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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Decomposing a San Francisco estuary microbiome using long-read metagenomics reveals species- and strain-level dominance from picoeukaryotes to viruses

ABSTRACT Although long-read sequencing has enabled obtaining high-quality and complete genomes from metagenomes, many challenges still remain to completely decompose a metagenome into its constituent prokaryotic and viral genomes. This study focuses on decomposing an estuarine metagenome to obtain a more accurate estimate of microbial diversity. To achieve this, we developed a new bead-based DNA extraction method, a novel bin refinement method, and obtained 150 Gbp of Nanopore sequencing. We estimate that there are ~500 bacterial and archaeal species in our sample and obtained 68 high-quality bins (>90% complete, <5% contamination, ≤5 contigs, contig length of >100 kbp, and all ribosomal and tRNA genes). We also obtained many contigs of picoeukaryotes, environmental DNA of larger eukaryotes such as mammals, and complete mitochondrial and chloroplast genomes and detected ~40,000 viral populations. Our analysis indicates that there are only a few strains that comprise most of the species abundances. IMPORTANCE Ocean and estuarine microbiomes play critical roles in global element cycling and ecosystem function. Despite the importance of these microbial communities, many species still have not been cultured in the lab. Environmental sequencing is the primary way the function and population dynamics of these communities can be studied. Long-read sequencing provides an avenue to overcome limitations of short-read technologies to obtain complete microbial genomes but comes with its own technical challenges, such as needed sequencing depth and obtaining high-quality DNA. We present here new sampling and bioinformatics methods to attempt decomposing an estuarine microbiome into its constituent genomes. Our results suggest there are only a few strains that comprise most of the species abundances from viruses to picoeukaryotes, and to fully decompose a metagenome of this diversity requires 1 Tbp of long-read sequencing. We anticipate that as long-read sequencing technologies continue to improve, less sequencing will be needed.

Lui, Lauren M.↗
DOE OSTI2023

Targeted rRNA depletion enables efficient mRNA sequencing in diverse bacterial species and complex co-cultures

Bacterial mRNA sequencing is inefficient due to the abundance of ribosomal RNA that is challenging to deplete. While commercial kits target rRNA from common bacterial species, they are frequently inefficient when applied to divergent species, including those from environmental isolates. Similarly, other methods typically employ large probe sets that tile the entire length of rRNAs; however, such approaches are infeasible when applied to many species. Therefore, we present EMBR-seq+, which requires fewer than 10 oligonucleotides per rRNA by combining rRNA blocking primers with RNase H-mediated depletion to achieve rRNA removal efficiencies of up to 99% in diverse bacterial species. Furthermore, in more complex microbial co-cultures between Fibrobacter succinogenes strain UWB7 and anaerobic fungi, EMBR-seq+ depleted both bacterial and fungal rRNA, with a fourfold improvement in bacterial rRNA depletion compared with a commercial kit, thereby demonstrating that the method can be applied to non-model microbial mixtures. Notably, for microbes with unknown rRNA sequences, EMBR-seq+ enables rapid iterations in probe design without requiring to start experiments from total RNA. Finally, efficient depletion of rRNA enabled systematic quantification of the reprogramming of the bacterial transcriptome when cultured in the presence of the anaerobic fungi Anaeromyces robustus or Caecomyces churrovis. We observed that F. succinogenes strain UWB7 downregulated several lignocellulose-degrading carbohydrate-active enzymes in the presence of anaerobic gut fungi, suggesting close interactions between two cellulolytic species that specialize in different aspects of biomass breakdown. Thus, EMBR-seq+ enables efficient, cost-effective, and rapid quantification of the transcriptome to gain insights into non-model microbial systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Mapping the soil microbiome functions shaping wetland methane emissions

