ABSTRACT
Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures. We recently developed a nucleic acid cytometry platform called 'focused interrogation of cells by nucleic acid detection and sequencing' (FIND-seq), capable of isolating rare cells on the basis of RNA or DNA markers, followed by bulk or single-cell transcriptomic analysis. This platform has previously been used to characterize the splicing-dependent activation of the transcription factor XBP1 in astrocytes and HIV persistence in memory CD4 T cells from people on long-term antiretroviral therapy. Here, we outline the molecular and microfluidic steps involved in performing FIND-seq, including protocol updates that allow detection and whole transcriptome sequencing of rare HIV-infected cells that harbor genetically intact virus genomes. FIND-seq requires knowledge of microfluidics, optics and molecular biology. We expect that FIND-seq, and this comprehensive protocol, will enable mechanistic studies of rare HIV+ cells, as well as other cell subsets that were previously difficult to recover and sequence.
ABSTRACT
Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.
Subject(s)
Astrocytes , Encephalomyelitis, Autoimmune, Experimental , Epigenetic Memory , Multiple Sclerosis , Animals , Female , Humans , Male , Mice , Acetyl Coenzyme A/metabolism , Astrocytes/enzymology , Astrocytes/metabolism , Astrocytes/pathology , ATP Citrate (pro-S)-Lyase/metabolism , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation Sequencing , CRISPR-Cas Systems , Encephalomyelitis, Autoimmune, Experimental/enzymology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Inflammation/enzymology , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Multiple Sclerosis/enzymology , Multiple Sclerosis/genetics , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Single-Cell Gene Expression Analysis , Transposases/metabolismABSTRACT
Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE) 1-8 . However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY + p300 + memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY + p300 + astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro ; scRNA-seq and immunohistochemistry studies detected increased ACLY + p300 + astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.
ABSTRACT
Mixed phenotype acute leukemia (MPAL) is a leukemia whose biologic drivers are poorly understood, therapeutic strategy remains unclear, and prognosis is poor. We performed multiomic single cell (SC) profiling of 14 newly diagnosed adult MPAL patients to characterize the immunophenotypic, genetic, and transcriptional landscapes of MPAL. We show that neither genetic profile nor transcriptome reliably correlate with specific MPAL immunophenotypes. However, progressive acquisition of mutations is associated with increased expression of immunophenotypic markers of immaturity. Using SC transcriptional profiling, we find that MPAL blasts express a stem cell-like transcriptional profile distinct from other acute leukemias and indicative of high differentiation potential. Further, patients with the highest differentiation potential demonstrated inferior survival in our dataset. A gene set score, MPAL95, derived from genes highly enriched in this cohort, is applicable to bulk RNA sequencing data and was predictive of survival in an independent patient cohort, suggesting utility for clinical risk stratification.
ABSTRACT
Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.
Subject(s)
Amphiregulin , Astrocytes , Autocrine Communication , Genetic Testing , Microfluidic Analytical Techniques , Microglia , Astrocytes/physiology , Genetic Testing/methods , High-Throughput Screening Assays , Microfluidic Analytical Techniques/methods , Microglia/physiology , Amphiregulin/genetics , Autocrine Communication/genetics , Gene Expression , HumansABSTRACT
Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.
Subject(s)
High-Throughput Nucleotide Sequencing , Microfluidics , Humans , Animals , Mice , Microfluidics/methods , High-Throughput Nucleotide Sequencing/methods , Single-Cell Analysis/methods , Genomics/methods , Transcriptome/geneticsABSTRACT
Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.
