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At least 73 records · Page 4

A sorghum pangenome reference improves global crop trait discovery

Although the green revolution adapted a handful of crops to homogeneous and high-input industrialized agriculture, much of the global population still relies on the local production of variable crop cultivars by low-input smallholder farms. This diversity of unhomogenized crops, like that of the grain and bioenergy crop sorghum, offers raw materials for genetic gain and cultivar improvement. However, breeding efforts can be constrained by highly specialized traits and breeding targets Here, to bridge this diversity, we constructed a 33-member pangenome reference and a diversity panel across 1,984 cultivars and landraces. We leveraged these resources to explore the complex interplay among historical contingency, ongoing adaptation and previously uncharacterized structural diversity. Specifically, our analyses conclusively demonstrated multiple nested and deeply diverged structural variants in the domestication gene SHATTERING1, which distinguish the previously established multicentric origin of sorghum. We then applied landscape genomics to reveal how gene flow and secondary contact created the complex genetic mosaic in contemporary breeding networks. As proof of concept for pangenome-accelerated trait discovery, we connected biosynthetic gene cluster structural variation to phenotypic leaf concentration of the cyanogenic glucoside dhurrin. Combined, these approaches will accelerate breeding and trait discovery and provide a framework for similar applications in other crops.

agricultural genetics↗

Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here, in this study, we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/β-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases. Cofactorless oxygenases are rare in nature and natural product biosynthesis. Here the authors describe the biochemical and structural characterization of two such oxygenases catalyzing deformylation, ring cleavage and epoxidation in the biosynthesis of the enediyne natural product tiancimycin A.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Structural and functional analyses of the echinomycin resistance conferring protein Ecm16 from Streptomyces lasalocidi

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.

59 BASIC BIOLOGICAL SCIENCES↗

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system

Abstract Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli . Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus , RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

59 BASIC BIOLOGICAL SCIENCES↗

Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics

Abstract Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.

59 BASIC BIOLOGICAL SCIENCES↗

Microevolution in the pansecondary metabolome of Aspergillus flavus and its potential macroevolutionary implications for filamentous fungi

Significance Secondary metabolites (SMs) produced by fungi mediate ecological interactions, define fungal niches, and are of profound pharmacological importance to humans. Most work on SMs has focused on a small number of individuals from each species, not fully reflecting the importance of intraspecific diversity. We demonstrate that even in one of the best-studied model fungi, the carcinogen-producing Aspergillus flavus , more than 25% of SM-producing biosynthetic gene clusters (BGCs) are novel and/or show population-specific variants. These results support the finding that the organization of BGC diversity into population-specific patterns may sometimes result from ecologically important interactions and may inform evolutionary and etiological inferences of SM capacities within a species. Importantly, our work also presents a vision of sources of potential pharmaceuticals.

59 BASIC BIOLOGICAL SCIENCES↗

Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Science & Technology - Other Topics↗

Strategies for community-sourced biocuration in bioinformatics: a case study on MIBiG 4.0

Biocuration is essential to transform molecular sequence data into standardized, machine-readable resources. Such curated datasets enable comparative analysis, predictive modeling, and data integration across bioinformatics platforms. While professional biocuration is resource-intensive and usually limited to institutional settings, community-driven approaches can mobilize large-scale annotation of specialized datasets and are more resilient to disruptions in scientific funding. Here, we present a model for community-powered curation applied to the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) repository. Through a framework of workflows for metadata capture, annotation validation, and contributor coordination, the MIBiG 4.0 initiative recruited 267 scientists across 178 institutions from 33 countries, volunteering an estimated 4000 h of work. These efforts expanded the MIBiG repository by 22% and enhanced its usability in downstream molecular data analyses in comparative genomic analyses, natural product discovery, and machine learning applications. We provide strategies and actionable lessons for adopting this model, supporting the sustainability of curated bioinformatics resources central to nucleic acid research and related fields.

biocuration↗

The Architecture of Metabolism Maximizes Biosynthetic Diversity in the Largest Class of Fungi

