Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida
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Biotechnology & Applied Microbiology↗
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Anaerobic methane oxidation coupled to manganese reduction by members of the Methanoperedenaceae
Abstract Anaerobic oxidation of methane (AOM) is a major biological process that reduces global methane emission to the atmosphere. Anaerobic methanotrophic archaea (ANME) mediate this process through the coupling of methane oxidation to different electron acceptors, or in concert with a syntrophic bacterial partner. Recently, ANME belonging to the archaeal family Methanoperedenaceae (formerly known as ANME-2d) were shown to be capable of AOM coupled to nitrate and iron reduction. Here, a freshwater sediment bioreactor fed with methane and Mn(IV) oxides (birnessite) resulted in a microbial community dominated by two novel members of the Methanoperedenaceae, with biochemical profiling of the system demonstrating Mn(IV)-dependent AOM. Genomic and transcriptomic analyses revealed the expression of key genes involved in methane oxidation and several shared multiheme c-type cytochromes (MHCs) that were differentially expressed, indicating the likely use of different extracellular electron transfer pathways. We propose the names “Candidatus Methanoperedens manganicus” and “Candidatus Methanoperedens manganireducens” for the two newly described Methanoperedenaceae species. This study demonstrates the ability of members of the Methanoperedenaceae to couple AOM to the reduction of Mn(IV) oxides, which suggests their potential role in linking methane and manganese cycling in the environment.
54 ENVIRONMENTAL SCIENCES↗
MetaPop: a pipeline for macro- and microdiversity analyses and visualization of microbial and viral metagenome-derived populations
Abstract Background Microbes and their viruses are hidden engines driving Earth’s ecosystems from the oceans and soils to humans and bioreactors. Though gene marker approaches can now be complemented by genome-resolved studies of inter-(macrodiversity) and intra-(microdiversity) population variation, analytical tools to do so remain scattered or under-developed. Results Here, we introduce MetaPop, an open-source bioinformatic pipeline that provides a single interface to analyze and visualize microbial and viral community metagenomes at both the macro - and microdiversity levels. Macrodiversity estimates include population abundances and α- and β-diversity. Microdiversity calculations include identification of single nucleotide polymorphisms, novel codon-constrained linkage of SNPs, nucleotide diversity ( π and θ ), and selective pressures (pN/pS and Tajima’s D ) within and fixation indices ( F ST ) between populations. MetaPop will also identify genes with distinct codon usage. Following rigorous validation, we applied MetaPop to the gut viromes of autistic children that underwent fecal microbiota transfers and their neurotypical peers. The macrodiversity results confirmed our prior findings for viral populations (microbial shotgun metagenomes were not available) that diversity did not significantly differ between autistic and neurotypical children. However, by also quantifying microdiversity, MetaPop revealed lower average viral nucleotide diversity ( π ) in autistic children. Analysis of the percentage of genomes detected under positive selection was also lower among autistic children, suggesting that higher viral π in neurotypical children may be beneficial because it allows populations to better “bet hedge” in changing environments. Further, comparisons of microdiversity pre- and post-FMT in autistic children revealed that the delivery FMT method (oral versus rectal) may influence viral activity and engraftment of microdiverse viral populations, with children who received their FMT rectally having higher microdiversity post-FMT. Overall, these results show that analyses at the macro level alone can miss important biological differences. Conclusions These findings suggest that standardized population and genetic variation analyses will be invaluable for maximizing biological inference, and MetaPop provides a convenient tool package to explore the dual impact of macro - and microdiversity across microbial communities.
