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DOE OSTI2020

Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems

The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

BONCAT-FACS-Seq reveals the active fraction of a biocrust community undergoing a wet-up event

Determining which microorganisms are active within soil communities remains a major technical endeavor in microbial ecology research. One promising method to accomplish this is coupling bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence activated cell sorting (FACS) which sorts cells based on whether or not they are producing new proteins. Combined with shotgun metagenomic sequencing (Seq), we apply this method to profile the diversity and potential functional capabilities of both active and inactive microorganisms in a biocrust community after being resuscitated by a simulated rain event. We find that BONCAT-FACS-Seq is capable of discerning the pools of active and inactive microorganisms, especially within hours of applying the BONCAT probe. The active and inactive components of the biocrust community differed in species richness and composition at both 4 and 21 h after the wetting event. The active fraction of the biocrust community is marked by taxa commonly observed in other biocrust communities, many of which play important roles in species interactions and nutrient transformations. Among these, 11 families within the Firmicutes are enriched in the active fraction, supporting previous reports indicating that the Firmicutes are key early responders to biocrust wetting. We highlight the apparent inactivity of many Actinobacteria and Proteobacteria through 21 h after wetting, and note that members of the Chitinophagaceae, enriched in the active fraction, may play important ecological roles following wetting. Based on the enrichment of COGs in the active fraction, predation by phage and other bacterial members, as well as scavenging and recycling of labile nutrients, appear to be important ecological processes soon after wetting. To our knowledge, this is the first time BONCAT-FACS-Seq has been applied to biocrust samples, and therefore we discuss the potential advantages and shortcomings of coupling metagenomics to BONCAT to intact soil communities such as biocrust. In all, by pairing BONCAT-FACS and metagenomics, we are capable of highlighting the taxa and potential functions that typifies the microbes actively responding to a rain event.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Computational Insights into Fuels and Chemicals Extraction from Microbial Biorefineries

Over the past two decades, substantial investments have been made in engineering microorganisms to produce specific fuels and chemicals as part of the global bioeconomy. Many target molecules accumulate intracellularly, and a challenge is how to effectively extract the product from the cells without needing to destroy them due to the barrier imposed by the cell membrane. For some hydrophobic compounds, an organic overlay is an effective strategy for nondestructive product extraction, although the relationship between functional groups on the product and the rate of extraction are not well understood. Through both biased and unbiased molecular dynamics simulations for a range of fatty acyl compounds and terpenoids, we directly compute permeability coefficients for different steps of the extraction process. Via comparative analysis between the calculated permeability coefficients and observed interactions between the compounds and the membrane, we determine how the rate limiting steps vary depending on product chemistry. For instance, fatty aldehydes are found to transfer very rapidly across the membrane bilayer relative to alcohols, although their comparable rate of extraction into the organic phase makes them equally effective at extraction from the cell. In assessing the terpenoids, it is found that in general a modestly hydrophilic product improves desorption rates into an organic phase sufficiently to make up for their lower bilayer crossing rate. With this new insight, we can more effectively engineer microorganisms towards the production of these modestly hydrophilic fuel precursors or chemicals.

09 BIOMASS FUELS↗

DOE OSTI2026

The HypA and HypB metallochaperones from Methanococcus maripaludis have unique metal-binding properties and a distinct nickel transfer mechanism

[NiFe] hydrogenases are widespread microbial metalloenzymes that catalyze the reversible conversion of hydrogen (H2) to protons and electrons, playing key roles in energy metabolism. The biosynthesis of the NiFe(CN) 2 CO cofactor involves a suite of maturation proteins, including the HypA and HypB nickel metallochaperones. Here, we define the metal-binding properties, nucleotide-dependent behavior, and functional interplay of HypA and HypB from the hydrogenotrophic methanogenic archaeon, Methanococcus maripaludis . Methanogens have multiple essential nickel-dependent enzymes, so they require efficient systems for nickel delivery that remain largely unexplored. Purified M. maripaludis HypA binds zinc or mononuclear iron at the C-terminal metal binding site, the latter of which has not been reported in other HypA proteins and may serve a unique regulatory role in methanogens. The G-protein metallochaperone HypB binds nickel at the G-domain, which stimulates GTPase activity. Size exclusion chromatography experiments reveal that HypA and HypB form complexes in the presence of nickel, and zinc-bound HypA is optimized for nickel transfer from HypB. The identity of the nucleotide bound to HypB (GDP or GTP) alters the oligomeric state of HypA-HypB complexes, supporting a GTPase-mediated nickel delivery pathway. The HypA-HypB 2 complex configuration is enriched and stable in the presence of GDP and nickel, indicating that this complex delivers nickel to the hydrogenase as opposed to HypA alone. Interestingly, affinity purification-mass spectrometry revealed that HypB interacts with several nickel-dependent proteins, suggesting that HypB may play a broader role in nickel homeostasis in M. maripaludis . Together, this work establishes a biochemical framework for HypAB-mediated nickel trafficking in methanogens.

