Gene Cluster Research Articles

Increasing prevalence of bacteriocin carriage in a 6-year hospital cohort of E. faecium.

Vancomycin-resistant enterococci (VRE) are important pathogens in hospitalized patients; however, the factors involved in VRE colonization of hospitalized patients are not well characterized. Bacteriocins provide a competitive advantage to enterococci in experimental models of colonization, but little is known about bacteriocin content in samples derived from humans and even less is known about their dynamics in the clinical setting. To identify bacteriocins which may be relevant in the transmission of VRE, we present a systematic analysis of bacteriocin content in the genomes of 2,248 patient-derived E. faecium isolates collected over a 6-year period from a single hospital system. We used computational methods to broadly search for bacteriocin structural genes and a functional assay to look for phenotypes consistent with bacteriocin expression. We identified homology to 15 different bacteriocins, with 2 having a high presence in this clinical cohort. Bacteriocin 43 (bac43) was found in a total of 58% of isolates, increasing from 8% to 91% presence over the 6-year collection period. There was little genetic variation in the bac43 structural or immunity genes across isolates. The enterocin A structural gene was found in 98% of isolates, but only 0.3% of isolates had an intact enterocin A gene cluster and displayed a bacteriocin-producing phenotype. This study presents a wide survey of bacteriocins from hospital isolates and identified bac43 as highly conserved, increasing in prevalence, and phenotypically functional. This makes bac43 an interesting target for future investigation for a potential role in E. faecium transmission.IMPORTANCEWhile enterococci are a normal inhabitant of the human gut, vancomycin-resistant E. faecalis and E. faecium are urgent public health threats responsible for hospital-associated infections. Bacteriocins are ribosomally synthesized antimicrobial proteins and are commonly used by bacteria to provide a competitive advantage in polymicrobial environments. Bacteriocins have the potential to be used by E. faecium to invade and dominate the human gut leading to a greater propensity for transmission. In this work, we explore bacteriocin content in a defined clinically derived population of E. faecium using both genetic and phenotypic studies. We show that one highly active bacteriocin is increasing in prevalence over time and demonstrates great potential relevance to E. faecium transmission.

Genetic variation within the arctic-alpine Calamagrostis stricta (Poaceae) species complex in Europe

The Calamagrostis stricta (Poaceae) species complex is a circumpolar, boreo-arctic and montane taxon that includes numerous subspecies and varieties. The recent discovery of Calamagrostis lonana in the Alps calls for a thorough assessment of relationships within C. stricta. The main aim of our study was to elucidate the phylogenetic position, genetic structure and ploidy level of C. lonana, as compared to the other members of the C. stricta species complex from Central Europe to the Arctic. Fifteen populations of the C. stricta species complex were sampled across Central and Northern Europe, and their ploidy level was estimated using flow cytometry. Genetic variation was characterized using double digest RAD sequencing reads (ddRAD-seq) on a total of 115 individuals genotyped at 1157 single-nucleotide polymorphisms. Based on flow cytometric measurements, tetraploidy was observed in Arctic populations from Northern Europe and C. lonana in the Alps, in contrast to other populations exhibiting higher ploidy levels. Calamagrostis lonana was genetically closely related to the arctic C. stricta subsp. groenlandica, while C. stricta subsp. stricta formed a second genetic cluster across Central Europe. A third, very distinct genetic cluster was observed in the northern Svalbard archipelago. Despite lacking evidence of sexual reproduction, substantially more genetic diversity than expected under asexual reproduction was detected within populations in C. lonana and other taxa. The distribution and genetic structure of the C. stricta species complex has been shaped by major post-glacial environmental changes having affected cold regions and specifically highlights C. lonana as a valuable relict taxon for the Alps.

Enzymatic Synthesis of a Polyketide/Nonribosomal Peptide Hybrid Antibiotic, Salivabactin.