59 BASIC BIOLOGICAL SCIENCES↗
Transformative Impacts of Laser-Induced Breakdown Spectroscopy on Environmental and Biological Research at Oak Ridge National Laboratory
This manuscript will present an advancement of transformative research that has been conducted at Oak Ridge National Laboratory (ORNL) over a 25-year period (2000–2025) on a variety of environmental and biological matrices. These investigations derived a fundamental understanding of how elemental detection and analysis of these matrices led to the knowledge and discovery of natural processes in plants and the environment. Each project led to the initiation of a new research area which unearthed awesome and novel breakthroughs. Highlights are listed below: 1. The preliminary research at ORNL centered on the detection of aerosols utilizing Laser-induced Breakdown Spectroscopy (LIBS) technology. The Clean Air Act Amendment (CAAA) of 1990 highlighted the importance of identifying hazardous air pollutants (HAPs) due to their impact on environmental and human health, thereby underscoring the need to detect various toxic elements. Research in aerosol chemistry aimed to identify these harmful elements released by factories during periods of increased emissions in their manufacturing processes. LIBS emerged as the most effective method for real-time, in situ measurements of metal species in both gaseous and aerosol phases. 2. An understanding of the presence of total carbon in soils gives perspective on how to develop carbon sequestration strategies. The recognition that carbon sinks can evolve back to carbon sources to emit back to the atmosphere was an important consideration. Also, the concentration of carbon in soil indicates the health of land areas for growing crops successfully. 3. The direct detection of most of the elements in a wood sample in a single emission spectrum, without sample preparation, encouraged the research to use the LIBS technique for preservative treated wood coupled with use of multivariate statistical methodology. Additionally, it encouraged the researchers to try to differentiate natural woods from different parts of the country, and it was successfully demonstrated that LIBS coupled with MVA analysis could differentiate wood of different species from each other and of similar species grown in different environments based on their elemental spectra. This was a breakthrough since it revealed a systematic approach to connect elemental scarcity and abundance to either drought or typical rainfall conditions for the hardwood trees grown in specific areas. 4. Furthermore, the research progressed to reveal physiological and developmental processes contributing to biomass production such that the variation in leaf elemental composition increases our understanding of terrestrial nutrient cycles, as well as tracking the transfer of toxic elements from soils to living organisms. 5. Recently another breakthrough viz., ionomics initiated the correlation of elements to specific genes, uncovering the function that the element performed in the plant. More recently, this has been extended from plants to fungi as well as fungi growing in symbiotic relations with plants.
09 BIOMASS FUELS↗
Respiratory Vinyl Chloride Reductive Dechlorination to Ethene in TceA-Expressing Dehalococcoides mccartyi
Bioremediation of chlorinated ethenes in anoxic aquifers hinges on organohalide-respiring Dehalococcoidia expressing vinyl chloride (VC) reductive dehalogenase (RDase). The tceA gene encoding the trichloroethene-dechlorinating RDase TceA is frequently detected in contaminated groundwater but not recognized as a biomarker for VC detoxification. We demonstrate that tceA-carrying Dehalococcoides mccartyi (Dhc) strains FL2 and 195 grow with VC as an electron acceptor when sufficient vitamin B 12 (B 12 ) is provided. Strain FL2 cultures that received 50 μg L –1 B 12 completely dechlorinated VC to ethene at rates of 14.80 ± 1.30 μM day –1 and attained 1.64 ± 0.11 × 10 8 cells per μmol of VC consumed. Strain 195 attained similar growth yields of 1.80 ± 1.00 × 10 8 cells per μmol of VC consumed, and both strains could be consecutively transferred with VC as the electron acceptor. Proteomic analysis demonstrated TceA expression in VC-grown strain FL2 cultures. Resequencing of the strain FL2 and strain 195 tceA genes identified non-synonymous substitutions, although their consequences for TceA function are currently unknown. The finding that Dhc strains expressing TceA respire VC can explain ethene formation at chlorinated solvent sites, where quantitative polymerase chain reaction analysis indicates that tceA dominates the RDase gene pool.
54 ENVIRONMENTAL SCIENCES↗
Structure of VanS from vancomycin-resistant enterococci: A sensor kinase with weak ATP binding
The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci. VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from vancomycin-resistant enterococci types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with K D values in the low millimolar range. Since these K D values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.
59 BASIC BIOLOGICAL SCIENCES↗
A New Pneumococcal Capsule Type, 10D, is the 100th Serotype and Has a Large cps Fragment from an Oral Streptococcus
Streptococcus pneumoniae (pneumococcus) is a major human pathogen producing structurally diverse capsular polysaccharides. Widespread use of highly successful pneumococcal conjugate vaccines (PCVs) targeting pneumococcal capsules has greatly reduced infections by the vaccine types but increased infections by nonvaccine serotypes. Herein, we report a new and the 100th capsule type, named serotype 10D, by determining its unique chemical structure and biosynthetic roles of all capsule synthesis locus (cps) genes. The name 10D reflects its serologic cross-reaction with serotype 10A and appearance of cross-opsonic antibodies in response to immunization with 10A polysaccharide in a 23-valent pneumococcal vaccine. Genetic analysis showed that 10D cps has three large regions syntenic to and highly homologous with cps loci from serotype 6C, serotype 39, and an oral streptococcus strain (S. mitis SK145). The 10D cps region syntenic to SK145 is about 6 kb and has a short gene fragment of wciNα at the 5' end. The presence of this nonfunctional wciNα fragment provides compelling evidence for a recent interspecies genetic transfer from oral streptococcus to pneumococcus. Since oral streptococci have a large repertoire of cps loci, widespread PCV usage could facilitate the appearance of novel serotypes through interspecies recombination.