[NiFe] hydrogenase↗

DOE OSTI2024

Reduction of Hg II by Mn II

The reduction of Hg II to Hg I or Hg 0 can lead to significant changes in Hg toxicity and mobility in the environment. Photochemical reduction is the primary process for the reduction of Hg II to Hg 0 in sunlit environments; however, dark reduction of HgII can occur via microbial metabolic processes and/or reduction by reduced natural organic matter, Fe II mineral phases, Fe II sorbed to minerals, or aqueous Fe II . Here, in this study, we demonstrate a novel Hg II reduction pathway involving another environmentally relevant reductant, Mn II . Abiotic reduction of Hg II O by Mn II was studied as a function of pH and anion environment (perchlorate, sulfate, chloride) using X-ray absorption spectroscopy to characterize the solid-phase Hg and Mn species. At circumneutral pH of 7.5, about 70% of Hg II was reduced to elemental Hg 0 within 2 h. In contrast, 12 h were needed to achieve the same extent of reduction at pH 6.9. In the presence of sulfate and chloride, Hg I species were formed. Hg II reduction was initially rapid and coupled with the oxidation of soluble Mn II -oxides to insoluble Mn IV -oxides, followed by a significantly slower reduction of Hg II during the Mn II -catalyzed transformation of the Mn IV -oxides to hydroxide and oxyhydroxide minerals. The observed reduction of Hg II by Mn II at circumneutral pH could be an important transformation pathway for environmental Hg, affecting its bioavailability and mobility under mildly reducing conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Moisture Effect on Chitin Decomposition Biogeochemistry

This dataset contains data files for multiple measurements of sample biogeochemistry and function collected for the Soils SFA Chitin Decomposition project in task 2.2. Samples were generated from soil incubated under different moisture levels, with and without chitin. Each sheet in the file refers to the preprocessed data collected. Sheet 1 "Respiration" measures CO2 production daily for the course of the incubation. Sheet 2 "Biomass" contains the microbial biomass and salt extractable measurements for carbon and nitrogen. Sheet 3 "Chitin" is for HPLC measured chitin from each sample. Sheet 4 "Extracellular Enzyme Assays" records the level of activity for several enzyme assays. Sheet 5 "Enzyme Kinetics" measures degradation of substrate over time for all samples.

Reichart, Nicholas J [Pacific Northwest National L↗

DOE OSTI2020

A comparison of lodgepole and spruce needle chemistry impacts on terrestrial biogeochemical processes during isolated decomposition

This study investigates the isolated decomposition of spruce and lodgepole conifer needles to enhance our understanding of how needle litter impacts near-surface terrestrial biogeochemical processes. Harvested needles were exported to a subalpine meadow to enable a discrete analysis of the decomposition processes over 2 years. Initial chemistry revealed the lodgepole needles to be less recalcitrant with a lower carbon to nitrogen (C:N) ratio. Total C and N fundamentally shifted within needle species over time with decreased C:N ratios for spruce and increased ratios for lodgepole. Differences in chemistry correlated with CO 2 production and soil microbial communities. The most pronounced trends were associated with lodgepole needles in comparison to the spruce and needle-free controls. Increased organic carbon and nitrogen concentrations associated with needle presence in soil extractions further corroborate the results with clear biogeochemical signatures in association with needle chemistry. Interestingly, no clear differentiation was observed as a function of bark beetle impacted spruce needles vs those derived from healthy spruce trees despite initial differences in needle chemistry. These results reveal that the inherent chemistry associated with tree species has a greater impact on soil biogeochemical signatures during isolated needle decomposition. By extension, biogeochemical shifts associated with bark beetle infestation are likely driven more by changes such as the cessation of rhizospheric processes than by needle litter decomposition.