Salivabactin is a newly identified polyketide/nonribosomal peptide (PK/NRP) from a human oral probiotic, possessing a unique chemical structure and outstanding antibiotic activities. Although the biosynthetic gene cluster for salivabactin is known, the enzymatic logic that governs the synthesis of salivabactin has not yet been fully studied. In this work, we dissected the biosynthetic pathway for salivabactin using biochemical analysis. We successfully reconstituted the enzymatic synthesis of salivabactin in vitro, identified the minimal set of enzymes required for its biosynthesis, and revealed an unusual thioesterase domain involved in terminal olefin formation.

A Comprehensive Genome Mining Analysis of Biosynthetic Gene Clusters in Pseudomonas sp. SXM-1

Very resistant pathogenic microorganisms have been reported to current antibiotics in the last decade. Therefore, there is a great need to understand not only resistance metabolism but also secondary metabolites of pathogenic microorganisms. Genome mining tools have so far been improved to understand secondary metabolites from biosynthetic gene clusters. Microorganisms predicted for their genomes and secondary metabolites using these tools are widely employed in pharmaceutical and industrial studies. Pseudomonas spp. are widely used in recombinant DNA technology to produce commercial products. Bioinformatics-based in silico tools significantly contribute to the discovery of new bioactive compounds for pharmacy and medicine. This study aims to conduct a comprehensive gene cluster analysis of the Pseudomonas sp. SXM-1 strain isolated from the coastal seawater of Xiamen Bay using antiSMASH (7.0.1). The accession number of Pseudomonas sp. SXM-1 strain was retrieved from NCBI. 14 regions were found, including non-ribosomal peptides metallophores (NRP-metallophore), nonribosomal peptide-synthetase (NRPS), NRPS-like, ribosomally synthesized and post-translationally modified peptide-like (RiPP-like), betalactone, nonribosomal peptide-synthetase (NRPS), ectoine and N-acetylglutaminylglutamine amide (NAGGN). Analysis of all 14 regions revealed secondary metabolites with potential applications in diverse fields. Microbiologists are strongly advised to conduct wet-lab experiments to validate the secondary metabolites discussed in this study.

Phylogeography, taxonomy, and conservation of the endangered brown howler monkey, Alouatta guariba (Primates, Atelidae), of the Atlantic Forest

The brown howler, Alouatta guariba, endemic to the Atlantic Forest of Brazil and Argentina, is threatened by habitat loss and fragmentation, hunting, and its susceptibility to yellow fever. Two subspecies have been recognized, but their names, validity, and geographic ranges have been controversial. We obtained samples covering the species' entire distribution in Brazil and Argentina to clarify these issues by investigating their genetic diversity and structure and assessing their evolutionary history. We analyzed, for the first time, a set of ten microsatellite markers (N = 153), plus mitochondrial DNA (mtDNA) segments of the control region (N = 207) and cytochrome b gene (N = 116). The microsatellite data support two to three genetic clusters with biological significance. The southern populations (Argentina, Santa Catarina, and Rio Grande do Sul) presented a homogeneous genetic component, and populations from São Paulo (SP) to the north presented another component, although most presented ∼20% of the southern component. With K = 3, SP emerged as a third component while sharing some ancestry with Rio de Janeiro and Argentina. The mtDNA phylogenies revealed three main clades that diverged almost simultaneously around 250 thousand years ago (kya). Clades A and B are from central SP to the north and east, while clade C is from SP to the south and southwest. Samples from SP presented haplotypes in all three clades, sometimes in the same population. The demographic history of the species estimated with the Bayesian skyline plot of the mtDNA showed a strong expansion ∼40–20 kya and a strong reduction over the last ∼4–2 kya. Although the genetic clusters identified here deserve appropriate management strategies as conservation units, the absence of (i) concordance between the mtDNA and microsatellite data, (ii) reciprocal monophyly in the mtDNA, and (iii) clear-cut non-genetic diagnostic characters advises against considering them as different taxonomic entities. None of the previous taxonomic proposals were corroborated by our data. Our results elucidate the taxonomy of the Atlantic Forest brown howler, indicating it should be considered a monotypic species, A. guariba. We also clarify the evolutionary history of the species regarding its intraspecific genetic diversity, which is crucial information for its conservation and population management.

