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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Evidence for autotrophic growth of purple sulfur bacteria using pyrite as electron and sulfur source

ABSTRACT Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that Allochromatium vinosum (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures (“py”’) demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various c - and b -type cytochromes was observed in “py,” pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells’ photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(−), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis. IMPORTANCE Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet’s history.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Significance of lysogeny for the metabolism of Desulfovibrio spp. strains isolated from aquatic environments of Georgia

Sulphate-reducing bacteria (SRB) are ecologically important group of anaerobic micro-organisms that can reduce sulphate to form hydrogen sulphide-a toxic gas causing iron corrosion on metal surfaces. In this work, SRB strains were isolated from aquatic environments in the country of Georgia to determine their lysogenicity and the role of temperate phages in host metabolism. SRB strains were isolated in samples from the Black Sea coast of Georgia. Based on their genetic, cytological and physiological properties of bacteria, 10 Georgian isolates were assigned to the genus Desulfovibrio. Temperate bacteriophages were induced from three out of ten strains by UV-exposure. Comparison of metal (Fe and Cr) reduction and utilization of various carbon sources by the wild-type (lysogenic) bacterial strains and their UV-irradiated counterparts was done. Temperate phage in the cells of SRB could alter significant functions of bacteria and may have a contribution in the acquisition of different traits by SRB. This article pointed to a significant role for temperate bacteriophages in the metabolism and metabolic potential of host strains of SRB, which were first isolated from the aquatic environment of Georgia.

temperate phage↗
DOE OSTI2024

Ecological Trait-Based Digital Categorization of Microbial Genomes for Denitrification Potential

Microorganisms encode proteins that function in the transformations of useful and harmful nitrogenous compounds in the global nitrogen cycle. The major transformations in the nitrogen cycle are nitrogen fixation, nitrification, denitrification, anaerobic ammonium oxidation, and ammonification. The focus of this report is the complex biogeochemical process of denitrification, which, in the complete form, consists of a series of four enzyme-catalyzed reduction reactions that transforms nitrate to nitrogen gas. Denitrification is a microbial strain-level ecological trait (characteristic), and denitrification potential (functional performance) can be inferred from trait rules that rely on the presence or absence of genes for denitrifying enzymes in microbial genomes. Despite the global significance of denitrification and associated large-scale genomic and scholarly data sources, there is lack of datasets and interactive computational tools for investigating microbial genomes according to denitrification trait rules. Therefore, our goal is to categorize archaeal and bacterial genomes by denitrification potential based on denitrification traits defined by rules of enzyme involvement in the denitrification reduction steps. We report the integration of datasets on genome, taxonomic lineage, ecosystem, and denitrifying enzymes to provide data investigations context for the denitrification potential of microbial strains. We constructed an ecosystem and taxonomic annotated denitrification potential dataset of 62,624 microbial genomes (866 archaea and 61,758 bacteria) that encode at least one of the twelve denitrifying enzymes in the four-step canonical denitrification pathway. Our four-digit binary-coding scheme categorized the microbial genomes to one of sixteen denitrification traits including complete denitrification traits assigned to 3280 genomes from 260 bacteria genera. The bacterial strains with complete denitrification potential pattern included Arcobacteraceae strains isolated or detected in diverse ecosystems including aquatic, human, plant, and Mollusca (shellfish). The dataset on microbial denitrification potential and associated interactive data investigations tools can serve as research resources for understanding the biochemical, molecular, and physiological aspects of microbial denitrification, among others. The microbial denitrification data resources produced in our research can also be useful for identifying microbial strains for synthetic denitrifying communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Global patterns of diversity and metabolism of microbial communities in deep-sea hydrothermal vent deposits

When deep-sea hydrothermal fluids mix with cold oxygenated fluids, minerals precipitate out of solution and form hydrothermal deposits. These actively venting deep-sea hydrothermal deposits support a rich diversity of thermophilic microorganisms which are involved in a range of carbon, sulfur, nitrogen, and hydrogen metabolisms. Global patterns of thermophilic microbial diversity in deep-sea hydrothermal ecosystems have illustrated the strong connectivity between geological processes and microbial colonization, but little is known about the genomic diversity and physiological potential of these novel taxa. Here we explore this genomic diversity in 42 metagenomes from four deep-sea hydrothermal vent fields and a deep-sea volcano collected from 2004 to 2018 and document their potential implications in biogeochemical cycles. Our dataset represents 3635 metagenome-assembled genomes encompassing 511 novel and recently identified genera from deep-sea hydrothermal settings. Some of the novel bacterial (107) and archaeal genera (30) that were recently reported from the deep-sea Brothers volcano were also detected at the deep-sea hydrothermal vent fields, while 99 bacterial and 54 archaeal genera were endemic to the deep-sea Brothers volcano deposits. We report some of the first examples of medium- (≥ 50% complete, ≤ 10% contaminated) to high-quality (> 90% complete, < 5% contaminated) MAGs from phyla and families never previously identified, or poorly sampled, from deep-sea hydrothermal environments. We greatly expand the novel diversity of Thermoproteia, Patescibacteria (Candidate Phyla Radiation, CPR), and Chloroflexota found at deep-sea hydrothermal vents and identify a small sampling of two potentially novel phyla, designated JALSQH01 and JALWCF01. Metabolic pathway analysis of metagenomes provides insights into the prevalent carbon, nitrogen, sulfur, and hydrogen metabolic processes across all sites and illustrates sulfur and nitrogen metabolic “handoffs” in community interactions. We confirm that Campylobacteria and Gammaproteobacteria occupy similar ecological guilds but their prevalence in a particular site is driven by shifts in the geochemical environment. Our study of globally distributed hydrothermal vent deposits provides a significant expansion of microbial genomic diversity associated with hydrothermal vent deposits and highlights the metabolic adaptation of taxonomic guilds. Collectively, our results illustrate the importance of comparative biodiversity studies in establishing patterns of shared phylogenetic diversity and physiological ecology, while providing many targets for enrichment and cultivation of novel and endemic taxa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

From wolves to humans: oral microbiome resistance to transfer across mammalian hosts

The mammalian mouth is colonized by complex microbial communities, adapted to specific niches, and in homeostasis with the host. Individual microbes interact metabolically and rely primarily on nutrients provided by the host, with which they have potentially co-evolved along the mammalian lineages. The oral environment is similar across mammals, but the diversity, specificity, and evolution of community structure in related or interacting mammals are little understood. Here, we compared the oral microbiomes of dogs with those of wild wolves and humans. In dogs, we found an increased microbial diversity relative to wolves, possibly related to the transition to omnivorous nutrition following domestication. This includes a larger diversity of Patescibacteria than previously reported in any other oral microbiota. The oral microbes are most distinct at bacterial species or strain levels, with few if any shared between humans and canids, while the close evolutionary relationship between wolves and dogs is reflected by numerous shared taxa. More taxa are shared at higher taxonomic levels including with humans, supporting their more ancestral common mammalian colonization followed by diversification. Phylogenies of selected oral bacterial lineages do not support stable human-dog microbial transfers but suggest diversification along mammalian lineages (apes and canids). Therefore, despite millennia of cohabitation and close interaction, the host and its native community controls and limits the assimilation of new microbes, even if closely related. Higher resolution metagenomic and microbial physiological studies, covering a larger mammalian diversity, should help understand how oral communities assemble, adapt, and interact with their hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Culturing of “Unculturable” Subsurface Microbes: Natural Organic Carbon Source Fuels the Growth of Diverse and Distinct Bacteria From Groundwater

Recovery and cultivation of diverse environmentally-relevant microorganisms from the terrestrial subsurface remain a challenge despite recent advances in modern molecular technology. Here, we applied complex carbon (C) sources, i.e., sediment dissolved organic matter (DOM) and bacterial cell lysate, to enrich groundwater microbial communities for 30 days. As comparisons, we also included enrichments amended with simple C sources including glucose, acetate, benzoate, oleic acid, cellulose, and mixed vitamins. Our results demonstrate that complex C is far more effective in enriching diverse and distinct microorganisms from groundwater than simple C. Simple C enrichments yield significantly lower biodiversity, and are dominated by few phyla (e.g., Proteobacteria and Bacteroidetes ), while microcosms enriched with complex C demonstrate significantly higher biodiversity including phyla that are poorly represented in published culture collections (e.g., Verrucomicrobia , Planctomycetes , and Armatimonadetes ). Subsequent isolation from complex C enrichments yielded 228 bacterial isolates representing five phyla, 17 orders, and 56 distinct species, including candidate novel, rarely cultivated, and undescribed organisms. Results from this study will substantially advance cultivation and isolation strategies for recovering diverse and novel subsurface microorganisms. Obtaining axenic representatives of “once-unculturable” microorganisms will enhance our understanding of microbial physiology and function in different biogeochemical niches of terrestrial subsurface ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Specific Bacterial Taxa and Their Metabolite, DHPS, May Be Linked to Gut Dyshomeostasis in Patients with Alzheimer’s Disease, Parkinson’s Disease, and Amyotrophic Lateral Sclerosis

Background: Neurodegenerative diseases (NDDs) are multifactorial disorders frequently associated with gut dysbiosis, oxidative stress, and inflammation; however, the pathophysiological mechanisms remain poorly understood. Methods: Using untargeted mass spectrometry-based metabolomics and 16S sequencing of human stool, we investigated bacterial and metabolic dyshomeostasis in the gut microbiome associated with early disease stages across three NDDs—amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD)—and healthy controls (HC). Results: We discovered a previously unrecognized link between a microbial-derived metabolite with an unknown role in human physiology, 2,3-dihydroxypropane-1-sulfonate (DHPS), and gut dysbiosis in NDDs. DHPS was downregulated in AD, ALS, and PD, while bacteria involved in DHPS metabolism, Eubacterium and Desulfovibrio, were increased in all disease cohorts. Additionally, select taxa within the Clostridia class had strong negative correlations to DHPS, suggesting a potential role in DHPS metabolism. A catabolic product of DHPS is hydrogen sulfide, and when in excess, it is known to promote inflammation, oxidative stress, mitochondrial damage, and gut dysbiosis, known hallmarks of NDDs. Conclusions: These findings suggest that cryptic sulfur metabolism via DHPS is a potential missing link in our current understanding of gut dysbiosis associated with NDD onset and progression. As this was a hypothesis generating study, more work is needed to elucidate the role of DHPS in gut dysbiosis and neurodegenerative diseases.

Nutrition & Dietetics↗
DOE OSTI2020

Structure of the essential inner membrane lipopolysaccharide–PbgA complex

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genomic Mysteries of Giant Bacteria: Insights and Implications

Bacteria and Archaea are traditionally regarded as organisms with a simple morphology constrained to a size of 2–3 µm. Nevertheless, the history of microbial research is rich in the description of giant bacteria exceeding tens and even hundreds of micrometers in length or diameter already from its early days, for example, Beggiatoa spp., to the present, for example, Candidatus Thiomargarita magnifica. While some of these giants are still being studied, some were lost to science, with merely drawings and photomicrographs as evidence for their existence. The physiology and biogeochemical role of giant bacteria have been studied, with a large focus on those involved in the sulfur cycle. With the onset of the genomic era, no special emphasis has been given to this group, in an attempt to gain a novel, evolutionary, and molecular understanding of the phenomenon of bacterial gigantism. The few existing genomic studies reveal a mysterious world of hyperpolyploid bacteria with hundreds to hundreds of thousands of chromosomes that are, in some cases, identical and in others, extremely different. These studies on giant bacteria reveal novel organelles, cellular compartmentalization, and novel mechanisms to combat the accumulation of deleterious mutations in polyploid bacteria. In this perspective paper, we provide a brief overview of what is known about the genomics of giant bacteria and build on that to highlight a few burning questions that await to be addressed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Severe and mild drought cause distinct phylogenetically linked shifts in the blue grama (Bouteloua gracilis) rhizobiome

Plants rely on a diverse rhizobiome to regulate nutrient acquisition and plant health. With increasing severity and frequency of droughts worldwide due to climate change, untangling the relationships between plants and their rhizobiomes is vital to maintaining agricultural productivity and protecting ecosystem diversity. While some plant physiological responses to drought are generally conserved, patterns of root exudation (release of small metabolites shown to influence microbes) and the consequential effects on the plant rhizobiome can differ widely across plant species under drought. To address this knowledge gap, we conducted a greenhouse study using blue grama ( Bouteloua gracilis ), a drought-tolerant C4 grass native to shortgrass prairie across North American plains, as a model organism to study the effect of increasing drought severity (ambient, mild drought, severe drought) on root exudation and the rhizobiome. Our previous results demonstrated physiological effects of increasing drought severity including an increase in belowground carbon allocation through root exudation and shifts in root exudate composition concurrent with the gradient of drought severity. This work is focused on the rhizobiome community structure using targeted sequencing and found that mild and severe drought resulted in unique shifts in the bacterial + archaeal and fungal communities relative to ambient, non-droughted controls. Specifically, using the change in relative abundance between ambient and drought conditions for each ZOTU as a surrogate for population-scale drought tolerance (e.g., as a response trait), we found that rhizobiome response to drought was non-randomly distributed across the phylogenies of both communities, suggesting that Planctomycetota , Thermoproteota (formerly Thaumarchaeota ), and the Glomeromycota were the primary clades driving these changes. Correlation analyses indicated weak correlations between droughted community composition and a select few root exudate compounds previously implicated in plant drought responses including pyruvic acid, D-glucose, and myoinositol. This study demonstrates the variable impacts of drought severity on the composition of the blue grama rhizobiome and provides a platform for hypothesis generation for targeted functional studies of specific taxa involved in plant-microbe drought responses.

Goemann, Hannah M.↗
DOE OSTI2021

Metapangenomics reveals depth-dependent shifts in metabolic potential for the ubiquitous marine bacterial SAR324 lineage

Abstract Background Oceanic microbiomes play a pivotal role in the global carbon cycle and are central to the transformation and recycling of carbon and energy in the ocean’s interior. SAR324 is a ubiquitous but poorly understood uncultivated clade of Deltaproteobacteria that inhabits the entire water column, from ocean surface waters to its deep interior. Although some progress has been made in elucidating potential metabolic traits of SAR324 in the dark ocean, very little is known about the ecology and the metabolic capabilities of this group in the euphotic and twilight zones. To investigate the comparative genomics, ecology, and physiological potential of the SAR324 clade, we examined the distribution and variability of key genomic features and metabolic pathways in this group from surface waters to the abyss in the North Pacific Subtropical Gyre, one of the largest biomes on Earth. Results We leveraged a pangenomic ecological approach, combining spatio-temporally resolved single-amplified genome, metagenomic, and metatranscriptomic datasets. The data revealed substantial genomic diversity throughout the SAR324 clade, with distinct depth and temporal distributions that clearly differentiated ecotypes. Phylogenomic subclade delineation, environmental distributions, genomic feature similarities, and metabolic capacities revealed strong congruence. The four SAR324 ecotypes delineated in this study revealed striking divergence from one another with respect to their habitat-specific metabolic potentials. The ecotypes living in the dark or twilight oceans shared genomic features and metabolic capabilities consistent with a sulfur-based chemolithoautotrophic lifestyle. In contrast, those inhabiting the sunlit ocean displayed higher plasticity energy-related metabolic pathways, supporting a presumptive photoheterotrophic lifestyle. In epipelagic SAR324 ecotypes, we observed the presence of two types of proton-pumping rhodopsins, as well as genomic, transcriptomic, and ecological evidence for active photoheterotrophy, based on xanthorhodopsin-like light-harvesting proteins. Conclusions Combining pangenomic and both metagenomic and metatranscriptomic profiling revealed a striking divergence in the vertical distribution, genomic composition, metabolic potential, and predicted lifestyle strategies of geographically co-located members of the SAR324 bacterial clade. The results highlight the utility of metapangenomic approaches employed across environmental gradients, to decipher the properties and variation in function and ecological traits of specific phylogenetic clades within complex microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bacterial xylan utilization regulons: systems for coupling depolymerization of methylglucuronoxylans with assimilation and metabolism

Abstract Bioconversion of lignocellulosic resources offers an economically promising path to renewable energy. Technological challenges to achieving bioconversion include the development of cost-effective processes that render the cellulose and hemicellulose components of these resources to fermentable hexoses and pentoses. Natural bioprocessing of the hemicellulose fraction of lignocellulosic biomass requires depolymerization of methylglucuronoxylans. This requires secretion of endoxylanases that release xylooligosaccharides and aldouronates. Physiological, biochemical, and genetic studies with selected bacteria support a process in which a cell-anchored multimodular GH10 endoxylanase catalyzes release of the hydrolysis products, aldotetrauronate, xylotriose, and xylobiose, which are directly assimilated and metabolized. Gene clusters encoding intracellular enzymes, including α-glucuronidase, endoxylanase, β-xylosidase, ABC transporter proteins, and transcriptional regulators, are coordinately responsive to substrate induction or repression. The rapid rates of glucuronoxylan utilization and microbial growth, along with the absence of detectable products of depolymerization in the medium, indicate that assimilation and depolymerization are coupled processes. Genomic comparisons provide evidence that such systems occur in xylanolytic species in several genera, including Clostridium, Geobacillus, Paenibacillus, and Thermotoga. These systems offer promise, either in their native configurations or through gene transfer to other organisms, to develop biocatalysts for efficient production of fuels and chemicals from the hemicellulose fractions of lignocellulosic resources.

Biotechnology & Applied Microbiology↗
DOE OSTI2022

One fold, many functions—M23 family of peptidoglycan hydrolases

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons.

Razew, Alicja↗
DOE OSTI2023

Physiological and genomic evidence of cysteine degradation and aerobic hydrogen sulfide production in freshwater bacteria

The sulfur-containing amino acid cysteine is abundant in the environment, including in freshwater lakes. Biological cysteine degradation can result in hydrogen sulfide (H 2 S), a toxic and ecologically relevant compound that is a central player in biogeochemical cycling in aquatic environments. Here, we investigated the ecological significance of cysteine in oxic freshwater, using isolated cultures, controlled experiments, and multiomics. We screened bacterial isolates enriched from natural lake water for their ability to produce H 2 S when provided cysteine. We identified 29 isolates (Bacteroidota, Proteobacteria, and Actinobacteria) that produced H 2 S. To understand the genomic and genetic basis for cysteine degradation and H 2 S production, we further characterized three isolates using whole-genome sequencing (using a combination of short-read and long-read sequencing) and tracked cysteine and H 2 S levels over their growth ranges: Stenotrophomonas maltophilia (Gammaproteobacteria), S. bentonitica (Gammaproteobacteria), and Chryseobacterium piscium (Bacteroidota). Cysteine decreased and H 2 S increased, and all three genomes had genes involved in cysteine degradation. Finally, to assess the presence of these organisms and genes in the environment, we surveyed a 5-year time series of metagenomic data from the same isolation source (Lake Mendota, Madison, WI, USA) and identified their presence throughout the time series. Overall, our study shows that diverse isolated bacterial strains can use cysteine and produce H 2 S under oxic conditions, and we show evidence using metagenomic data that this process may occur more broadly in natural freshwater lakes. Future considerations of sulfur cycling and biogeochemistry in oxic environments should account for H 2 S production from the degradation of organosulfur compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Changes in microbial community and network structure precede shrub degradation in a desert ecosystem

Large-scale restoration is intended to promote ecological recovery. Improvements in plant and microbial conditions, however, may slow or even reverse in late succession. To better understand long-term restoration outcomes and underlying drivers of successional pathways, we tracked plant, bacterial and fungal, and soil conditions across a 40-year shrub plantation that was intended to stabilize desertified land in northern China. Here, we found that planted Haloxylon ammodendron shrubs developed and then subsequently became degraded after 30–40 years. Bacterial abundance and α-diversity were much higher than those of fungi, but no significant differences in composition and structure were found in different plantation ages. In contrast, the dominant taxa of fungal communities shifted from symbiotroph and saprotroph species towards pathotroph species with increased soil nutrients in the plantation chronosequence after two decades. The changes in fungal dominant species led to a transition in microbial network structure and function, with an increase in negative linkages among taxa that began in the middle stages of succession. Changes in fungal community structure had direct and indirect negative effects on shrub leaf physiology, root activity, and biomass. Our results highlight the preceding role of a breakdown in soil microbial community composition and network structure on the degradation of shrub performance in long-term desert succession. Our study emphasizes the importance of understanding soil-microbial-plant linkages on restoration outcomes, and mechanisms that can slow or reverse the recovery of ecosystems.

Bacterial and fungal community composition↗
DOE OSTI2025

In vitro demonstration and in planta characterization of a condensed, reverse TCA (crTCA) cycle

Introduction Plants employ the Calvin-Benson cycle (CBC) to fix atmospheric CO 2 for the production of biomass. The flux of carbon through the CBC is limited by the activity and selectivity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO). Alternative CO 2 fixation pathways that do not use RuBisCO to fix CO 2 have evolved in some anaerobic, autotrophic microorganisms. Methods Rather than modifying existing routes of carbon metabolism in plants, we have developed a synthetic carbon fixation cycle that does not exist in nature but is inspired by metabolisms of bacterial autotrophs. In this work, we build and characterize a condensed, reverse tricarboxylic acid (crTCA) cyclein vitroandin planta. Results We demonstrate that a simple, synthetic cycle can be used to fix carbon in vitro under aerobic and mesophilic conditions and that these enzymes retain activity whenexpressed transientlyin planta. We then evaluate stable transgenic lines ofCamelina sativathat have both phenotypic and physiologic changes. TransgenicC. sativaare shorter than controls with increased rates of photosynthetic CO 2 assimilation and changes in photorespiratory metabolism. Discussion This first iteration of a build-test-learn phase of the crTCA cycle provides promising evidence that this pathway can be used to increase photosynthetic capacity in plants.

Plant Sciences↗
DOE OSTI2021

Unifying themes and distinct features of carbon and nitrogen assimilation by polysaccharide-degrading bacteria: a summary of four model systems

Our current understanding of enzymatic polysaccharide degradation has come from a huge number of in vitro studies with purified enzymes. While this vast body of work has been invaluable in identifying and characterizing novel mechanisms of action and engineering desirable traits into these enzymes, a comprehensive picture of how these enzymes work as part of a native in vivo system is less clear. Recently, several model bacteria have emerged with genetic systems that allow for a more nuanced study of carbohydrate active enzymes (CAZymes) and how their activity affects bacterial carbon metabolism. With these bacterial model systems it is now possible to not only study a single nutrient system in isolation (i.e. carbohydrate degradation and carbon metabolism), but also how multiple systems are integrated. Given that most environmental polysaccharides are carbon rich but nitrogen poor (e.g. lignocellulose), the interplay between carbon and nitrogen metabolism in polysaccharide-degrading bacteria can now be studied in a physiologically relevant manner. Therefore, in this review we have summarized what has been experimentally determined for CAZyme regulation, production, and export in relation to nitrogen metabolism for two Gram-positive (Caldicellulosiruptor bescii and Clostridium thermocellum) and two Gram-negative (Bacteroides thetaiotaomicron and Cellvibrio japonicus) polysaccharide-degrading bacteria. By comparing and contrasting these four bacteria we have highlighted the shared and unique features of each, with a focus on in vivo studies, in regard to carbon and nitrogen assimilation. Finally, we conclude with what we believe are two important questions that can act as guideposts for future work to better understand the integration of carbon and nitrogen metabolism in polysaccharide-degrading bacteria.

Cellvibrio japonicus↗
DOE OSTI2025

Exploring phage–host interactions in Burkholderia cepacia complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics

Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage–host interactions, revealing active phage resistance and host factor genes in Burkholderia cenocepacia K56-2. Burkholderia cepacia complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of B. cenocepacia K56-2 with Bcep176, a temperate phage isolated from Burkholderia multivorans. A genome-wide dCas9 knockdown library targeting B. cenocepacia K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of B. cenocepacia K56-2 during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of B. cenocepacia and its phages, and for designing phage therapies.

Bacterial Pathogenesis↗