Accounting for only 8% of Earth’s land cover, freshwater wetlands remain the foremost contributors to global methane emissions. Yet the microorganisms and processes underlying methane emissions from wetland soils remain poorly understood. Over a five-year period, we surveyed the microbial membership and in situ methane measurements from over 700 samples in one of the most prolific methane-emitting wetlands in the United States. We constructed a catalog of 2,502 metagenome-assembled genomes (MAGs), with more than half of the 70 bacterial and archaeal phyla sampled containing novel lineages. Integration of these data with 133 soil metatranscriptomes provided a genome-resolved view of the biogeochemical specialization and versatility expressed over wetland soil spatial and temporal gradients. Centimeter-scale depth differences best explained patterns of microbial community structure and transcribed functionalities, even more than land cover or temporal information. Moreover, while extended flooding restructured soil redox, this perturbation failed to reconfigure the transcriptional profiles of methane-cycling microorganisms, contrasting with theoretically expected responses to hydrological perturbations. Co-expression analyses, coupled with depth-resolved methane measurements, revealed the metabolisms and trophic structures most predictive of methane hotspots. Mapping the spatiotemporal transcriptional patterns on this compendium of biogeochemically classified soil-derived genomes begins to untangle the microbial carbon, energy, and nutrient processing contributing to wetland methane production.

MAG↗
DOE OSTI2025

Microbes display broad diversity in cobamide preferences

ABSTRACT Cobamides, the vitamin B 12 (cobalamin) family of cofactors, are used by most organisms but produced by only a fraction of prokaryotes, and are thus considered key shared nutrients among microbes. Cobamides are structurally diverse, with multiple different cobamides found in most microbial communities. The ability to use different cobamides has been tested for several bacteria and microalgae, and nearly all show preferences for certain cobamides. This approach is limited by the commercial unavailability of cobamides other than cobalamin. Here, we have extracted and purified seven commercially unavailable cobamides to characterize bacterial cobamide preferences based on growth in specific cobamide-dependent conditions. The tested bacteria include engineered strains of Escherichia coli , Sinorhizobium meliloti , and Bacillus subtilis expressing native or heterologous cobamide-dependent enzymes, cultured under conditions that functionally isolate specific cobamide-dependent processes such as methionine synthesis. Comparison of these results to those of previous studies of diverse bacteria and microalgae revealed that a broad diversity of cobamide preferences exists not only across different organisms but also between different cobamide-dependent metabolic pathways within the same organism. The microbes differed in the cobamides that support growth most efficiently, cobamides that do not support growth, and the minimum cobamide concentrations required for growth. The latter differ by up to four orders of magnitude across organisms from different environments and by up to 20-fold between cobamide-dependent enzymes within the same organism. Given that cobamides are shared, required for use of specific growth substrates, and essential for central metabolism in certain organisms, cobamide preferences likely impact community structure and function. IMPORTANCE Nearly all bacteria are found in microbial communities with tens to thousands of other species. Molecular interactions such as metabolic cooperation and competition are key factors underlying community assembly and structure. Cobamides, the vitamin B 12 family of enzyme cofactors, are one such class of nutrients, produced by only a minority of prokaryotes but required by most microbes. A unique aspect of cobamides is their broad diversity, with nearly 20 structural forms identified in nature. Importantly, this structural diversity impacts growth as most bacteria that have been tested show preferences for specific cobamide forms. We measured cobamide-dependent growth in several model bacteria and compared the results to those of previous analyses of cobamide preference. We found that cobamide preferences vary widely across bacteria, showing the importance of characterizing these aspects of cobamide biology to understand the impact of cobamides on microbial communities.

Mok, Kenny C. (ORCID:0000000252276987)↗
DOE OSTI2025

Patchy burn severity explains heterogeneous soil viral and prokaryotic responses to fire in a mixed conifer forest