Cell-free supernatant of Lactobacillus gasseri 1A-TV shows a promising activity to eradicate carbapenem-resistant Klebsiella pneumoniae colonization.

The use of beneficial bacteria like Lactobacillus spp. is a potential innovative approach to fight antibiotic-resistant pathogens. Klebsiella pneumoniae is one of the most concerning multi drug-resistant (MDR) pathogens, and its ability to colonize the human gut is considered to be the main reason for recurrent infections in critically ill patients. In this study, Lactobacillus gasseri 1A-TV, already described for its probiotic activity, was characterized at the genomic level. Moreover, its cell-free supernatant (CFS) was tested for antimicrobial activity against extended-spectrum β-lactamase (ESBL)- and carbapenemase (KPC)-producing K. pneumoniae clinical isolates. Whole-genome sequencing showed that the L. gasseri 1A-TV genome was of 2,018,898 bp in size with 34.9% GC content, containing 1,937 putative protein coding sequences, 55 tRNA, and 4 rRNA detected by RAST and classified in 20 functional groups by Cluster of Orthologous Genes (COG). BAGEL4 (BActeriocin GEnome minimal tooL) and the antiSMASH 7.0 pipeline identified two bacteriocin biosynthetic gene clusters (BBGCs), namely, BBGC1 that comprises two class IIc bacteriocins including gassericin A-like bacteriocin, and BBGC2 carrying the class III bacteriocin helveticin J. Strikingly, 1A-TV CFS inhibited the growth of all K. pneumoniae isolates only after 8 h of incubation, showing a bactericidal effect at 24 h and interfering, even at lower concentrations, with the biofilm production of biofilm-producer strains independently of a bactericidal effect. NMR analysis of CFS identified and quantified several metabolites involved in carbohydrate metabolism and amino acid metabolism, and organic acids like ethanol, lactate, acetate, and succinate. Finally, in vitro assays of 1A-TV showed significant co-aggregation effects against carbapenem-resistant K. pneumoniae, namely, strains 1, 2, 3, and 7. Our findings highlight the antimicrobial activity of 1A-TV as a probiotic candidate or its CFS as a natural bioproduct active against MDR K. pneumoniae strains, underlining the importance of novel therapeutic strategies for prevention and control of ESBL- and carbapenemase-producing K. pneumoniae colonization.

Long-read sequencing sheds light on key bacteria contributing to deadwood decomposition processes

BackgroundDeadwood decomposition is an essential ecological process in forest ecosystems, playing a key role in nutrient cycling and carbon sequestration by enriching soils with organic matter. This process is driven by diverse microbial communities encompassing specialized functions in breaking down organic matter, but the specific roles of individual microorganisms in this process are still not fully understood.ResultsHere, we characterized the deadwood microbiome in a natural mixed temperate forest in Central Europe using PacBio HiFi long-read sequencing and a genome-resolved transcriptomics approach in order to uncover key microbial contributors to wood decomposition. We obtained high quality assemblies, which allowed attribution of complex microbial functions such as nitrogen fixation to individual microbial taxa and enabled the recovery of metagenome-assembled genomes (MAGs) from both abundant and rare deadwood bacteria. We successfully assembled 69 MAGs (including 14 high-quality and 7 single-contig genomes) from 4 samples, representing most of the abundant bacterial phyla in deadwood. The MAGs exhibited a rich diversity of carbohydrate-active enzymes (CAZymes), with Myxococcota encoding the highest number of CAZymes and the full complement of enzymes required for cellulose decomposition. For the first time we observed active nitrogen fixation by Steroidobacteraceae, as well as hemicellulose degradation and chitin recycling by Patescibacteria. Furthermore, PacBio HiFi sequencing identified over 1000 biosynthetic gene clusters, highlighting a vast potential for secondary metabolite production in deadwood, particularly in Pseudomonadota and Myxococcota.ConclusionsPacBio HiFi long-read sequencing offers comprehensive insights into deadwood decomposition processes by advancing the identification of functional features involving multiple genes. It represents a robust tool for unraveling novel microbial genomes in complex ecosystems and allows the identification of key microorganisms contributing to deadwood decomposition.

