimethylation and NNMT protein levels were measured by western blot analysis. Expressions of NNMT mRNA and the H3k9me2-associated genes, mitogen-activated protein kinase 3 (MAP2K3) and dickkopf1 (DKK1), were determined by qPCR analysis. The cellular ratio of nicotinamide adenine dinucleotide (NAD+) to reduced NAD (NADH) and SAM/SAH ratio were determined. RESULTS: Exposure of BEAS-2B cells to nickel increased H3K9 dimethylation (H3K9me2), suppressed the expressions of H3K9me2-associated genes (MAP2K3 and DKK1), and induced NNMT repression at both the protein and mRNA levels. Furthermore, over-expression of NNMT inhibited nickel-induced H3K9me2 and altered the cellular SAM/SAH ratio. Additionally, the NADH oxidant phenazine methosulfate (PMS) not only reversed the nickel-induced reduction in NAD+/NADH but also inhibited the increase in H3K9me2. CONCLUSIONS: These findings indicate that the repression of NNMT may underlie nickel-induced H3K9 dimethylation by altering the cellular SAM/SAH ratio.