Biology subjects

Lee, J.

Publications and source records attributed to Lee, J..

At least 757 records · Page 42Linked to original sources

bioRxiv2020

Combined assessment of MHC binding and antigen expression improves T cell epitope predictions

MHC class I antigen processing consists of multiple steps that result in the presentation of MHC bound peptides that can be recognized as T cell epitopes. Many of the pathway steps can be predicted using computational methods, but one is often neglected: mRNA expression of the epitope source proteins. In this study, we improve epitope prediction by taking into account both peptide-MHC binding affinities and expression levels of the peptides source protein. Specifically, we utilized biophysical principles and existing MHC binding prediction tools in concert with RNA expression to derive a function that estimates the likelihood of a peptide being presented on a given MHC class I molecule. Our combined model of Antigen eXpression based Epitope Likelihood-Function (AXEL-F) outperformed predictions based only on binding or based only on antigen expression for discriminating eluted ligands from random background peptides as well as in predicting neoantigens that are recognized by T cells. We also showed that in cases where cancer patient-specific RNA-Seq data is not available, cancer-type matched expression data from TCGA can be used to accurately estimate patient-specific gene expression. Using AXEL-F together with TGCA expression data we were able to more accurately predict neoantigens that are recognized by T cells. The method is available in the IEDB Analysis Resource and free to use for the academic community. Significance statementEpitope prediction tools have been used to call epitopes in viruses and other pathogens for almost 30 years, and more recently, to call cancer neoantigens. Several such tools have been developed, however most of them ignore the mRNA expression of the epitope source proteins. In the present study, we have, to our knowledge for the first time, developed a biophysically motivated model to combine peptide-MHC binding and abundance of the peptides source protein to improve epitope predictions. Our novel tool AXEL-F is freely available on the IEDB and presents a clear opportunity for predicting and selecting epitopes more efficiently.

bioinformatics↗

bioRxiv2020

Coexpression reveals conserved mechanisms of transcriptional cell identity

What makes a mouse a mouse, and not a hamster? The answer lies in the genome, and more specifically, in differences in gene regulation between the two organisms: where and when each gene is expressed. To quantify differences, a typical study will either compare functional genomics data from homologous tissues, limiting the approach to closely related species; or compare gene repertoires, limiting the resolution of the analysis to gross correlations between phenotypes and gene family size. As an alternative, gene coexpression networks provide a basis for studying the evolution of gene regulation without these constraints. By incorporating data from hundreds of independent experiments, meta-analytic coexpression networks reflect the convergent output of species-specific transcriptional regulation. In this work, we develop a measure of regulatory evolution based on gene coexpression. Comparing data from 14 species, we quantify the conservation of coexpression patterns 1) as a function of evolutionary time, 2) across orthology prediction algorithms, and 3) with reference to cell- and tissue-specificity. Strikingly, we uncover deeply conserved patterns of gradient-like expression across cell types from both the animal and plant kingdoms. These results suggest that ancient genes contribute to transcriptional cell identity through mechanisms that are independent of duplication and divergence.

bioinformatics↗

bioRxiv2020

Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate

BackgroundCoronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. MethodWe constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Results and conclusionsThese three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. General significanceBy genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

immunology↗

bioRxiv2020

Phylogenetic reconstruction of myeloproliferative neoplasm reveals very early origins and lifelong evolution

Mutations in cancer-associated genes drive tumour outgrowth. However, the timing of driver mutations and dynamics of clonal expansion that lead to human cancers are largely unknown. We used 448,553 somatic mutations from whole-genome sequencing of 843 clonal haematopoietic colonies to reconstruct the phylogeny of haematopoiesis, from embryogenesis to clinical disease, in 10 patients with myeloproliferative neoplasms which are blood cancers more common in older age. JAK2V617F, the pathognomonic mutation in these cancers, was acquired in utero or childhood, with upper estimates of age of acquisition ranging between 4.1 months and 11.4 years across 5 patients. DNMT3A mutations, which are associated with age-related clonal haematopoiesis, were also acquired in utero or childhood, by 7.9 weeks of gestation to 7.8 years across 4 patients. Subsequent driver mutation acquisition was separated by decades. The mean latency between JAK2V617F acquisition and clinical presentation was 31 years (range 12-54 years). Rates of clonal expansion varied substantially (<10% to >200% expansion/year), were affected by additional driver mutations, and predicted latency to clinical presentation. Driver mutations and rates of expansion would have been detectable in blood one to four decades before clinical presentation. This study reveals how driver mutation acquisition very early in life with life-long growth and evolution drive adult blood cancer, providing opportunities for early detection and intervention, and a new paradigm for cancer development.