Bark Beetle Disturbance↗

DOE OSTI2022

Patterns and ecological drivers of viral communities in acid mine drainage sediments across Southern China

Recent advances in environmental genomics have provided unprecedented opportunities for the investigation of viruses in natural settings. Yet, our knowledge of viral biogeographic patterns and the corresponding drivers is still limited. Here, we perform metagenomic deep sequencing on 90 acid mine drainage (AMD) sediments sampled across Southern China and examine the biogeography of viruses in this extreme environment. The results demonstrate that prokaryotic communities dictate viral taxonomic and functional diversity, abundance and structure, whereas other factors especially latitude and mean annual temperature also impact viral populations and functions. In silico predictions highlight lineage-specific virus-host abundance ratios and richness-dependent virus-host interaction structure. Further functional analyses reveal important roles of environmental conditions and horizontal gene transfers in shaping viral auxiliary metabolic genes potentially involved in phosphorus assimilation. Our findings underscore the importance of both abiotic and biotic factors in predicting the taxonomic and functional biogeographic dynamics of viruses in the AMD sediments.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Global Diversity and Biogeography of the Zostera marina Mycobiome

Seagrasses are marine flowering plants that provide critical ecosystem services in coastal environments worldwide. Marine fungi are often overlooked in microbiome and seagrass studies, despite terrestrial fungi having critical functional roles as decomposers, pathogens, or endophytes in global ecosystems. Here, we characterize the distribution of fungi associated with the seagrass Zostera marina, using leaves, roots, and rhizosphere sediment from 16 locations across its full biogeographic range. Using high-throughput sequencing of the ribosomal internal transcribed spacer (ITS) region and 18S rRNA gene, we first measured fungal community composition and diversity. We then tested hypotheses of neutral community assembly theory and the degree to which deviations suggested that amplicon sequence variants (ASVs) were plant selected or dispersal limited. Finally, we identified a core mycobiome and investigated the global distribution of differentially abundant ASVs. We found that the fungal community is significantly different between sites and that the leaf mycobiome follows a weak but significant pattern of distance decay in the Pacific Ocean. Generally, there was evidence for both deterministic and stochastic factors contributing to community assembly of the mycobiome, with most taxa assembling through stochastic processes. The Z. marina core leaf and root mycobiomes were dominated by unclassified Sordariomycetes spp., unclassified Chytridiomycota lineages (including Lobulomycetaceae spp.), unclassified Capnodiales spp., and Saccharomyces sp. It is clear from the many unclassified fungal ASVs and fungal functional guilds that knowledge of marine fungi is still rudimentary. Further studies characterizing seagrass-associated fungi are needed to understand the roles of these microorganisms generally and when associated with seagrasses. Fungi have important functional roles when associated with land plants, yet very little is known about the roles of fungi associated with marine plants, like seagrasses. In this study, we report the results of a global effort to characterize the fungi associated with the seagrass Zostera marina across its full biogeographic range. Although we defined a putative global core fungal community, it is apparent from the many fungal sequences and predicted functional guilds that had no matches to existing databases that general knowledge of seagrass-associated fungi and marine fungi is lacking. This work serves as an important foundational step toward future work investigating the functional ramifications of fungi in the marine ecosystem.

ITS2↗

DOE OSTI2017

Lake Bacterial Assemblage Composition Is Sensitive to Biological Disturbance Caused by an Invasive Filter Feeder

One approach to improve forecasts of how global change will affect ecosystem processes is to better understand how anthropogenic disturbances alter bacterial assemblages that drive biogeochemical cycles. Species invasions are important contributors to global change, but their impacts on bacterial community ecology are rarely investigated. Here, we studied direct impacts of invasive dreissenid mussels (IDMs), one of many invasive filter feeders, on freshwater lake bacterioplankton. We demonstrated that direct effects of IDMs reduced bacterial abundance and altered assemblage composition by preferentially removing larger and particle-associated bacteria. While this increased the relative abundances of many free-living bacterial taxa, some were susceptible to filter feeding, in line with efficient removal of phytoplankton cells of <2 μm. This selective removal of particle-associated and larger bacteria by IDMs altered inferred bacterial functional group representation, defined by carbon and energy source utilization. Specifically, we inferred an increased relative abundance of chemoorganoheterotrophs predicted to be capable of rhodopsin-dependent energy generation. In contrast to the few previous studies that have focused on the longer-term combined direct and indirect effects of IDMs on bacterioplankton, our study showed that IDMs act directly as a biological disturbance to which freshwater bacterial assemblages are sensitive. The negative impacts on particle-associated bacteria, which have been shown to be more active than free-living bacteria, and the inferred shifts in functional group representation raise the possibility that IDMs may directly alter bacterially mediated ecosystem functions.Freshwater bacteria play fundamental roles in global elemental cycling and are an intrinsic part of local food webs. Human activities are altering freshwater environments, and much has been learned regarding the sensitivity of bacterial assemblages to a variety of these disturbances. Yet, relatively few studies have focused on how species invasion, which is one of the most important aspects of anthropogenic global change, affects freshwater bacterial assemblages. This study focuses on the impact of invasive dreissenid mussels (IDMs), a globally distributed group of invasive species with large impacts on freshwater phyto- and zooplankton assemblages. Here, we show that IDMs have direct effects on lake bacterioplankton abundance, taxonomic composition, and inferred bacterial functional group representation.

16S rRNA gene↗

DOE OSTI2025

Biotechnological advances in algae-based foods: applications in nutrition and microbiome health

Algae are a sustainable, nutrient-rich resource with growing potential in food biotechnology. Their ability to thrive in diverse environments makes them a promising alternative to conventional crops. Rich in proteins, essential fatty acids, and bioactive compounds, algae support the development of functional foods, including plant-based meat and seafood alternatives. Advances in synthetic biology and fermentation have enhanced algal nutrient profiles and enabled novel applications. Algae-derived polysaccharides, such as alginate, fucoidan, laminarin, and porphyran, exhibit prebiotic effects by modulating the gut microbiota and promoting SCFA production. Enzymatic hydrolysis efficiently produces bioactive oligosaccharides, while engineered microbial systems support scalable production. Algae also enable synbiotic food development by serving as both prebiotic substrates and probiotic carriers.

Yu, Sora↗

DOE OSTI2026

GenomeDepot: data management system for microbial comparative genomics

Summary GenomeDepot is an open-source web-based platform for annotation, management, and comparative analysis of microbial genomic sequences and associated data including ortholog families, protein domains, operons, regulatory interactions, strain taxonomy, and sample metadata. GenomeDepot supports rapid creation of websites for user-defined genome collections that include bioinformatic tools for interactive genome browsing, Basic Local Alignment Search Tool (BLAST) search, annotation search, comparative genomic neighborhood visualization, and sequence download. Gene function annotations are generated by a customizable annotation pipeline. The pipeline runs annotation tools in Conda environments and can be easily extended with additional user-specified tools. Availability and implementation GenomeDepot is open source and distributed under the GNU General Public License via GitHub (https://github.com/aekazakov/genome-depot). GenomeDepot is implemented in Python and was tested in Ubuntu Linux. Full installation instructions and documentation are available at https://aekazakov.github.io/genome-depot/. GenomeDepot demo server is freely accessible at https://iseq.lbl.gov/demogd/.

Kazakov, Alexey [Lawrence Berkeley National Labora↗

DOE OSTI2022

Biochemical and Regulatory Analyses of Xylanolytic Regulons in Caldicellulosiruptor bescii Reveal Genus-Wide Features of Hemicellulose Utilization

Microbial deconstruction of lignocellulose for the production of biofuels and chemicals requires the hydrolysis of heterogeneous hemicelluloses to access the microcrystalline cellulose portion. This work extends previous in vivo and in vitro efforts to characterize hemicellulose utilization by integrating genomic reconstruction, transcriptomic data, operon structures, and biochemical characteristics of key enzymes to understand the deployment and functionality of hemicellulases by the extreme thermophile Caldicellulosiruptor bescii .

Biotechnology & Applied Microbiology↗

DOE OSTI2018

Metatranscriptomic and Thermodynamic Insights into Medium-Chain Fatty Acid Production Using an Anaerobic Microbiome

Mixed communities of microbes play important roles in health, the environment, agriculture, and biotechnology. While tapping the combined activities of organisms within microbiomes may allow the utilization of a wider range of substrates in preference to the use of pure cultures for biomanufacturing, harnessing the metabolism of these mixed cultures remains a major challenge. Here, we predicted metabolic functions of bacteria in a microbiome that produces medium-chain fatty acids from a renewable feedstock. Our findings lay the foundation for efforts to begin addressing how to engineer and control microbiomes for improved biomanufacturing, how to build synthetic mixtures of microbes that produce valuable chemicals from renewable resources, and how to better understand the microbial communities that contribute to health, agriculture, and the environment.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Single-cell RNA sequencing reveals plasmid constrains bacterial population heterogeneity and identifies a non-conjugating subpopulation

Transcriptional heterogeneity in isogenic bacterial populations can play various roles in bacterial evolution, but its detection remains technically challenging. Here, we use microbial split-pool ligation transcriptomics to study the relationship between bacterial subpopulation formation and plasmid-host interactions at the single-cell level. We find that single-cell transcript abundances are influenced by bacterial growth state and plasmid carriage. Moreover, plasmid carriage constrains the formation of bacterial subpopulations. Plasmid genes, including those with core functions such as replication and maintenance, exhibit transcriptional heterogeneity associated with cell activity. Notably, we identify a cell subpopulation that does not transcribe conjugal plasmid transfer genes, which may help reduce plasmid burden on a subset of cells. Our study advances the understanding of plasmid-mediated subpopulation dynamics and provides insights into the plasmid-bacteria interplay.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

The Thermodynamic Links between Substrate, Enzyme, and Microbial Dynamics in Michaelis-Menten-Monod Kinetics

Accurate prediction of the temperature response of the velocity v of a biochemical reaction has wide applications in cell biology, reaction design, and biomass yield enhancement. Here in this study, we introduce a simple but comprehensive mechanistic approach that uses thermodynamics and biochemical kinetics to describe and link the reaction rate and Michaelis–Menten constants (k T and K T ) with the biomass yield and mortality rate (Y T and δ T ) as explicit functions of T . The temperature control is exerted by catabolic enthalpy at low temperatures and catabolic entropy at high temperatures, whereas changes in cell and enzyme–substrate heat capacity shift the anabolic electron use efficiency e A and the maximum reaction velocity v max . We show that cells have optimal growth when the catabolic (differential) free energy of activation decreases the cell free energy harvest required to duplicate their internal structures as long as electrons for anabolism are available. Lastly, with the described approach, we accurately predicted observed glucose fermentation and ammonium nitrification dynamics across a wide temperature range with a minimal number of thermodynamics parameters, and we highlight how kinetic parameters are linked to each other using first principles.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Microbial community dynamics in the soil-root continuum are linked with plant species turnover during secondary succession

Grazing exclusion and land abandonment are commonly adopted to restore degraded ecosystems in semiarid and arid regions worldwide. However, the temporal variation in the soil- versus root-associated microbiome over plant species turnover during secondary succession has rarely been quantified. Using the chronosequence restored from fenced grassland and abandoned farmlands on the Loess Plateau of China, we characterized the dynamics of the soil- and root-associated microbiome of host plant with different dominance statuses during secondary succession from 0 to 40 years. Our results revealed that the root microhabitat, the host plant and their interactions were the main contributors to the bacterial community shift (R 2 = 15.5%, 8.1%, and 22.3%, respectively), and plant interspecies replacement had a greater effect on the shift in the root-associated microbial community than intraspecies replacement did during succession. The root-associated bacterial community of pioneer plants was particularly responsive to succession, especially the endosphere community. Endosphere microbial diversity was positively correlated with host plant coverage change, and the diversity and abundance of taxon recruitment into the endosphere of pioneer plants from the surrounding environment decreased as succession progressed. The community assembly processes also indicated that the endosphere microbiota are strongly selected in younger host plants, whereas stochastic processes dominate in aged host plants. Our study provides evidence of the unique response of the root-associated microbiome to the replacement of plant species during secondary succession, and the function of endosphere microbes should be considered when studying plant–microbe feedback.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Unexpected host dependency of Antarctic Nanohaloarchaeota

In hypersaline environments, Nanohaloarchaeota (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaeota [DPANN] superphylum) are thought to be free-living microorganisms. We report cultivation of 2 strains of Antarctic Nanohaloarchaeota and show that they require the haloarchaeon Halorubrum lacusprofundi for growth. By performing growth using enrichments and fluorescence-activated cell sorting, we demonstrated successful cultivation of Candidatus Nanohaloarchaeum antarcticus, purification of Ca. Nha. antarcticus away from other species, and growth and verification of Ca. Nha. antarcticus with Hrr. lacusprofundi ; these findings are analogous to those required for fulfilling Koch’s postulates. We use fluorescent in situ hybridization and transmission electron microscopy to assess cell structures and interactions; metagenomics to characterize enrichment taxa, generate metagenome assembled genomes, and interrogate Antarctic communities; and proteomics to assess metabolic pathways and speculate about the roles of certain proteins. Metagenome analysis indicates the presence of a single species, which is endemic to Antarctic hypersaline systems that support the growth of haloarchaea. The presence of unusually large proteins predicted to function in attachment and invasion of hosts plus the absence of key biosynthetic pathways (e.g., lipids) in metagenome assembled genomes of globally distributed Nanohaloarchaeota indicate that all members of the lineage have evolved as symbionts. Our work expands the range of archaeal symbiotic lifestyles and provides a genetically tractable model system for advancing understanding of the factors controlling microbial symbiotic relationships.

Science & Technology - Other Topics↗