Distribution of C30 carotenoid biosynthesis genes suggests habitat adaptation function in insect-adapted and nomadic Lactobacillaceae

Carotenoids are membrane-bound pigments that are essential for photosynthesizing plants and algae, widely applied in food, feed and cosmetics due to their antioxidant and anti-inflammatory properties. The production of carotenoids, particularly C30 forms, has been documented in some non-photosynthetic prokaryotes. However, their function, distribution and ecology beyond photosynthesizing organisms remains understudied. In this study, we performed an eco-evolutionary analysis of terpenoid biosynthetic gene clusters in the Lactobacillaceae family, screening 4203 dereplicated genomes for terpenoid biosynthesis genes, and detected crtMN genes in 28/361 (7.7%) species across 14/34 (41.2%) genera. These genes encode key enzymes for producing the C30 carotenoid 4,4′-diaponeurosporene. crtMN genes appeared to be convergently gained within Fructilactobacillus and horizontally transferred across species and genera, including Lactiplantibacillus to Levilactobacillus. The phenotype was confirmed in 87% of the predicted crtMN gene carriers (27/31). Nomadic and insect-adapted species, particularly those isolated from vegetable fermentations, e.g., Lactiplantibacillus, and floral habitats, e.g., Fructilactobacillus, contained crtMN genes, while vertebrate-associated species, including vaginal associated species, lacked this trait. This habitat association aligned with the observations that C30 carotenoid-producing strains were more resistant to UV-stress. In summary, C30 carotenoid biosynthesis plays a role in habitat adaptation and is scattered across Lactobacillaceae in line with this habitat adaptation.

The Most Common Types of 3.7 Kilobase Deletion in the Iranian Population

The 3.7 kb deletion is the most common known mutation in the α-globin gene cluster worldwide. The aim of this study is to investigate the most common types of 3.7 kb deletions in the Iranian population and, on the other hand, to compare the extent of deletion of the different reported types. In this study, 50 Iranian α-thalassemia carriers in whom the 3.7 kb deletion had been previously identified by multiplex gap PCR, were further investigated by MLPA. A map of the region where the 3.7 kb deletion occurs was also created and the extents of the reported types were compared Approximately 90% of chromosomes with 3.7 kb deletion in this study had MLPA type D and 10% had MLPA type F. This study showed that subtype I of the 3.7 kb deletion reported by Higgs and his coworkers can be classified into at least 5 MLPA types. The results of this study can be used to complete the information on the distribution of the 3.7 kb deletion subtypes in different populations. Investigation of further populations using higher resolution methods may lead to more information being obtained in this field.

Patterns of siderophore production and utilization at Station ALOHA from the surface to mesopelagic waters

AbstractThe North Pacific subtropical gyre is a globally important contributor to carbon uptake despite being a persistently oligotrophic ecosystem. Supply of the micronutrient iron to the upper ocean varies seasonally to episodically, and when coupled with rapid biological consumption, results in low iron concentrations. In this study, we examined changes in iron uptake rates, along with siderophore concentrations and biosynthesis potential at Station ALOHA across time (2013–2016) and depth (surface to 500 m) to observe changes in iron acquisition and internal cycling by the microbial community. The genetic potential for siderophore biosynthesis was widespread throughout the upper water column, and biosynthetic gene clusters peaked in spring and summer along with siderophore concentrations, suggesting changes in nutrient delivery, primary production, and carbon export seasonally impact iron acquisition. Dissolved iron turnover times, calculated from iron‐amended experiments in surface (15 m) and mesopelagic (300 m) waters, ranged from 9 to 252 d. The shortest average turnover times at both depths were associated with inorganic iron additions (14 9 d) and the longest with iron bound to strong siderophores (148 225 d). Uptake rates of siderophore‐bound iron were faster in mesopelagic waters than in the surface, leading to high Fe : C uptake ratios of heterotrophic bacteria in the upper mesopelagic. The rapid cycling and high demand for iron at 300 m suggest differences in microbial metabolism and iron acquisition in the mesopelagic compared to surface waters. Together, changes in siderophore production and consumption over the seasonal cycle suggest organic carbon availability impacts iron cycling at Station ALOHA.