ABSTRACT Effects of fire on soil viruses and virus–host dynamics are largely unexplored, despite known microbial contributions to biogeochemical processes and ecosystem recovery. Here, we assessed how viral and prokaryotic communities responded to a prescribed burn in a mixed conifer forest. We sequenced 91 viral-size fraction metagenomes (viromes) and 115 16S rRNA gene amplicon libraries from 120 samples: four samples at five timepoints (two before fire and three after fire) at six sites (four treatment, two control). We hypothesized that compositional differences would be most significant between burned and unburned soils, but instead, plot location best distinguished viral communities, more than treatment (burned or not), depth (0–3 or 3–6 cm), or timepoint. For both viruses and prokaryotes, some burned communities resembled unburned controls, while others were significantly different, revealing heterogeneous responses to fire. These patterns were explained by burn severity, here defined by soil chemistry. Viral but not prokaryotic richness decreased significantly with burn severity, and low viromic DNA yields indicated substantial loss of viral biomass at higher severity. The relative abundances of Firmicutes, Actinobacteriota, and the viruses predicted to infect them increased significantly with burn severity, suggesting survival and viral infection of these fire-responsive and potentially spore-forming taxa. The degree of burn severity experienced by each patch of soil, rather than burn status alone, differed over mere meters in the same fire. Therefore, our analyses highlight the importance of high-resolution, paired biogeochemical data to explain soil community responses to fire. IMPORTANCE The impact of fire on the soil microbiome, particularly on understudied soil viral communities, warrants investigation, given known microbial contributions to biogeochemical processes and ecosystem recovery. Here, we collected 120 soil samples before and after a prescribed burn in a mixed conifer forest to assess the impacts of this disturbance on soil viral and prokaryotic communities. We show that simple categorical comparisons of burned and unburned areas were insufficient to reveal the underlying community response patterns. The patchy nature of the fire (indicated by soil chemistry data) led to significant changes in viral and prokaryotic community composition in areas of high burn severity, while communities that experienced lower burn severity were indistinguishable from those in unburned controls. Our results highlight the importance of considering highly resolved burn severity and biogeochemical measurements, even in nearby soils after the same fire, in order to understand soil microbial responses to prescribed burns.

Microbiology↗
DOE OSTI2024

Identification and characterization of a skin microbiome on Caenorhabditis elegans suggests environmental microbes confer cuticle protection

ABSTRACT In the wild, C. elegans are emersed in environments teeming with a veritable menagerie of microorganisms. The C. elegans cuticular surface serves as a barrier and first point of contact with their microbial environments. In this study, we identify microbes from C. elegans natural habitats that associate with its cuticle, constituting a simple “skin microbiome.” We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on the C. elegans surface. Furthermore, we conduct 16S rRNA gene sequencing studies to identify relative shifts in the proportion of mCeMbio bacteria upon surface-sterilization, implying distinct skin- and gut-microbiomes. We find that some strains of bacteria, including Enterobacter sp. JUb101 , are primarily found on the nematode skin, while others like Stenotrophomonas indicatrix JUb19 and Ochrobactrum vermis MYb71 are predominantly found in the animal’s gut. Finally, we show that this skin microbiome promotes host cuticle integrity in harsh environments. Together, we identify a skin microbiome for the well-studied nematode model and propose its value in conferring host fitness advantages in naturalized contexts. IMPORTANCE The genetic model organism C. elegans has recently emerged as a tool for understanding host–microbiome interactions. Nearly all of these studies either focus on pathogenic or gut-resident microbes. Little is known about the existence of native, nonpathogenic skin microbes or their function. We demonstrate that members of a modified C. elegans model microbiome, mCeMbio, can adhere to the animal's cuticle and confer protection from noxious environments. We combine a novel micro-swab tool, the first 16S microbial sequencing data from relatively unperturbed C. elegans , and physiological assays to demonstrate microbially mediated protection of the skin. This work serves as a foundation to explore wild C. elegans skin microbiomes and use C. elegans as a model for skin research.