Abstract Ecological diversity in fungi is largely defined by metabolic traits, including the ability to produce secondary or “specialized” metabolites (SMs) that mediate interactions with other organisms. Fungal SM pathways are frequently encoded in biosynthetic gene clusters (BGCs), which facilitate the identification and characterization of metabolic pathways. Variation in BGC composition reflects the diversity of their SM products. Recent studies have documented surprising diversity of BGC repertoires among isolates of the same fungal species, yet little is known about how this population-level variation is inherited across macroevolutionary timescales. Here, we applied a novel linkage-based algorithm to reveal previously unexplored dimensions of diversity in BGC composition, distribution, and repertoire across 101 species of Dothideomycetes, which are considered the most phylogenetically diverse class of fungi and known to produce many SMs. We predicted both complementary and overlapping sets of clustered genes compared with existing methods and identified novel gene pairs that associate with known secondary metabolite genes. We found that variation among sets of BGCs in individual genomes is due to nonoverlapping BGC combinations and that several BGCs have biased ecological distributions, consistent with niche-specific selection. We observed that total BGC diversity scales linearly with increasing repertoire size, suggesting that secondary metabolites have little structural redundancy in individual fungi. We project that there is substantial unsampled BGC diversity across specific families of Dothideomycetes, which will provide a roadmap for future sampling efforts. Our approach and findings lend new insight into how BGC diversity is generated and maintained across an entire fungal taxonomic class.

59 BASIC BIOLOGICAL SCIENCES↗

A glimpse into the fungal metabolomic abyss: Novel network analysis reveals relationships between exogenous compounds and their outputs

Fungal specialized metabolites are a major source of beneficial compounds that are routinely isolated, characterized, and manufactured as pharmaceuticals, agrochemical agents, and industrial chemicals. The production of these metabolites is encoded by biosynthetic gene clusters that are often silent under standard growth conditions. There are limited resources for characterizing the direct link between abiotic stimuli and metabolite production. Herein, we introduce a network analysis-based, data-driven algorithm comprising two routes to characterize the production of specialized fungal metabolites triggered by different exogenous compounds: the direct route and the auxiliary route. Both routes elucidate the influence of treatments on the production of specialized metabolites from experimental data. The direct route determines known and putative metabolites induced by treatments and provides additional insight over traditional comparison methods. The auxiliary route is specific for discovering unknown analytes, and further identification can be curated through online bioinformatic resources. We validated our algorithm by applying chitooligosaccharides and lipids at two different temperatures to the fungal pathogen Aspergillus fumigatus. After liquid chromatography–mass spectrometry quantification of significantly produced analytes, we used network centrality measures to rank the treatments’ ability to elucidate these analytes and confirmed their identity through fragmentation patterns or in silico spiking with commercially available standards. Later, we examined the transcriptional regulation of these metabolites through real-time quantitative polymerase chain reaction. Our data-driven techniques can complement existing metabolomic network analysis by providing an approach to track the influence of any exogenous stimuli on metabolite production. Our experimental-based algorithm can overcome the bottlenecks in elucidating novel fungal compounds used in drug discovery.

59 BASIC BIOLOGICAL SCIENCES↗

Holo-Omics disentangle drought response and biotic interactions among plant, endophyte and pathogen

Holo-omics provide a novel opportunity to study the interactions among fungi from different functional guilds in host plants in field conditions. We address the entangled responses of plant pathogenic and endophytic fungi associated with sorghum when droughted through the assembly of the most abundant fungal, endophyte genome from rhizospheric metagenomic sequences followed by a comparison of its metatranscriptome with the host plant metabolome and transcriptome. The rise in relative abundance of endophytic Acremonium persicinum (operational taxonomic unit 5 (OTU5)) in drought co-occurs with a rise in fungal membrane dynamics and plant metabolites, led by ethanolamine, a key phospholipid membrane component. The negative association between endophytic A. persicinum (OTU5) and plant pathogenic fungi co-occurs with a rise in expression of the endophyte's biosynthetic gene clusters coding for secondary compounds. Endophytic A. persicinum (OTU5) and plant pathogenic fungi are negatively associated under preflowering drought but not under postflowering drought, likely a consequence of variation in fungal fitness responses to changes in the availability of water and niche space caused by plant maturation over the growing season. Our findings suggest that the dynamic biotic interactions among host, beneficial and harmful microbiota in a changing environment can be disentangled by a blending of field observation, laboratory validation, holo-omics and ecological modelling.