cancer biology↗

bioRxiv2020

In-vivo histology of iron and myelin in the brain using magnetic susceptibility source separation in MRI

Obtaining a histological fingerprint from the in-vivo brain has been a long-standing target of magnetic resonance imaging (MRI). In particular, non-invasive imaging of iron and myelin, which are involved in normal brain functions and are histopathological hallmarks in neurodegenerative diseases, has practical utilities in neuroscience and medicine. Here, we propose a biophysical model that describes the individual contribution of paramagnetic (e.g., iron) and diamagnetic (e.g., myelin) susceptibility sources to the frequency shift and transverse relaxation of MRI signals. Using this model, we develop a method,{chi} -separation, that generates the voxel-wise distributions of the two sources. The method is validated using computer simulation and phantom experiments, and applied to ex-vivo and in-vivo brains. The results delineate the well-known histological features of iron and myelin in the specimen, healthy volunteers, and multiple sclerosis patients. This new technology may serve as a practical tool for exploring the microstructural information of the brain.

neuroscience↗

bioRxiv2020

SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel(R) were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-{gamma}, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel(R) containing formulation and possibly in combination with other immunostimulants.

immunology↗

bioRxiv2020

Bromodomain protein inhibition protects β-cells from cytokine-induced death and dysfunction via antagonism of NF-κB pathway.

Cytokine induced {beta}-cell apoptosis is the major pathogenic mechanism in type 1 diabetes (T1D). Despite significant advances in understanding underlying mechanisms, few drugs have been translated to protect {beta}-cells in T1D. Epigenetic modulators such as bromodomain-containing BET (Bromo- and Extra-Terminal) proteins are important regulators of immune responses. Pre-clinical studies have demonstrated a protective effect of BET inhibitors in NOD (non-obese diabetes) mouse model of T1D. However, the role of BET proteins in {beta}-cell function in response to cytokines is unknown. Here we demonstrate that I-BET, a BET protein inhibitor, protected {beta}-cells from cytokine induced dysfunction and death. In vivo administration of I-BET to mice exposed to low-dose STZ (streptozotocin), a model of T1D, significantly reduced {beta}-cell apoptosis and preserved {beta}-cell mass, suggesting a cytoprotective function of I-BET. Furthermore, human islets treated with I-BET displayed better glucose stimulated insulin secretion compared to controls, when exposed to cytokines. Mechanistically, RNA-Seq analysis revealed I-BET treatment suppressed pathways involved in apoptosis, including NF-kB signaling, while maintaining the expression of genes critical for {beta}-cell function, such as Pdx1 and Ins1. Taken together, this study demonstrates that I-BET is effective in protecting {beta}-cells from cytokine-induced dysfunction and apoptosis, and may have potential therapeutic values in T1D.

molecular biology↗

bioRxiv2020

Yeast Nuak1 phosphorylates histone H3 threonine 11 in low glucose stress conditions by the cooperation of AMPK and CK2 signaling

Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress responsive genes. Understanding how this histone modification senses external glucose changes remains elusive. Here, we show that Tda1, the yeast orthologue of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMPK directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation.

cell biology↗

bioRxiv2020

LH LepR neurons directly drive sustained food seeking behavior after AgRP neuronal deactivation via neuromodulation of NPY

The physiological need for energy evokes motivated feeding behaviours that help to ensure survival. However, the neural mechanisms underlying the generation of food motivation remain poorly understood. We investigated these mechanisms by subdividing feeding-related motivated behaviours into food-seeking, evaluating, and swallowing. Micro-endoscopic results indicated that neurons containing leptin receptors (LepRs) in the lateral hypothalamus (LH) are the major food-specific subpopulation of LHGABA neurons. Optogenetic manipulation of LHLepR neurons bidirectionally regulated both food-seeking and evaluating. Furthermore, micro-endoscope data revealed that distinct LHLepR neurons encode seeking and evaluating. Computational modelling analysis demonstrated that LHLepR neurons encode motivation, whereas neurons containing agouti-related peptide and neuropeptide Y (AgRP/NPY) encode the need for food. Additionally, slice studies revealed that NPY decreases inhibitory input to LHLepR neurons via LHGABA interneurons. This mechanism explains the permissive gate role of hunger (food need) in seeking/evaluating motivation. Together, the present study provides a comprehensive neural mechanism of how physiological needs drive distinct motivated behaviours.

neuroscience↗

bioRxiv2020

Chlamydia trachomatis Encodes a Dynamic, Ring-Forming Bactofilin Critical for Maintaining Cell Size and Shape

Bactofilins are polymer-forming cytoskeletal proteins that are widely conserved in bacteria. Members of this protein family have diverse functional roles such as orienting subcellular molecular processes, establishing cell polarity, and aiding in cell shape maintenance. Chlamydia species are obligate intracellular bacteria that undergo a developmental cycle alternating between an infectious, non-dividing EB and a non-infectious, dividing RB. As Chlamydia divides by a polarized division process, we hypothesized that BacACT may function to establish polarity in these unique bacteria. Using sequence alignment to the conserved bactofilin domain, we identified a bactofilin ortholog, BacACT, in the obligate intracellular pathogen Chlamydia trachomatis. Utilizing a combination of fusion constructs and high-resolution fluorescence microscopy, we determined that BacACT forms a dynamic, membrane-associated, ring-like structure in Chlamydias replicative RB form. Contrary to our hypothesis, this filamentous ring structure is distinct from the microbes cell division machinery and does not colocalize with septal peptidoglycan or MreB, the major organizer of the bacteriums division complex. Bacterial two-hybrid assays demonstrated BacACT interacts homotypically but does not directly interact with proteins involved in cell division or peptidoglycan biosynthesis. To investigate the function of BacACT in chlamydial development, we constructed a conditional knockdown strain using a newly developed CRISPR interference system. We observed that reducing bacACT expression significantly impacted chlamydial cell size and morphology. Normal RB morphology was restored when an additional copy of BacACT was expressed in trans during knockdown. These data reveal a novel function for chlamydial bactofilin in maintaining cell shape in this obligate intracellular bacterium. IMPORTANCEChlamydia is an ancient, obligate intracellular bacterium with a unique biphasic developmental cycle. As a result of its evolution within the osmotically stable environment of a host cell, Chlamydia has lost its dependence on side-wall peptidoglycan, and maintains only a fraction of the components thought to be required for regulating bacterial cell size and division. As such, very little is known about how Chlamydia species carry out these critical processes in the absence of a stabilizing peptidoglycan layer. In the current study, we identify a novel cytoskeletal element, termed a bactofilin, that is critical for maintaining the morphology of the bacteria. Using state-of-the-art genetic techniques for this organism, we demonstrate that chlamydial bactofilin forms a dynamic ring structure independent of the microbes division machinery and that abrogating its expression level using CRISPR interference results in abnormal morphologic forms. These findings enhance our understanding of chlamydial biology and bactofilins more generally.

microbiology↗

bioRxiv2020

Inverse oculomotor responses of achiasmatic mice expressing a transfer-defective Vax1 mutant