16S RNA↗
DOE OSTI2024

Changes in environmental and engineered conditions alter the plasma membrane lipidome of fractured shale bacteria

ABSTRACT Microorganisms that persist in fractured shale reservoirs cause several problems including secreting foul gases and forming biofilms. Current biocontrol measures often fail due to limited knowledge of their in situ activities. The plasma membrane protects the cell, mediates many of its critical functions, and responds to intracellular cues and ecological perturbations through physicochemical modifications. As such, it provides valuable insight into the physiological adaptation of microorganisms in disturbed environmental systems. Here, we (i) demonstrate how changes in salinity and hydraulic retention time (HRT) influence the plasma membrane intact polar lipid (IPL) chemistry of model bacterium, Halanaerobium congolense WG10, and mixed microbial consortia enriched from shale-produced fluids and (ii) elucidate adjustments in membrane IPL chemistry during biofilm growth relative to planktonic cells. We incubated H. congolense WG10 in chemostats under three salinities (7%, 13%, and 20% NaCl), operated under three HRTs (19.2, 24, and 48 h), and in drip flow biofilm reactors under the same salinity gradients. Also, mixed microbial consortia in produced fluids were enriched in triplicate chemostat vessels under three HRTs (19.2, 24, and 72 h) and biofilm reactors. Lipids were analyzed by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Our results show that phosphatidylglycerols, cardiolipins, and phosphatidylethanolamines were predominantly enriched in planktonic H. congolense WG10 cells grown at hypersalinity (20%) compared to optimum (13%). In addition, several zwitterionic phosphatidylcholines and phosphatidylethanolamines were higher in abundance during biofilm growth. These observations suggest that microbial adaptation and biofilm formation in fractured shale are enabled by strategic plasma membrane IPL chemistry adjustments. IMPORTANCE Microorganisms inadvertently introduced into the shale reservoir during fracturing face multiple stressors including brine-level salinities and starvation. However, some anaerobic halotolerant bacteria adapt and persist for long periods of time. They produce hydrogen sulfide, which sours the reservoir and corrodes engineering infrastructure. In addition, they form biofilms on rock matrices, which decrease shale permeability and clog fracture networks. These reduce well productivity and increase extraction costs. Under stress, microbes remodel their plasma membrane to optimize its roles in protection and mediating cellular processes such as signaling, transport, and energy metabolism. Hence, by observing changes in the membrane lipidome of model shale bacteria, Halanaerobium congolense WG10, and mixed consortia enriched from produced fluids under varying subsurface conditions and growth modes, we provide insight that advances our knowledge of the fractured shale biosystem. We also offer data-driven recommendations for improving biocontrol efficacy and the efficiency of energy recovery from unconventional formations.

03 NATURAL GAS↗
DOE OSTI2022

BioSTEAMDevelopmentGroup/Bioindustrial-Park/TAL

Triacetic acid lactone (TAL) can be microbially produced and further chemically upgraded to several high-value chemicals. In this work, several acidic and basic ion-exchange resins and activated charcoal were evaluated for their ability to adsorb microbially produced TAL. Activated charcoal and a weak base resin, Dowex 66, showed similar TAL adsorption capacity of 0.18 ± 0.002 g/g. At 15% w/v activated charcoal, about 98% of TAL present in fermentation broth could be adsorbed. Further, ethanol washing allowed recovery of 72% of adsorbed TAL. A biorefinery producing TAL from sucrose was designed, simulated, and evaluated (through technoeconomic analysis) under uncertainty, for an estimated TAL minimum product selling price (MPSP) of $4.27/kg [$3.71−4.94/kg; 5th-95th percentiles] for the current state of technology and $2.83/kg [$2.46–3.29/kg] following potential near-term improvements to fermentation. Thus, this work provides an adsorptive process to recover microbially produced TAL that can be chemically upgraded to several industrial products.

Cortes-Peña, Yoel↗
DOE OSTI2021

Feature selection and causal analysis for microbiome studies in the presence of confounding using standardization