Subject(s)
CD4-Positive T-Lymphocytes , Gene Expression Regulation, Viral , HIV Infections , HIV-1 , Virus Latency , Humans , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA, Viral/isolation & purification , Gene Expression Regulation, Viral/drug effects , HIV Infections/drug therapy , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/virology , HIV-1/drug effects , HIV-1/genetics , HIV-1/isolation & purification , HIV-1/pathogenicity , Immunologic Memory , Microfluidics , Necroptosis/drug effects , Signal Transduction/drug effects , Transcriptome/drug effects , Virus Latency/drug effects , Anti-Retroviral Agents/pharmacology , Anti-Retroviral Agents/therapeutic useABSTRACT
Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.
Subject(s)
Astrocytes , Encephalomyelitis, Autoimmune, Experimental , Microfluidics , Multiple Sclerosis , Nucleic Acids , Single-Cell Gene Expression Analysis , Animals , Humans , Mice , Astrocytes/metabolism , Astrocytes/pathology , Gene Expression Regulation , Mice, Knockout , Multiple Sclerosis/pathology , Microfluidics/methods , Single-Cell Gene Expression Analysis/methods , Nucleic Acids/analysis , Gene EditingABSTRACT
Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets.
Subject(s)
Astrocytes/physiology , Cell Communication , Central Nervous System/pathology , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Microglia/physiology , Multiple Sclerosis/physiopathology , Single-Cell Analysis , Animals , Antigens, CD/metabolism , Brain/pathology , Brain/physiopathology , Central Nervous System/physiopathology , Encephalomyelitis, Autoimmune, Experimental/pathology , Ephrin-B3/metabolism , Herpesvirus 1, Suid/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Multiple Sclerosis/pathology , NF-kappa B/metabolism , Nerve Tissue Proteins/metabolism , RNA-Seq , Reactive Oxygen Species/metabolism , Receptor, EphB3/antagonists & inhibitors , Receptor, EphB3/metabolism , Receptors, Cell Surface/metabolism , Semaphorins/metabolism , Signal Transduction , T-Lymphocytes/physiology , TOR Serine-Threonine Kinases/metabolismABSTRACT
The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence-function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant "S helix" located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , DNA/chemistry , DNA/metabolism , Microfluidics , Protein Engineering , Protein Interaction Domains and Motifs , Protein Kinases/chemistry , Protein Kinases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Enzyme Activation , Gene Expression , Gene Library , Microfluidics/instrumentation , Microfluidics/methods , Models, Molecular , Molecular Conformation , Protein Engineering/methods , Protein Kinases/genetics , Structure-Activity RelationshipABSTRACT
Analyzing every cell in a diverse sample provides insight into population-level heterogeneity, but abundant cell types dominate the analysis and rarer populations are scarcely represented in the data. To focus on specific cell types, the current paradigm is to physically isolate subsets of interest prior to analysis; however, it remains difficult to isolate and then single-cell sequence such populations because of compounding losses. Here, we describe an alternative approach that selectively merges cells with reagents to achieve enzymatic reactions without having to physically isolate cells. We apply this technique to perform single-cell transcriptome and genome sequencing of specific cell subsets. Our method for analyzing heterogeneous populations obviates the need for pre- or post-enrichment and simplifies single-cell workflows, making it useful for other applications in single-cell biology, combinatorial chemical synthesis, and drug screening.
Subject(s)
Sequence Analysis, DNA/methods , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Cell Line, Tumor , Fluorescence , High-Throughput Nucleotide Sequencing , HumansABSTRACT
Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.
Subject(s)
Astrocytes/pathology , Central Nervous System/pathology , Inflammation/pathology , MafG Transcription Factor/genetics , Repressor Proteins/genetics , Animals , Antioxidants/metabolism , Astrocytes/metabolism , Central Nervous System/metabolism , DNA Methylation , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Inflammation/genetics , Male , Methionine Adenosyltransferase/genetics , Mice , Multiple Sclerosis/genetics , Multiple Sclerosis/pathology , NF-E2-Related Factor 2/genetics , Sequence Analysis, RNA , Signal Transduction , Transcription, GeneticABSTRACT
Microsphere beads are functionalized with oligonucleotides, antibodies, and other moieties to enable specific detection of analytes. Droplet microfluidics leverages this for single-molecule or -cell analysis by pairing beads and targets in water-in-oil droplets. Pairing is achieved with devices operating in the dripping regime, limiting throughput. Here, we describe a pairing method that uses beads to trigger the breakup of a jet into monodispersed droplets. We use the method to pair 105 Human T cells with polyacrylamide beads ten times faster than methods operating in the dripping regime. Our method improves the throughput of bead-based droplet workflows, enabling analysis of large populations and the detection of rare events.