Chen, Peilin↗

Horizontal Gene Transfer to a Defensive Symbiont with a Reduced Genome in a Multipartite Beetle Microbiome

Symbiotic mutualisms of bacteria and animals are ubiquitous in nature, running a continuum from facultative to obligate from the perspectives of both partners. The loss of functions required for living independently but not within a host gives rise to reduced genomes in many symbionts. Although the phenomenon of genome reduction can be explained by existing evolutionary models, the initiation of the process is not well understood. Here, we describe the microbiome associated with the eggs of the beetle Lagria villosa, consisting of multiple bacterial symbionts related to Burkholderia gladioli, including a reduced-genome symbiont thought to be the exclusive producer of the defensive compound lagriamide. We show that the putative lagriamide-producing symbiont is the only member of the microbiome undergoing genome reduction and that it has already lost the majority of its primary metabolism and DNA repair pathways. The key step preceding genome reduction in the symbiont was likely the horizontal acquisition of the putative lagriamide lga biosynthetic gene cluster. Unexpectedly, we uncovered evidence of additional horizontal transfers to the symbiont’s genome while genome reduction was occurring and despite a current lack of genes needed for homologous recombination. These gene gains may have given the genome-reduced symbiont a selective advantage in the microbiome, especially given the maintenance of the large lga gene cluster despite ongoing genome reduction.

59 BASIC BIOLOGICAL SCIENCES↗

Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity

ABSTRACT Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) potentially involved in fungal competition and communication. However, the functionality of these BGCs is not known. This study employed co-culture techniques to activate BGC expression and then used metabolomics to investigate the diversity of metabolic products produced by three Suillus species ( Suillus hirtellus EM16, Suillus decipiens EM49, and Suillus cothurnatus VC1858), core members of the pine microbiome. After 28 days of growth on solid media, liquid chromatography–tandem mass spectrometry identified a diverse range of extracellular metabolites (exometabolites) along the interaction zone between Suillus co-cultures. Prenol lipids were among the most abundant chemical classes. Out of the 62 unique terpene BGCs predicted by genome mining, 41 putative prenol lipids (includes 37 putative terpenes) were identified across the three Suillus species using metabolomics. Notably, some terpenes were significantly more abundant in co-culture conditions. For example, we identified a metabolite matching to isomers isopimaric acid, sandaracopimaric acid, and abietic acid, which can be found in pine resin and play important roles in host defense mechanisms and Suillus spore germination. This research highlights the importance of combining genomics and metabolomics to advance our understanding of the chemical diversity underpinning fungal signaling and communication. IMPORTANCE Using a combination of genomics and metabolomics, this study’s findings offer new insights into the chemical diversity of Suillus fungi, which serve a critical role in forest ecosystems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Multifactorial genetic control and magnesium levels govern the production of a Streptomyces antibiotic with unusual cell density dependence

Streptomyces bacteria are renowned both for their antibiotic production capabilities and for their cryptic metabolic potential. Their metabolic repertoire is subject to stringent genetic control, with many of the associated biosynthetic gene clusters being repressed by the conserved nucleoid-associated protein Lsr2. In an effort to stimulate new antibiotic production in wild Streptomyces isolates, we leveraged the activity of an Lsr2 knockdown construct and successfully enhanced antibiotic production in the wild Streptomyces isolate WAC07094. We determined that this new activity stemmed from increased levels of the angucycline-like family member saquayamycin. Saquayamycin has both antibiotic and anti-cancer activities, and intriguingly, beyond Lsr2-mediated repression, we found saquayamycin production was also suppressed at high density on solid or in liquid growth media; its levels were greatest in low-density cultures. This density-dependent control was exerted at the level of the cluster-situated regulatory gene sqnR and was mediated in part through the activity of the PhoRP two-component regulatory system, where deleting phoRP led to both constitutive antibiotic production and sqnR expression. This suggests that PhoP functions to repress the expression of sqnR at high cell density. We further discovered that magnesium supplementation could alleviate this density dependence, although its action was independent of PhoP. Finally, we revealed that the nitrogen-responsive regulators GlnR and AfsQ1 could relieve the repression exerted by Lsr2 and PhoP. Intriguingly, we found that this low density-dependent production of saquayamycin was not unique to WAC07094; saquayamycin production by another wild isolate also exhibited low-density activation, suggesting that this spatial control may serve an important ecological function in their native environments.

59 BASIC BIOLOGICAL SCIENCES↗

Salt supplementation-induced metabolic reprogramming in Streptomyces coelicolor

Members of the genus Streptomyces are major producers of a wide variety of secondary metabolites that serve as bioactive compounds. Many secondary metabolites are produced in response to environmental signals such as biotic and abiotic stresses. In this study, we identified salt supplementation as one of the stimuli activating secondary metabolism in the model Streptomyces species, Streptomyces coelicolor. Comparative metabolomics revealed overproduction of several known secondary metabolites, most notably undecylprodigiosin and coelimycin P1, in addition to their biosynthetic intermediates and derivatives, as well as many unknown metabolites. Transcriptomic analysis revealed activation of diverse biological processes including cation uptake, compatible solute production, and the phosphate limitation stress response through conserved and species-specific mechanisms, presumably to overcome the increased salinity. This response leads to activation of a variety of regulatory and metabolic pathways required for production of secondary metabolites including activation of conserved metabolic pathways for energy and substrate supply and species-specific secondary metabolite biosynthetic gene clusters. Furthermore, several promoter sequences contributing to upregulation of secondary metabolism induced by salt supplementation were identified. Overall, our data show how S. coelicolor copes with the increased salinity and tailors the cellular metabolism toward secondary metabolism in a conserved and species-specific manner.

Otani, Hiroshi [USDOE Joint Genome Institute (JGI)↗

Genome-based analysis for the bioactive potential of Streptomyces yeochonensis CN732, an acidophilic filamentous soil actinobacterium

Background: Acidophilic members of the genus Streptomyces can be a good source for novel secondary metabolites and degradative enzymes of biopolymers. In this study, a genome-based approach on Streptomyces yeochonensis CN732, a representative neutrotolerant acidophilic streptomycete, was employed to examine the biosynthetic as well as enzymatic potential, and also presence of any genetic tools for adaptation in acidic environment. Results: A high quality draft genome (7.8Mb) of S. yeochonensis CN732 was obtained with a G+C content of 73.53% and 6549 protein coding genes. The in silico analysis predicted presence of multiple biosynthetic gene clusters (BGCs), which showed similarity with those for antimicrobial, anticancer or antiparasitic compounds. However, the low levels of similarity with known BGCs for most cases suggested novelty of the metabolites from those predicted gene clusters. The production of various novel metabolites was also confirmed from the combined high performance liquid chromatography-mass spectrometry analysis. Through comparative genome analysis with related Streptomyces species, genes specific to strain CN732 and also those specific to neutrotolerant acidophilic species could be identified, which showed that genes for metabolism in diverse environment were enriched among acidophilic species. In addition, the presence of strain specific genes for carbohydrate active enzymes (CAZyme) along with many other singletons indicated uniqueness of the genetic makeup of strain CN732. The presence of cysteine transpeptidases (sortases) among the BGCs was also observed from this study, which implies their putative roles in the biosynthesis of secondary metabolites. Conclusions: This study highlights the bioactive potential of strain CN732, an acidophilic streptomycete with regard to secondary metabolite production and biodegradation potential using genomics based approach. The comparative genome analysis revealed genes specific to CN732 and also those among acidophilic species, which could give some insights into the adaptation of microbial life in acidic environment.

59 BASIC BIOLOGICAL SCIENCES↗

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation

ABSTRACT Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales) , respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 ( Hypocreales , Ascomycota ) was isolated from the sponge Stelletta normani . A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica . Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica . The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics.

59 BASIC BIOLOGICAL SCIENCES↗

Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T . atroviride exudates. In contrast, P . simiae and P . putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δ tmk 3) gene of T . atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T . atroviride , bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δ tmk3 mutant. Unexpectedly, exudates from wild-type T . atroviride and the Δ tmk 3 mutant rescued purine auxotrophic mutants of H . seropedicae , K . michiganensis and P . simiae . Metabolomic analysis on exudates from wild-type T . atroviride and the Δ tmk 3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma -metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.

59 BASIC BIOLOGICAL SCIENCES↗