Abstract Background Microbiome studies have uncovered associations between microbes and human, animal, and plant health outcomes. This has led to an interest in developing microbial interventions for treatment of disease and optimization of crop yields which requires identification of microbiome features that impact the outcome in the population of interest. That task is challenging because of the high dimensionality of microbiome data and the confounding that results from the complex and dynamic interactions among host, environment, and microbiome. In the presence of such confounding, variable selection and estimation procedures may have unsatisfactory performance in identifying microbial features with an effect on the outcome. Results In this manuscript, we aim to estimate population-level effects of individual microbiome features while controlling for confounding by a categorical variable. Due to the high dimensionality and confounding-induced correlation between features, we propose feature screening, selection, and estimation conditional on each stratum of the confounder followed by a standardization approach to estimation of population-level effects of individual features. Comprehensive simulation studies demonstrate the advantages of our approach in recovering relevant features. Utilizing a potential-outcomes framework, we outline assumptions required to ascribe causal, rather than associational, interpretations to the identified microbiome effects. We conducted an agricultural study of the rhizosphere microbiome of sorghum in which nitrogen fertilizer application is a confounding variable. In this study, the proposed approach identified microbial taxa that are consistent with biological understanding of potential plant-microbe interactions. Conclusions Standardization enables more accurate identification of individual microbiome features with an effect on the outcome of interest compared to other variable selection and estimation procedures when there is confounding by a categorical variable.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

Abstract Background Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. Results We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. Conclusions Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

Chun, Kayla↗
DOE OSTI2023

Engineered Escherichia coli platforms for tyrosine-derivative production from phenylalanine using phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system

Aromatic compounds derived from tyrosine are important and diverse chemicals that have industrial and commercial applications. Although these aromatic compounds can be obtained by extraction from natural producers, their growth is slow, and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes. To facilitate development of microbial cell factories for producing tyrosine derivatives, we developed simple and convenient tyrosine-producing Escherichia coli platforms with a bacterial phenylalanine hydroxylase, which converted phenylalanine to tyrosine with tetrahydromonapterin as a cofactor, using a synthetic biology approach. By introducing a tetrahydrobiopterin-regeneration system, the tyrosine titer of the plasmid-based engineered strain was 4.63 g/L in a medium supplemented with 5.00 g/L phenylalanine with a test tube. The strains were successfully used to produce industrially attractive compounds, such as tyrosol with a yield of 1.58 g/L by installing a tyrosol-producing module consisting of genes encoding tyrosine decarboxylase and tyramine oxidase on a plasmid. Gene integration into E. coli chromosomes has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and enables more flexible pathway engineering by accepting more plasmids with artificial pathway genes. Therefore, we constructed a plasmid-free tyrosine-producing platform by integrating five modules, comprising genes encoding the phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system, into the chromosome. The platform strain could produce 1.04 g/L of 3,4-dihydroxyphenylalanine, a drug medicine, by installing a gene encoding tyrosine hydroxylase and the tetrahydrobiopterin-regeneration system on a plasmid. Moreover, by installing the tyrosol-producing module, tyrosol was produced with a yield of 1.28 g/L. We developed novel E. coli platforms for producing tyrosine from phenylalanine at multi-gram-per-liter levels in test-tube cultivation. The platforms allowed development and evaluation of microbial cell factories installing various designed tyrosine-derivative biosynthetic pathways at multi-grams-per-liter levels in test tubes.

09 BIOMASS FUELS↗
DOE OSTI2020

Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes

Background: The ability of blood-feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered with, or enhanced by, the arthropod’s native microbiome. Mosquitoes transmit viruses, protozoan and filarial nematodes, the majority of which contribute to the 17% of infectious disease cases worldwide. Diroflaria immitis, a mosquito-transmitted filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti. Methods: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female Ae. aegypti. Metagenomic analysis of the V3–V4 variable region of the microbial 16S RNA gene was used for identification of the microbial differences down to species level. Results: We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis-infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship, and has been previously shown to indirectly compete for nutrients with fungi on domestic housefly eggs and larvae. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacterial genera and phyla between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacterial species when commonly identified bacteria were compared. Conclusions: To the best of our knowledge, this is the first study to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies are required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of Ae. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Demographic drivers of gut microbiome diversity

Abstract The gut microbiome plays a central role in orchestrating metabolic, immune, and neurological functions essential for human health. While extensive research has explored the effects of diseases and pathological conditions on gut microbiome composition, the influence of demographic factors remains underexplored, limiting our understanding of microbiome variations in disease states. This study addresses this gap by investigating the impact of demographic variables, including age, sex, and geography, on gut microbiome diversity in healthy individuals. Using the American Gut Project’s extensive dataset and the QIIME2 bioinformatics pipeline, we conducted a comprehensive analysis of microbial profiles across diverse demographic groups. Our results revealed significant age-related shifts in microbial richness and composition, and geographic location strongly influenced phylogenetic diversity. In contrast, sex exhibited limited impact on microbial diversity within healthy BMI ranges. These findings highlight the critical role of demographic factors in shaping gut microbiome diversity, providing a foundational framework to better contextualize disease-related microbiome variations and advance personalized healthcare approaches.

Biotechnology & Applied Microbiology↗
DOE OSTI2021

Holo-omics for deciphering plant-microbiome interactions

Host-microbiome interactions are recognized for their importance to host health. An improved understanding of the molecular underpinnings of host-microbiome relationships will advance our capacity to accurately predict host fitness and manipulate interaction outcomes. Within the plant microbiome research field, unlocking the functional relationships between plants and their microbial partners is the next step to effectively using the microbiome to improve plant fitness. We propose that strategies that pair host and microbial datasets—referred to here as holo-omics—provide a powerful approach for hypothesis development and advancement in this area. We discuss several experimental design considerations and present a case study to highlight the potential for holo-omics to generate a more holistic perspective of molecular networks within the plant microbiome system. In addition, we discuss the biggest challenges for conducting holo-omics studies; specifically, the lack of vetted analytical frameworks, publicly available tools, and required technical expertise to process and integrate heterogeneous data. Finally, we conclude with a perspective on appropriate use-cases for holo-omics studies, the need for downstream validation, and new experimental techniques that hold promise for the plant microbiome research field. We argue that utilizing a holo-omics approach to characterize host-microbiome interactions can provide important opportunities for broadening system-level understandings and significantly inform microbial approaches to improving host health and fitness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Data from A Mass Spectrometry-Based High-Throughput Screening Method for Engineering Fatty Acid Synthases with Improved Production of Medium Chain Fatty Acids

Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs) as key components of crucial nutrients, soaps, industrial chemicals, and fuels. However, our ability to control the composition of microbially synthesized FFAs is still limited, particularly, for producing medium‐chain fatty acids (MCFAs). This is mainly due to the lack of high‐throughput approaches for FFA analysis to engineer enzymes with desirable product specificity. Here we report a mass spectrometry (MS)‐based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. In particular, matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and a higher m/z peak ratio at 730 and 758 was used as an indication for improved MCFA production. This colony‐based method can be performed at a rate of ~2 s per sample, representing a substantial improvement over gas chromatography‐MS (typically >30 min per sample) as the gold standard method for FFA detection. To demonstrate the power of this method, we performed site‐saturation mutagenesis of the yeast fatty acid synthase and identified nine missense mutations that resulted in improved MCFA production relative to the wild‐type strain. Colony‐based MALDI‐ToF MS screening provides an effective approach for engineering microbial fatty acid compositions in a high‐throughput manner.

Conversion↗
DOE OSTI2021

Searching for fat tails in CRISPR-Cas systems: Data analysis and mathematical modeling

Understanding CRISPR-Cas systems—the adaptive defence mechanism that about half of bacterial species and most of archaea use to neutralise viral attacks—is important for explaining the biodiversity observed in the microbial world as well as for editing animal and plant genomes effectively. The CRISPR-Cas system learns from previous viral infections and integrates small pieces from phage genomes called spacers into the microbial genome. The resulting library of spacers collected in CRISPR arrays is then compared with the DNA of potential invaders. One of the most intriguing and least well understood questions about CRISPR-Cas systems is the distribution of spacers across the microbial population. Here, using empirical data, we show that the global distribution of spacer numbers in CRISPR arrays across multiple biomes worldwide typically exhibits scale-invariant power law behaviour, and the standard deviation is greater than the sample mean. We develop a mathematical model of spacer loss and acquisition dynamics which fits observed data from almost four thousand metagenomes well. In analogy to the classical ‘rich-get-richer’ mechanism of power law emergence, the rate of spacer acquisition is proportional to the CRISPR array size, which allows a small proportion of CRISPRs within the population to possess a significant number of spacers. Our study provides an alternative explanation for the rarity of all-resistant super microbes in nature and why proliferation of phages can be highly successful despite the effectiveness of CRISPR-Cas systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A genome-scale metabolic model for the denitrifying bacterium Thauera sp. MZ1T accurately predicts degradation of pollutants and production of polymers