59 BASIC BIOLOGICAL SCIENCES↗
Ligand dependent interaction between PC-TP and PPARδ mitigates diet-induced hepatic steatosis in male mice
Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is a soluble lipid-binding protein that transports phosphatidylcholine (PC) between cellular membranes. To better understand the protective metabolic effects associated with hepatic PC-TP, we generated a hepatocyte-specific PC-TP knockdown (L-Pctp -/- ) in male mice, which gains less weight and accumulates less liver fat compared to wild-type mice when challenged with a high-fat diet. Hepatic deletion of PC-TP also reduced adipose tissue mass and decreases levels of triglycerides and phospholipids in skeletal muscle, liver and plasma. Gene expression analysis suggest that the observed metabolic changes are related to transcriptional activity of peroxisome proliferative activating receptor (PPAR) family members. An in-cell protein complementation screen between lipid transfer proteins and PPARs uncovered a direct interaction between PC-TP and PPARδ that was not observed for other PPARs. We confirmed the PC-TP– PPARδ interaction in Huh7 hepatocytes, where it was found to repress PPARδ-mediated transactivation. Mutations of PC-TP residues implicated in PC binding and transfer reduce the PC-TP-PPARδ interaction and relieve PC-TP-mediated PPARδ repression. Reduction of exogenously supplied methionine and choline reduces the interaction while serum starvation enhances the interaction in cultured hepatocytes. Together our data points to a ligand sensitive PC-TP– PPARδ interaction that suppresses PPAR activity.
59 BASIC BIOLOGICAL SCIENCES↗
Evaluation of large volume yeast interfering RNA lure-and-kill ovitraps for attraction and control of Aedes mosquitoes
Aedes mosquitoes (Diptera: Culicidae), principle vectors of several arboviruses, typically lay eggs in man-made water-filled containers located near human dwellings. Given the widespread emergence of insecticide resistance, stable and biofriendly alternatives for mosquito larviciding are needed. Laboratory studies have demonstrated that inactivated yeast interfering RNA tablets targeting key larval developmental genes can be used to facilitate effective larvicidal activity while also promoting selective gravid female oviposition behaviour. In this work, we examined the efficacy of transferring this technology toward development of lure-and-kill ovitraps targeting Aedes aegypti (L.) and Aedes albopictus (Skuse) female mosquitoes. Insectary, simulated field and semi-field experiments demonstrated that two mosquito-specific yeast interfering RNA pesticides induce high levels of mortality among larvae of both species in treated large volume containers. Small-scale field trials conducted in Trinidad, West Indies demonstrated that large volume ovitrap containers baited with inactivated yeast tablets lure significantly more gravid females than traps containing only water and were highly attractive to both A. aegypti and A. albopictus females. Finally, these studies indicate that development of biorational yeast interfering RNA-baited ovitraps may represent a new tool for control of Aedes mosquitoes, including deployment in existing lure-and-kill ovitrap technologies or traditional container larviciding programs.