In binocular animals that exhibit stereoscopic visual responses, the axons of retinal ganglion cells (RGCs) connect to brain areas bilaterally by forming a commissure called the optic chiasm (OC). Ventral anterior homeobox 1 (Vax1) contributes to formation of the OC, acting endogenously in optic pathway cells and exogenously in growing RGC axons. Here, we generated Vax1AA/AA mice expressing the Vax1AA mutant, which is incapable of intercellular transfer. We found that RGC axons cannot take up Vax1AA protein from Vax1AA/AA mouse optic stalk (OS) cells, of which maturation is delayed, and fail to access the midline. Consequently, RGC axons of Vax1AA/AA mice connect exclusively to ipsilateral brain areas, resulting in the reduced visual acuity and the abnormal oculomotor responses. Together, our study provides physiological evidences for the necessity of intercellular transfer of Vax1 and the importance of the bilateral RGC axon projection in visuomotor responses.

developmental biology↗

bioRxiv2020

Differential in vitro activity of individual drugs and bedaquiline-rifabutin combinations against actively multiplying and nutrient-starved Mycobacterium abscessus

Current treatment options for lung disease caused by Mycobacterium abscessus complex infections have limited effectiveness. To maximize the use of existing antibacterials and to help inform regimen design for treatment, we assessed the in vitro bactericidal activity of single drugs against actively multiplying and net non-replicating M. abscessus populations in nutrient-rich and nutrient starvation conditions, respectively. As single drugs, bedaquiline and rifabutin exerted bactericidal activity only against nutrient-starved and actively growing M. abscessus, respectively. However, when combined, both bedaquiline and rifabutin were able to specifically contribute bactericidal activity at relatively low, clinically relevant concentrations against both replicating and non-replicating bacterial populations. The addition of a third drug, amikacin, further enhanced the bactericidal activity of the bedaquiline-rifabutin combination against nutrient-starved M. abscessus. Overall, these in vitro data suggest that bedaquiline-rifabutin may be a potent backbone combination to support novel treatment regimens for M. abscessus infections. This rich dataset of differential time-and concentration-dependent activity of drugs, alone and together, against M. abscessus also highlights several issues affecting interpretation and translation of in vitro findings.

microbiology↗

bioRxiv2020

Comparison of Extracellular Vesicle Isolation Methods from Human Biofluids using Single Molecule Arrays

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

biochemistry↗

bioRxiv2020

Single cell transcriptome analysis defines novel heterogeneity within the pancreatic ductal tree

Lineage tracing using genetically engineered mouse models is an essential tool for investigating cell-fate decisions of progenitor cells and biology of mature cell types, with relevance to physiology and disease progression. To study disease development, an inventory of an organs cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify SPP1 as a regulator of this fate decision as well as human duct cell de-differentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for Geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis. SIGNIFICANCEMurine models are extensively used for pancreatic lineage tracing experiments and investigation of pancreatic disease progression. Here, we describe the transcriptome of murine pancreatic duct cells, intrapancreatic bile duct cells, and pancreatobiliary cells at single cell resolution. Our analysis defines novel heterogeneity within the pancreatic ductal tree and supports the paradigm that more than one population of pancreatic duct cells harbors progenitor capacity. We identify and validate unique functional properties of subpopulations of pancreatic duct cells including an epithelial-mesenchymal transcriptomic axis and roles in chronic pancreatic inflammation.

bioinformatics↗

bioRxiv2020

Negative controls of chemical probes can be misleading

Chemical probes are selective modulators that are used in cell assays to link a phenotype to a gene and have become indispensable tools to explore gene function and discover therapeutic targets. While binding to off-targets can be acceptable or beneficial for drugs, it is a confounding factor for chemical probes, as the observed phenotype may be driven by inhibition of an unknown off-target instead of the targeted protein. A negative control - a close chemical analog of the chemical probe that is inactive against the intended target - is typically used to verify that the phenotype is indeed driven by targeted protein. Here, we compare the selectivity profiles of four unrelated chemical probes and their respective negative controls and find that the control is sometimes inactive against up to 80% of known off-targets, suggesting that a lost phenotype upon treatment with the negative control may be driven by loss of inhibition of the off-target. To extend this analysis, we inspect the crystal structures of 90 pairs of unrelated proteins, where both proteins within each pair is in complex with the same drug-like ligand, and estimate that in 50% of cases, methylation (a simple chemical modification often used to generate negative controls) of the ligand at a position that will preclude binding to one protein (intended target) will also preclude binding to the other (off-target). These results uncover a risk associated with the use of negative controls to confirm gene-phenotype associations. We propose that a best practice should rather be to verify that two chemically unrelated chemical probes targeting the same protein lead to the same phenotype. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/320465v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@119bc09org.highwire.dtl.DTLVardef@8828c7org.highwire.dtl.DTLVardef@12d2d66org.highwire.dtl.DTLVardef@ff8d5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2020

Brain Connectivity and Behavioral Changes in aSpaceflight Analog Environment with Elevated CO2

Astronauts are exposed to microgravity and elevated CO2 levels onboard the International Space Station. Little is known about how microgravity and elevated CO2 combine to affect the brain and sensorimotor performance during and after spaceflight. Here we examined changes in resting-state functional connectivity (FC) and sensorimotor behavior associated with a spaceflight analog environment. Participants underwent 30 days of strict 6{degrees} head-down tilt bed rest with elevated ambient CO2 (HDBR+CO2). Resting-state functional magnetic resonance imaging and sensorimotor assessments were collected 13 and 7 days prior to bed rest, on days 7 and 29 of bed rest, and 0, 5, 12, and 13 days following bed rest. We assessed the time course of FC changes from before, during, to after HDBR+CO2. We then compared the observed connectivity changes with those of a HDBR control group, which underwent HDBR in standard ambient air. Moreover, we assessed associations between post-HDBR+CO2 FC changes and alterations in sensorimotor performance. HDBR+CO2 was associated with significant changes in functional connectivity between vestibular, visual, somatosensory and motor brain areas. Several of these sensory and motor regions showed post-HDBR+CO2 FC changes that were significantly associated with alterations in sensorimotor performance. We propose that these FC changes reflect multisensory reweighting associated with adaptation to the HDBR+CO2 microgravity analog environment. This knowledge will further improve HDBR as a model of microgravity exposure and contribute to our knowledge of brain and performance changes during and after spaceflight.

neuroscience↗

bioRxiv2020

Striatal indirect pathway mediates action switching via modulation of collicular dynamics

Type 2 dopamine receptor-expressing, or indirect pathway striatal projection (iSPNs), neurons comprise one of two major pathways through the basal ganglia1, and are a major drug target for treatment of neuropsychiatric disorders2-4. The function of iSPNs is unclear with proposed roles in suppression of unwanted actions and in refining selection actions or their kinematics5-12. Here, we show that iSPNs can simultaneously suppress and facilitate conflicting motor actions in a lateralized licking task. Activation of iSPNs suppresses contraversive while promoting ipsiversive licking, allowing mice to rapidly switch between alternative motor programs. iSPN activity is prokinetic even when mice are not cued to perform an action. Activity in lateral superior colliculus (lSC), a basal ganglia target, is necessary for performing the task and predicts action. Furthermore, iSPN activation suppresses ipsilateral lSC, but surprisingly, excites contralateral lSC. iSPN activity has neuron-specific effects that, at the population level, steers the neural trajectory towards that associated with ipsiversive licking. Thus, our results reveal a previously unknown specificity of iSPNs effects on downstream brain regions, including the ability to excite contralateral regions and trigger motor programs. These results suggest a general circuit mechanism for flexible action switching during competitive selection of lateralized actions.

neuroscience↗

bioRxiv2020

Enhancement of nitrous oxide emissions in soil microbial consortia via copper competition between proteobacterial methanotrophs and denitrifiers

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers in situ and thereby enhance N2O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soils native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Lastly, with microcosms amended with varying cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH4 enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration while implying the presence of alternative copper abstraction mechanisms for Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit. ImportanceProteobacterial methanotrophs, groups of microorganisms that utilize methane as source of energy and carbon, have been known to utilize unique mechanisms to scavenge copper, namely utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas N2O has been suggested in axenic and co-culture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments co-habited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allow for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N2O production may account for significant proportion of N2O efflux from agricultural soils.

microbiology↗