The denitrifying bacterium Thauera sp . MZ1T, a common member of microbial communities in wastewater treatment facilities, can produce different compounds from a range of carbon (C) and nitrogen (N) sources under aerobic and anaerobic conditions. In these different conditions, Thauera modifies its metabolism to produce different compounds that influence the microbial community. In particular, Thauera sp . MZ1T produces different exopolysaccharides with floc-forming properties, impacting the physical disposition of wastewater consortia and the efficiency of nutrient assimilation by the microbial community. Under N-limiting conditions, Thauera sp . MZ1T decreases its growth rate and accelerates the accumulation of polyhydroxyalkanoate-related (PHA) compounds including polyhydroxybutyrate (PHB), which plays a fundamental role as C and energy storage in this β-proteobacterium. However, the metabolic mechanisms employed by Thauera sp . MZ1T to assimilate and catabolize many of the different C and N sources under aerobic and anaerobic conditions remain unknown. Systems biology approaches such as genome-scale metabolic modeling have been successfully used to unveil complex metabolic mechanisms for various microorganisms. Here, we developed a comprehensive metabolic model (M-model) for Thauera sp . MZ1T ( i Thauera861), consisting of 1,744 metabolites, 2,384 reactions, and 861 genes. We validated the model experimentally using over 70 different C and N sources under both aerobic and anaerobic conditions. i Thauera861 achieved a prediction accuracy of 95% for growth on various C and N sources and close to 85% for assimilation of aromatic compounds under denitrifying conditions. The M-model was subsequently deployed to determine the effects of substrates, oxygen presence, and the C:N ratio on the production of PHB and exopolysaccharides (EPS), showing the highest polymer yields are achieved with nucleotides and amino acids under aerobic conditions. This comprehensive M-model will help reveal the metabolic processes by which this ubiquitous species influences communities in wastewater treatment systems and natural environments.

Tec-Campos, Diego (ORCID:0000000188194150)↗
DOE OSTI2020

Geology and climate influence rhizobiome composition of the phenotypically diverse tropical tree Tabebuia heterophylla

Plant-associated microbial communities have diverse phenotypic effects on their hosts that are only beginning to be revealed. We hypothesized that morpho-physiological variations in the tropical tree Tabebuia heterophylla, observed on different geological substrates, arise in part due to microbial processes in the rhizosphere. We characterized the microbiota of the rhizosphere and soil communities associated with T. heterophylla trees in high and low altitude sites (with varying temperature and precipitation) of volcanic, karst and serpentine geologies across Puerto Rico. We sampled 6 areas across the island in three geological materials including volcanic, serpentine and karst soils. Collection was done in 2 elevations (>450m and 0-300m high), that included 3 trees for each site and 4 replicate soil samples per tree of both bulk and rhizosphere. Genomic DNA was extracted from 144 samples, and 16S rRNA V4 sequencing was performed on the Illumina MiSeq platform. Proteobacteria, Actinobacteria, and Verrucomicrobia were the most dominant phyla, and microbiomes clustered by geological substrate and elevation. Volcanic samples were enriched in Verrucomicrobia; karst was dominated by nitrogen-fixing Proteobacteria, and serpentine sites harbored the most diverse communities, with dominant Cyanobacteria. Sites with similar climates but differing geologies showed significant differences on rhizobiota diversity and composition demonstrating the importance of geology in shaping the rhizosphere microbiota, with implications for the plant's phenotype. Our study sheds light on the combined role of geology and climate in the rhizosphere microbial consortia, likely contributing to the phenotypic plasticity of the trees.

59 BASIC BIOLOGICAL SCIENCES↗