Subject(s)
Lab-On-A-Chip Devices , Microspheres , Capsules , Elasticity , Equipment Design , Single-Cell AnalysisABSTRACT
Microfluidic droplet sorting allows selection of subpopulations of cells, nucleic acids, and biomolecules with soluble assays. Dielectrophoresis is widely used for sorting because it generates strong forces on droplets, actuates rapidly, and is easy to integrate into microfluidic chips. However, existing device designs apply a short force, limiting the deflection of droplets, and therefore the speed and reliability of sorting. We describe a concentric design that applies a long force, allowing large deflections and increased reliability. We demonstrate the utility of this design by sorting polydisperse emulsions, which are typically difficult to sort with high purity.
Subject(s)
Microfluidic Analytical Techniques/instrumentation , Adsorption , Electrodes , Equipment Design , Particle Size , Surface PropertiesABSTRACT
Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.
Subject(s)
Microfluidic Analytical Techniques/instrumentation , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Single-Cell Analysis/instrumentation , Equipment Design , Gene Expression , Humans , Jurkat Cells , MCF-7 CellsABSTRACT
Nucleic acids encode the information of life, programming cellular functions and dictating many biological outcomes. Differentiating between cells based on their nucleic acid programs is, thus, a powerful way to unravel the genetic bases of many phenotypes. This is especially important considering that most cells exist in heterogeneous populations, requiring them to be isolated before they can be studied. Existing flow cytometry techniques, however, are unable to reliably recover specific cells based on nucleic acid content. Nucleic acid cytometry is a new field built on droplet microfluidics that allows robust identification, sorting, and sequencing of cells based on specific nucleic acid biomarkers. This review highlights applications that immediately benefit from the approach, biological questions that can be addressed for the first time with it, and considerations for building successful workflows.
Subject(s)
Flow Cytometry , Microfluidic Analytical Techniques , Nucleic Acids , Animals , Cells, Cultured , Humans , Mice , Nucleic Acids/analysis , Nucleic Acids/chemistry , Nucleic Acids/isolation & purificationABSTRACT
Uniform amplification of low input DNA is important for applications across biology, including single-cell genomics, forensic science, and microbial and viral sequencing. However, the requisite biochemical amplification methods are prone to bias, skewing sequence proportions and obscuring signals relating to copy number. Digital droplet multiple displacement amplification enables uniform amplification, but requires expert knowledge of microfluidics to generate monodisperse emulsions. In addition, existing microfluidic methods are tedious and labor intensive for preparing many samples. Here, we introduce rapid emulsification multiple displacement amplification, a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter. While conventional microfluidic devices require >10 minutes to emulsify a sample, our system takes tens of seconds and yields data of equivalent quality. We demonstrate the approach by using it to accurately measure copy number variation in single cancer cells.
ABSTRACT
Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 µm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.
Subject(s)
Bacterial Proteins/chemistry , DNA Helicases/chemistry , Oxidoreductases/chemistry , Perchlorates/chemistry , Rhodocyclaceae/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , DNA Helicases/genetics , DNA Helicases/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Perchlorates/metabolism , Rhodocyclaceae/geneticsABSTRACT
Genes important for growth of Pseudomonas stutzeriâ PDA on chlorate were identified using a randomly DNA bar-coded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB and clrC were essential for chlorate reduction, while clrD, clrE and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillumâ PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment.