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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Enemy or ally: a genomic approach to elucidate the lifestyle of Phyllosticta citrichinaensis

Members of the fungal genus Phyllosticta can colonize a variety of plant hosts, including several Citrus species such as Citrus sinensis (orange), Citrus limon (lemon), and Citrus maxima (pomelo). Some Phyllosticta species have the capacity to cause disease, such as Citrus Black Spot, while others have only been observed as endophytes. Thus far, genomic differences underlying lifestyle adaptations of Phyllosticta species have not yet been studied. Furthermore, the lifestyle of Phyllosticta citrichinaensis is ambiguous, as it has been described as a weak pathogen but Koch’s postulates may not have been established and the presence of this species was never reported to cause any crop or economic losses. Here, we examined the genomic differences between pathogenic and endophytic Phyllosticta spp. colonizing Citrus and specifically aimed to elucidate the lifestyle of Phyllosticta citrichinaensis. We found several genomic differences between species of different lifestyles, including groups of genes that were only present in pathogens or endophytes. We also observed that species, based on their carbohydrate active enzymes, group independent of their phylogenetic association, and this clustering correlated with trophy prediction. Phyllosticta citrichinaensis shows an intermediate lifestyle, sharing genomic and phenotypic attributes of both pathogens and endophytes. We thus present the first genomic comparison of multiple citrus-colonizing pathogens and endophytes of the genus Phyllosticta, and therefore provide the basis for further comparative studies into the lifestyle adaptations within this genus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Mycoparasites, Gut Dwellers, and Saprotrophs: Phylogenomic Reconstructions and Comparative Analyses of Kickxellomycotina Fungi

Improved sequencing technologies have profoundly altered global views of fungal diversity and evolution. High-throughput sequencing methods are critical for studying fungi due to the cryptic, symbiotic nature of many species, particularly those that are difficult to culture. However, the low coverage genome sequencing (LCGS) approach to phylogenomic inference has not been widely applied to fungi. Here we analyzed 171 Kickxellomycotina fungi using LCGS methods to obtain hundreds of marker genes for robust phylogenomic reconstruction. Additionally, we mined our LCGS data for a set of nine rDNA and protein coding genes to enable analyses across species for which no LCGS data were obtained. The main goals of this study were to: 1) evaluate the quality and utility of LCGS data for both phylogenetic reconstruction and functional annotation, 2) test relationships among clades of Kickxellomycotina, and 3) perform comparative functional analyses between clades to gain insight into putative trophic modes. In opposition to previous studies, our nine-gene analyses support two clades of arthropod gut dwelling species and suggest a possible single evolutionary event leading to this symbiotic lifestyle. Furthermore, we resolve the mycoparasitic Dimargaritales as the earliest diverging clade in the subphylum and find four major clades of Coemansia species. Finally, functional analyses illustrate clear variation in predicted carbohydrate active enzymes and secondary metabolites (SM) based on ecology, that is biotroph versus saprotroph. Saprotrophic Kickxellales broadly lack many known pectinase families compared with saprotrophic Mucoromycota and are depauperate for SM but have similar numbers of predicted chitinases as mycoparasitic.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Bacterial population-level trade-offs between drought tolerance and resource acquisition traits impact decomposition

Microbes drive fundamental ecosystem processes, such as decomposition. Environmental stressors are known to affect microbes, their fitness, and the ecosystem functions that they perform; yet, understanding the causal mechanisms behind this influence has been difficult. We used leaf litter on soil surface as a model in situ system to assess changes in bacterial genomic traits and decomposition rates for 18 months with drought as a stressor. We hypothesized that genome-scale trade-offs due to investment in stress tolerance traits under drought reduce the capacity for bacterial populations to carry out decomposition, and that these population-level trade-offs scale up to impact emergent community traits, thereby reducing decomposition rates. We observed drought tolerance mechanisms that were heightened in bacterial populations under drought, identified as higher gene copy numbers in metagenome-assembled genomes. A subset of populations under drought had reduced carbohydrate-active enzyme genes that suggested—as a trade-off—a decline in decomposition capabilities. These trade-offs were driven by community succession and taxonomic shifts as distinct patterns appeared in populations. We show that trait–trade-offs in bacterial populations under drought could scale up to reduce overall decomposition capabilities and litter decay rates. Using a trait-based approach to assess the population ecology of soil bacteria, we demonstrate genome-level trade-offs in response to drought with consequences for decomposition rates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genome-wide association of the metabolic shifts underpinning dark-induced senescence in Arabidopsis

Dark-induced senescence provokes profound metabolic shifts to recycle nutrients and to guarantee plant survival. To date, research on these processes has largely focused on characterizing mutants deficient in individual pathways. Here, we adopted a time-resolved genome-wide association-based approach to characterize dark-induced senescence by evaluating the photochemical efficiency and content of primary and lipid metabolites at the beginning, or after 3 or 6 days in darkness. We discovered six patterns of metabolic shifts and identified 215 associations with 81 candidate genes being involved in this process. Among these associations, we validated the roles of four genes associated with glycine, galactinol, threonine, and ornithine levels. We also demonstrated the function of threonine and galactinol catabolism during dark-induced senescence. Intriguingly, we determined that the association between tyrosine contents and TYROSINE AMINOTRANSFERASE 1 influences enzyme activity of the encoded protein and transcriptional activity of the gene under normal and dark conditions, respectively. Moreover, the single-nucleotide polymorphisms affecting the expression of THREONINE ALDOLASE 1 and the amino acid transporter gene AVT 1 B, respectively, only underlie the variation in threonine and glycine levels in the dark. Taken together, these results allow us to present a very detailed model of the metabolic aspects of dark-induced senescence, as well as the process itself.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

H-lignin can be deposited independently of CINNAMYL ALCOHOL DEHYDROGENASE C and D in Arabidopsis

Abstract Lignin contributes substantially to the recalcitrance of biomass toward saccharification. To circumvent this problem, researchers have genetically altered lignin, although, in a number of cases, these efforts have resulted in an undesirable yield penalty. Recent findings have shown that by knocking out two subunits (MED5A and MED5B) of the transcriptional regulatory complex Mediator, the stunted growth phenotype of mutants in p-coumaroyl shikimate 3′-hydroxylase, reduced epidermal fluorescence 8-1 (ref8-1), can be alleviated. Furthermore, these plants synthesize a lignin polymer almost entirely derived from p-coumaryl alcohol. Plants deficient in cinnamyl alcohol dehydrogenase (CAD) are notable in that they primarily incorporate coniferaldehyde and sinapaldehyde into their lignin. We tested the hypothesis that by stacking mutations in the genes encoding for the CAD paralogs C and D on an Arabidopsis (Arabidopsis thaliana) med5a/5b ref8-1 genetic background, the biosynthesis of p-coumaryl alcohol would be blocked, making p-coumaraldehyde available for polymerization into a novel kind of lignin. The med5a/5b ref8-1 cadc cadd plants are viable, but lignin analysis demonstrated that they continue to synthesize p-hydroxyphenyl lignin despite being mutated for the CADs typically considered to be required for monolignol biosynthesis. In addition, enzyme activity tests showed that even in the absence of CADC and CADD, there is high CAD activity in stems. We tested the potential involvement of other CADs in p-coumaraldehyde biosynthesis in the quintuple mutant by mutating them using the CRISPR/Cas9 system. Lignin analysis demonstrated that the resulting hextuple mutant plants continue to deposit p-coumaryl alcohol-derived lignin, demonstrating a route for the synthesis of p-hydroxyphenyl lignin in Arabidopsis independent of four CAD isoforms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

SPOTTED-LEAF7 targets the gene encoding β-galactosidase9, which functions in rice growth and stress responses

Abstract β-Galactosidases (Bgals) remove terminal β-D-galactosyl residues from the nonreducing ends of β-D-galactosidases and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite the many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) β-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast 1-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical β-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Functional succession of actively growing soil microorganisms during rewetting is shaped by precipitation history

Rewetting of seasonally dry soils induces a burst of microbial activity and carbon mineralization that changes nutrient availability and leads to succession. Yet the microbial functions that underpin this succession are not well described. Further, it’s unclear how previous precipitation frames microbial capacities after rewetting and how long these effects persist. We used isotopically-labeled water to rewet seasonally dry annual grassland soil that experienced either mean annual or reduced precipitation during the previous two years, and sampled at five subsequent time points. We used quantitative stable isotope probing (qSIP)-informed genome- resolved metagenomics to identify growing microorganisms, predict their capabilities, and analyze how these traits differed over time and between precipitation treatments. Organisms that grew after wetup were more abundant than non-growing organisms prior to the wet-up, suggesting that traits that initiate succession are pre-defined at the end of the prior plant growing season or via survival over the summer. Fast-growing organisms had fewer carbohydrate active enzyme (CAZy) genes per genome than slower-growing organisms, suggesting that although fast-growers were capable of degrading complex C, they may not specialize in this process. Differential abundance of CAZy genes in growing organisms throughout the succession implies that substrate availability varied with time. In contrast, the abundance of peptidases in growing organisms differed between precipitation treatments, but not over time following wet-up. Before wet-up, the soil organisms’ gene inventories were different between the two precipitation treatments. Surprisingly, this legacy effect waned after just one week. Thus, pre-wetup differences in microbial functional capacity converged shortly after rewetting.

Sieradzki, E T↗
DOE OSTI2025

Hydrogen density mapping in biomolecular crystals through dynamic nuclear polarization

Many fundamental biological processes, including those in photosynthetic reaction centers and enzyme active sites, involve charge and energy transfer, bond cleavage, protonation and hydrogen bonding. Because H atoms play such central roles in these reactions, accurately determining their positions is essential. Yet, conventional X-ray crystallography primarily resolves the heavy atoms in biological structures and provides limited insight into hydrogen, even at atomic resolution. Neutron macromolecular crystallography (NMC) overcomes this limitation by offering exceptional sensitivity to hydrogen and deuterium. Here, we present a theoretical framework for the development of dynamic nuclear polarization NMC (DNP-NMC) techniques, which exploit the alignment of neutron and proton nuclear spins to enhance and tune the hydrogen signal contribution. The DNP-NMC approach advances the resolution of H atoms within biomolecular crystals, whether bound to protein residues or present in solvent. The method establishes key relationships for the coherent structure factor of polarized neutron scattering from hydrogenous matter. It theoretically achieves full accuracy in phase reconstruction and offers a path to improve neutron structure determination, achieving accuracies exceeding ≳80% by incorporating titration states. Using a variant of the hybrid input/output phase-retrieval algorithm, it allows recovery of the hydrogen density with ≳90% phase accuracy. In conclusion, we further discuss sources of experimental uncertainty for the upcoming DNP-enabled, quasi-Laue IMAGINE-X experiment at Oak Ridge National Laboratory's High Flux Isotope Reactor.

dynamic nuclear polarization↗
DOE OSTI2023

Warming decreases desert ecosystem functioning by altering biocrusts in drylands

Warming and precipitation fluctuations are changing desert ecosystems in global drylands. However, the effects of climate change on keystone species such as cryptogamic biocrust in drylands remain relatively under-investigated, even though biocrusts play a vital role in desert ecosystems. Here we conducted a long-term experiment (14 years) to simulate the responses of two main types of biocrusts to warming coupled with reduction in precipitation that was achieved by open-top chambers (OTCs) to simulate the predicted warming and precipitation decreasing under climate change scenario. We also conduct a structural analysis to evaluate the resulting changes in desert ecosystem functioning (carbon and nitrogen cycling). Neither warming and corresponding rainfall reduction treatments had a negative effect on lichen species richness, but both treatments reduced lichen cover and biomass. The negative effects of warming on moss-dominated crusts were much greater than those on lichen-dominated crusts. Although mosses and lichens had varying degree responses to warming, the loss of mosses and decreased lichen cover and biomass, as well as the shortening of the wet time, resulted in a reduction in carbon and nitrogen fixation, soil enzyme activity and water-holding capacity of biocrusts and topsoil. These impacts collectively change the water balance of drylands and weaken the hydrological and biogeochemical function of biocrusts. Synthesis and applications: Results from this long-term experiment suggest that the ecosystem C and N cycling and water balance of global drylands may be highly impacted by climate change, in part because of the response of biocrusts, which contribute an important implication for both dryland restoration and earth system dynamics.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Substrate availability and not thermal acclimation controls microbial temperature sensitivity response to long-term warming

Microbes are responsible for cycling carbon (C) through soils, and predicted changes in soil C stocks under climate change are highly sensitive to shifts in the mechanisms assumed to control the microbial physiological response to warming. Two mechanisms have been suggested to explain the long-term warming impact on microbial physiology: microbial thermal acclimation and changes in the quantity and quality of substrates available for microbial metabolism. Yet studies disentangling these two mechanisms are lacking. To resolve the drivers of changes in microbial physiology in response to long-term warming, we sampled soils from 13- and 28-year-old soil warming experiments in different seasons. We performed short-term laboratory incubations across a range of temperatures to measure the relationships between temperature sensitivity of physiology (growth, respiration, carbon use efficiency, and extracellular enzyme activity) and the chemical composition of soil organic matter. We observed apparent thermal acclimation of microbial respiration, but only in summer, when warming had exacerbated the seasonally-induced, already small dissolved organic matter pools. Irrespective of warming, greater quantity and quality of soil carbon increased the extracellular enzymatic pool and its temperature sensitivity. We propose that fresh litter input into the system seasonally cancels apparent thermal acclimation of C-cycling processes to decadal warming. Our findings reveal that long-term warming has indirectly affected microbial physiology via reduced C availability in this system, implying that earth system models including these negative feedbacks may be best suited to describe long-term warming effects on these soils.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Wildfire‐Induced Losses of Soil Particulate and Mineral‐Associated Organic Carbon Persist for Over 4 Years in a Chaparral Ecosystem

ABSTRACT Wildfires can lower soil carbon (C) stocks directly through combustion, but also indirectly during post‐fire recovery if microbial C demands outpace photosynthetic C inputs. However, how much C is respired by soil microorganisms post‐fire may depend on wildfire effects on particulate organic carbon (POC; mostly plant material accessible to microbes) and/or mineral‐associated organic carbon (MAOC; considered C protected by minerals from decomposers), meaning assessment of wildfire impacts on these pools is necessary to predict microbial decomposition rates and, thus, the fate of soil C. Here, we measured POC, MAOC, pyrogenic organic matter C, plant cover, extracellular enzyme activity (EEA), and microbial community abundance and composition 17 days, and 1, 3, and 4 years after the Holy Fire burned 94 km 2 of fire‐adapted chaparral. The wildfire immediately decreased POC by 50% (from 51 ± 21 to 26 ± 6 g C kg −1 ) and MAOC by 33% (from 9.3 ± 0.9 to 6.3 ± 0.9 g C kg −1 ), consistent with MAOC being less vulnerable to loss than POC. POC decreased by another 38% 1 year post‐fire, consistent with increases in microbial abundance and EEA suggesting increased microbial decomposition. Between 1 and 4 years after the fire, cover of the dominant shrub (Arctostaphylos glandulosa) increased from 3.9% ± 1.6% to 16% ± 5.4% (compared to 58% ± 4.6% in unburned plots), marking the end of net soil C losses. Still, soil C did not increase between 1 and 4 years post‐fire, suggesting plant C inputs did not outpace microbial respiration, a finding consistent with isotopically heavier C from microorganisms raising bulk soil δ 13 C values. As global changes favor increases in wildfire frequency and severity, C losses via combustion and decomposition may outpace plant C inputs during the first 4 years post‐fire in chaparral, slowing the replenishment of soil C stocks.

Biodiversity & Conservation↗
DOE OSTI2025

Discovery of an autoinhibited conformation in mesotrypsin reveals a strategy for selective serine protease inhibition

Selective inhibition of the more than 100 S1 family serine proteases is a long-standing challenge due to their active site similarity. Mesotrypsin, implicated in cancer progression, exemplifies these difficulties; no current inhibitors achieve selectivity over other human trypsins. We found an unexpected autoinhibited conformation of mesotrypsin via x-ray crystallography, revealing a cryptic pocket adjacent to the active site. Using high-throughput virtual screening targeting this cryptic pocket, we identified a conformationally selective small-molecule inhibitor that stabilizes the inactive state of mesotrypsin. This inhibitor demonstrates selectivity for mesotrypsin over other trypsins. Our findings challenge the accepted view of digestive trypsins as constitutively active enzymes lacking potential for allosteric regulation. Furthermore, analyses of other structures suggest that dynamic sampling of closed states with analogous allosteric cryptic pockets appears widespread among S1 serine proteases. These observations point to a potentially generalizable strategy to achieve selective inhibition, offering broad implications for drug development targeting serine proteases in cancer and other diseases.

Coban, Matt↗
DOE OSTI2020

Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. In this work, we investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts. While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Enrichable consortia of microbial symbionts degrade macroalgal polysaccharides in Kyphosus fish

ABSTRACT Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce genomic information about the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) from previously described Kyphosus gut metagenomes and newly sequenced bioreactor enrichments reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzyme collections able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea ) and Verrucomicrobiota (order Kiritimatiellales ) highlight the importance of metabolic contributions from multiple phyla to broaden polysaccharide degradation capabilities. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors. IMPORTANCE Seaweed has long been considered a promising source of sustainable biomass for bioenergy and aquaculture feed, but scalable industrial methods for decomposing terrestrial compounds can struggle to break down seaweed polysaccharides efficiently due to their unique sulfated structures. Fish of the genus Kyphosus feed on seaweed by leveraging gastrointestinal bacteria to degrade algal polysaccharides into simple sugars. This study reconstructs metagenome-assembled genomes for these gastrointestinal bacteria to enhance our understanding of herbivorous fish digestion and fermentation of algal sugars. Investigations at the gene level identify Kyphosus guts as an untapped source of seaweed-degrading enzymes ripe for further characterization. These discoveries set the stage for future work incorporating marine enzymes and microbial communities in the industrial degradation of algal polysaccharides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative genomics and stable isotope analysis reveal the saprotrophic-pathogenic lifestyle of a neotropical fungus

In terrestrial forested ecosystems, fungi may interact with trees in at least three distinct ways: (i) associated with roots as symbionts; (ii) as pathogens in roots, trunks, leaves, flowers, and fruits; or (iii) decomposing dead tree tissues on soil or even on dead tissues in living trees. Distinguishing the latter two nutrition modes is rather difficult in Hymenochaetaceae (Basidiomycota) species. Herein, we have used an integrative approach of comparative genomics, stable isotopes, host tree association, and bioclimatic data to investigate the lifestyle ecology of the scarcely known neotropical genus Phellinotus, focusing on the unique species Phellinotus piptadeniae. This species is strongly associated with living Piptadenia gonoacantha (Fabaceae) trees in the Atlantic Forest domain on a relatively high precipitation gradient. Phylogenomics resolved P. piptadeniae in a clade that also includes both plant pathogens and typical wood saprotrophs. Furthermore, both genome-predicted Carbohydrate-Active Enzymes (CAZy) and stable isotopes (δ 13 C and δ 15 N) revealed a rather flexible lifestyle for the species. Altogether, our findings suggest that P. piptadeniae has been undergoing a pathotrophic specialization in a particular tree species while maintaining all the metabolic repertoire of a wood saprothroph.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Regulation of bacterial stringent response by an evolutionarily conserved ribosomal protein L11 methylation

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics data set from 48 bacterial strains representing six phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. Comparative proteomics analysis combined with inorganic polyphosphate and guanosine tetra/pentaphosphate assays of the ΔprmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. In the stationary phase, we found that the ΔprmA mutant has impaired guanosine tetra/pentaphosphate production. This leads to a reduction in inorganic polyphosphate levels, accumulation of RNA and ribosomal proteins, and an abnormal polysome profile. Overall, our investigation demonstrates that the evolutionarily conserved bL11 methylation is important for stringent response signaling and ribosomal activity regulation and turnover.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Complete Genome Sequences of Caldicellulosiruptor acetigenus DSM 7040, Caldicellulosiruptor morganii DSM 8990 (RT8.B8), and Caldicellulosiruptor naganoensis DSM 8991 (NA10)

The genome sequences of three extremely thermophilic, lignocellulolytic Caldicellulosiruptor species were closed, improving previously reported multiple-contig assemblies. All 14 classified Caldicellulosiruptor spp. now have closed genomes. Genome closure will enhance bioinformatic analysis of the species, including identification of carbohydrate-active enzymes (CAZymes) and comparison against other Caldicellulosiruptor species and lignocellulolytic microorganisms.

Bing, Ryan G.↗
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↗