59 BASIC BIOLOGICAL SCIENCES↗
In Situ Optical Quantification of Extracellular Electron Transfer Using Plasmonic Metal Oxide Nanocrystals**
Extracellular electron transfer (EET) is a critical form of microbial metabolism that enables respiration on a variety of inorganic substrates, including metal oxides. However, quantifying current generated by electroactive bacteria has been predominately limited to biofilms formed on electrodes. To address this, we developed a platform for quantifying EET flux from cell suspensions using aqueous dispersions of infrared plasmonic tin-doped indium oxide nanocrystals. Furthermore, tracking the change in optical extinction during electron transfer enabled quantification of current generated by planktonic Shewanella oneidensis cultures. Using this method, we differentiated between starved and actively respiring cells, cells of varying genotype, and cells engineered to differentially express a key EET gene using an inducible genetic circuit. Overall, our results validate the utility of colloidally stable plasmonic metal oxide nanocrystals as quantitative biosensors in aqueous environments and contribute to a fundamental understanding of planktonic S. oneidensis electrophysiology using simple in situ spectroscopy.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Phylogenomic discovery and engineering of nitrogen fixation into the bioenergy woody crop poplar
Biological nitrogen fixation (BNF) is a key process enabling plants in specific lineages to convert atmospheric dinitrogen (N₂) into bioavailable ammonia through symbioses with diazotrophic microbes. Expanding this capability beyond native nitrogen-fixing clades into non-nodulating crops would reduce synthetic fertilizer use, lowering energy inputs and environmental impacts in agriculture. Supported by DOE Funding Award DE-SC0018247, the NitFix project advanced foundational knowledge required to engineer root-nodule symbioses in new host species. The team generated the most comprehensive phylogenomic analysis to date of all known nodulating lineages, resolving the evolutionary history of nitrogen-fixing symbiosis and identifying core gene suites retained across nodulating taxa. Through multimodal genomics, transcriptomics, and functional analyses in Medicago truncatula and related species, the project mapped regulatory networks underlying nodule organogenesis, bacterial infection, and nitrogen-fixation efficiency. Key discoveries include the identification of conserved signaling modules for rhizobial recognition, transcription factors controlling nodule differentiation, and metabolic pathways integrating fixed nitrogen into plant growth. The project also developed enabling tools—including optimized transformation pipelines, gene-editing workflows, and imaging-based phenotyping—to accelerate engineering efforts in emerging models. Together, these results refine the mechanistic framework of symbiotic nitrogen fixation and highlight transferable components essential for rewiring these traits into non-nodulating crops.
59 BASIC BIOLOGICAL SCIENCES↗
DOE new players Carbon cycle (2016-2021)
The overarching scientific goals of our multidisciplinary grant was to further expand understanding about the key microorganisms (players), metabolic strategies (processes), and interspecies relationships (interactions) involved in the formation and oxidation of methane in the environment. This research applied novel environmental metagenomics, transcriptomics, and proteomic techniques, state-of-the-art analytical imaging, stable isotope geochemistry, and reaction-transport modeling to address these goals and develop an ‘ecosystems level’ understanding of the factors which regulate microbial methane cycling in anoxic sedimentary ecosystems. For decades, it was believed that the obligate step in methanogenesis catalyzed by methyl coenzyme M reductase (Mcr) was limited to a specific branch of the archaeal Domain, formerly known as the Euryarchaeota. Less than a decade ago co-I Tyson’s team published a surprising metagenomic-based discovery of divergent Mcr genes in a novel uncultured phylum (Bathyarchaeota), catalyzing a major shift in thinking about the diversity of microorganisms that encode the potential for methane (or higher alkane) metabolism in anoxic environments (Evans et al., 2015). In our work here, we further expand on the groups of archaea harboring the genomic potential for methane or hydrocarbon metabolism using environmental metagenomics and new gene targeted bioinformatics techniques. We additionally advanced understanding about the terminal electron acceptors and metabolic potential supporting the anaerobic oxidation of methane (AOM) in terrestrial ecosystems, specifically focused on new lineages of ANME archaea capable of respiring manganese oxides with methane presumably using large extracellular multi-heme cytochrome complexes. New details about specific syntrophic mechanisms underlying the exchange of electrons during sulfate-coupled methane oxidation between ANME-2 archaea and their sulfate-reducing bacterial partners were also elucidated as part of this funded project. Through a series of experimental ‘omics and single cell stable isotope probing studies with incubated environmental sediment samples and a cultured model electrogenic microorganism combined with model-based predictions. Combined, this work provides strong support for the hypothesis of direct interspecies electron transfer (DIET) is the dominant syntrophic mechanism controlling the anaerobic oxidation of methane with sulfate over other proposed mechanisms and additionally illustrates important spatial constraints and the underlying physico-chemical factors influencing AOM syntrophic consortia structure for DIET and extracellular metal respiration. This collaborative multi-institutional project successfully advanced several of our milestone goals including the identification of new microbial players containing methyl coenzyme M reductases hypothesized to be central to methane or hydrocarbon cycling in anoxic environments and enhancing fundamental knowledge about the role extracellular electron transfer plays in the ecophysiology of methanotrophic archaea respiring metal oxides and in the physical and metabolic structuring of syntrophic interactions in methane-rich sedimentary ecosystems.
03 NATURAL GAS↗
Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria
Synechococcus elongatus PCC 7942 is a model cyanobacterium for study of the circadian clock, photosynthesis, and bioproduction of chemicals, yet nearly 40% of its gene identities and functions remain unknown, in part due to limitations of the existing genetic toolkit. While classical techniques for the study of genes (e.g., deletion or mutagenesis) can yield valuable information about the absence of a gene and its associated protein, there are limits to these approaches, particularly in the study of essential genes. In this research, we developed a tool for inducible degradation of target proteins in S. elongatus by adapting a method using degron tags from the Mesoplasma florum transfer-messenger RNA (tmRNA) system. We observed that M. florum lon protease can rapidly degrade exogenous and native proteins tagged with the cognate sequence within hours of induction. We used this system to inducibly degrade the essential cell division factor, FtsZ, as well as shell protein components of the carboxysome. Our results have implications for carboxysome biogenesis and the rate of carboxysome turnover during cell growth. Lon protease control of proteins offers an alternative approach for the study of essential proteins and protein dynamics in cyanobacteria.
59 BASIC BIOLOGICAL SCIENCES↗
Identification of xylan arabinosyl 2- O -xylosyltransferases catalyzing the addition of 2- O -xylosyl residue onto arabinosyl side chains of xylan in grass species
Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Further, consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.
59 BASIC BIOLOGICAL SCIENCES↗
Expression of a bacterial 3-dehydroshikimate dehydratase (QsuB) reduces lignin and improves biomass saccharification efficiency in switchgrass (Panicum virgatum L.)
Abstract Background Lignin deposited in plant cell walls negatively affects biomass conversion into advanced bioproducts. There is therefore a strong interest in developing bioenergy crops with reduced lignin content or altered lignin structures. Another desired trait for bioenergy crops is the ability to accumulate novel bioproducts, which would enhance the development of economically sustainable biorefineries. As previously demonstrated in the model plant Arabidopsis, expression of a 3-dehydroshikimate dehydratase in plants offers the potential for decreasing lignin content and overproducing a value-added metabolic coproduct (i.e., protocatechuate) suitable for biological upgrading. Results The 3-dehydroshikimate dehydratase QsuB from Corynebacterium glutamicum was expressed in the bioenergy crop switchgrass ( Panicum virgatum L.) using the stem-specific promoter of an O-methyltransferase gene ( pShOMT ) from sugarcane. The activity of pShOMT was validated in switchgrass after observation in-situ of beta-glucuronidase (GUS) activity in stem nodes of plants carrying a pShOMT::GUS fusion construct. Under controlled growth conditions, engineered switchgrass lines containing a pShOMT::QsuB construct showed reductions of lignin content, improvements of biomass saccharification efficiency, and accumulated higher amount of protocatechuate compared to control plants. Attempts to generate transgenic switchgrass lines carrying the QsuB gene under the control of the constitutive promoter pZmUbi-1 were unsuccessful, suggesting possible toxicity issues associated with ectopic QsuB expression during the plant regeneration process. Conclusion This study validates the transfer of the QsuB engineering approach from a model plant to switchgrass. We have demonstrated altered expression of two important traits: lignin content and accumulation of a co-product. We found that the choice of promoter to drive QsuB expression should be carefully considered when deploying this strategy to other bioenergy crops. Field-testing of engineered QsuB switchgrass are in progress to assess the performance of the introduced traits and agronomic performances of the transgenic plants.
59 BASIC BIOLOGICAL SCIENCES↗
Two related families of metal transferases, ZNG1 and ZNG2, are involved in acclimation to poor Zn nutrition in Arabidopsis
Metal homeostasis has evolved to tightly modulate the availability of metals within the cell, avoiding cytotoxic interactions due to excess and protein inactivity due to deficiency. Even in the presence of homeostatic processes, however, low bioavailability of these essential metal nutrients in soils can negatively impact crop health and yield. While research has largely focused on how plants assimilate metals, acclimation to metal-limited environments requires a suite of strategies that are not necessarily involved in metal transport across membranes. The identification of these mechanisms provides a new opportunity to improve metal-use efficiency and develop plant foodstuffs with increased concentrations of bioavailable metal nutrients. Here, we investigate the function of two distinct subfamilies of the nucleotide-dependent metallochaperones (NMCs), named ZNG1 and ZNG2, that are found in plants, using Arabidopsis thaliana as a reference organism. AtZNG1 (AT1G26520) is an ortholog of human and fungal ZNG1, and like its previously characterized eukaryotic relatives, localizes to the cytosol and physically interacts with methionine aminopeptidase type I (AtMAP1A). Analysis of At ZNG1 , At MAP1A , At MAP2A , and At MAP2B transgenic mutants are consistent with the role of Arabidopsis ZNG1 as a Zn transferase for AtMAP1A, as previously described in yeast and zebrafish. Structural modeling reveals a flexible cysteine-rich loop that we hypothesize enables direct transfer of Zn from AtZNG1 to AtMAP1A during GTP hydrolysis. Based on proteomics and transcriptomics, loss of this ancient and conserved mechanism has pleiotropic consequences impacting the expression of hundreds of genes, including those involved in photosynthesis and vesicle transport. Members of the plant-specific family of NMCs, ZNG2A1 (AT1G80480) and ZNG2A2 (AT1G15730), are also required during Zn deficiency, but their target protein(s) remain to be discovered. In conclusion, RNA-seq analyses reveal wide-ranging impacts across the cell when the genes encoding these plastid-localized NMCs are disrupted.
59 BASIC BIOLOGICAL SCIENCES↗
Catabolism and interactions of uncultured organisms shaped by eco-thermodynamics in methanogenic bioprocesses
We currently understand the carbon cycle in methanogenic environments as trophic interactions revolving around interspecies H 2 transfer between organotrophs and methanogens. However, many H 2 -generating processes are thermodynamically sensitive to H 2 accumulation and can be inhibited by high H 2 concentrations produced by other metabolisms. To uncover how anaerobes combat this H 2 conflict in situ, we employ metagenomics and metatranscriptomics to revisit a model ecosystem that has inspired many foundational discoveries in anaerobic ecology – methanogenic bioreactors. Through analysis of 15 anaerobic digesters, we recover 1343 high-quality metagenome-assembled genomes (MAGs) and corresponding gene expression profiles for diverse uncultured lineages spanning 66 phyla and reconstruct their individual metabolic capacities. We discover that diverse uncultured organotrophic populations can drive H 2 -sensitive degradation through (i) metabolic coupling with concurrent H 2 -tolerant catabolism, (ii) unprecedented shifts from H 2 generation to CO 2 -reducing formate transfer and cytochrome/pili-mediated direct interspecies electron transfer for avoiding thermodynamic conflict, and (iii) integration of low-concentration O 2 as an ancillary thermodynamics-enhancing electron sink. Archaeal populations in situ support the above processes through novel metabolisms distinct from conventional methanogenesis – high-affinity H 2 oxidation driven by methyl-reducing methanogenesis and concomitant uptake of formate, electrons, and acetate from HS metabolism by Methanothrix. In total, synthesis of omics analyses and eco-thermodynamics reveals overlooked metabolic behavior and interactions of uncultured organisms that address a central issue in methanogenic carbon cycling, thermodynamic conflict among diverse concurrent metabolic processes.
59 BASIC BIOLOGICAL SCIENCES↗
Genetic and behavioral adaptation of Candida parapsilosis to the microbiome of hospitalized infants revealed by in situ genomics, transcriptomics, and proteomics
Background Candida parapsilosis is a common cause of invasive candidiasis, especially in newborn infants, and infections have been increasing over the past two decades. C. parapsilosis has been primarily studied in pure culture, leaving gaps in understanding of its function in a microbiome context. Results. Here, we compare five unique C. parapsilosis genomes assembled from premature infant fecal samples, three of which are newly reconstructed, and analyze their genome structure, population diversity, and in situ activity relative to reference strains in pure culture. All five genomes contain hotspots of single nucleotide variants, some of which are shared by strains from multiple hospitals. A subset of environmental and hospital-derived genomes share variants within these hotspots suggesting derivation of that region from a common ancestor. Four of the newly reconstructed C. parapsilosis genomes have 4 to 16 copies of the gene RTA3, which encodes a lipid translocase and is implicated in antifungal resistance, potentially indicating adaptation to hospital antifungal use. Time course metatranscriptomics and metaproteomics on fecal samples from a premature infant with a C. parapsilosis blood infection revealed highly variable in situ expression patterns that are distinct from those of similar strains in pure cultures. For example, biofilm formation genes were relatively less expressed in situ, whereas genes linked to oxygen utilization were more highly expressed, indicative of growth in a relatively aerobic environment. In gut microbiome samples, C. parapsilosis co-existed with Enterococcus faecalis that shifted in relative abundance over time, accompanied by changes in bacterial and fungal gene expression and proteome composition. Conclusions The results reveal potentially medically relevant differences in Candida function in gut vs. laboratory environments, and constrain evolutionary processes that could contribute to hospital strain persistence and transfer into premature infant microbiomes.
59 BASIC BIOLOGICAL SCIENCES↗