Molecular Decoration and Unconventional Double Bond Migration in Irumamycin Biosynthesis

Irumamycin (Iru) is a complex polyketide with pronounced antifungal activity produced by a type I polyketide (PKS) synthase. Iru features a unique hemiketal ring and an epoxide group, making its biosynthesis and the structural diversity of related compounds particularly intriguing. In this study, we performed a detailed analysis of the iru biosynthetic gene cluster (BGC) to uncover the mechanisms underlying Iru formation. We examined the iru PKS, including the domain architecture of individual modules and the overall spatial structure of the PKS, and uncovered discrepancies in substrate specificity and iterative chain elongation. Two potential pathways for the formation of the hemiketal ring, involving either an olefin shift or electrocyclization, were proposed and assessed using 18O-labeling experiments and reaction activation energy calculations. Based on our findings, the hemiketal ring is likely formed by PKS-assisted double bond migration and TE domain-mediated cyclization. Furthermore, putative tailoring enzymes mediating epoxide formation specific to Iru were identified. The revealed Iru biosynthetic machinery provides insight into the complex enzymatic processes involved in Iru production, including macrocycle sculpting and decoration. These mechanistic details open new avenues for a targeted architecture of novel macrolide analogs through synthetic biology and biosynthetic engineering.

Environmental fungi target thiol homeostasis to compete with Mycobacterium tuberculosis.

Mycobacterial species in nature are found in abundance in sphagnum peat bogs where they compete for nutrients with a variety of microorganisms including fungi. We screened a collection of fungi isolated from sphagnum bogs by co-culture with Mycobacterium tuberculosis (Mtb) to look for inducible expression of antitubercular agents and identified 5 fungi that produced cidal antitubercular agents upon exposure to live Mtb. Whole genome sequencing of these fungi followed by fungal RNAseq after Mtb exposure allowed us to identify biosynthetic gene clusters induced by co-culture. Three of these fungi induced expression of patulin, one induced citrinin expression and one induced the production of nidulalin A. The biosynthetic gene clusters for patulin and citrinin have been previously described but the genes involved in nidulalin A production have not been described before. All 3 of these potent electrophiles react with thiols and treatment of Mtb cells with these agents followed by Mtb RNAseq showed that these natural products all induce profound thiol stress suggesting a rapid depletion of mycothiol. The induction of thiol-reactive mycotoxins through 3 different systems in response to exposure to Mtb suggests that fungi have identified this as a highly vulnerable target in a similar microenvironment to that of the caseous human lesion.

Integrating genetic and morphological data to assess species delimitation of two Japanese large abalones: Haliotis discus discus and H. madaka

Abstract Two large abalone species prevalent in the Japanese archipelago, Haliotis discus discus and H. madaka, are considered to have different morphological characteristics, and previous population genomics analyses have suggested that they have distinct genomic features. However, integrated analyses comparing the genetic and morphological data from these species are lacking; thus, it remains unclear whether these two species are distinct biological species. In this study, we performed integrated genetic and morphological analyses on individuals of the two species captured in Tokushima Prefecture, Japan. Genetic analysis based on 118 species-diagnostic single-nucleotide polymorphism (SNP) loci showed that H. discus discus and H. madaka were clearly assigned to distinct genetic clusters corresponding to the two species, with the exception of one H. madaka individual showing evidence of genomic introgression from H. discus discus. Multiple statistical analyses based on quantitative characteristics of shell shape and foot muscle color suggested that this H. madaka individual with genomic introgression from H. discus discus had H. discus discus-like quantitative characteristics consistently. In summary, this study suggests that H. discus discus and H. madaka are distinct biological species reproductively isolated in natural environments, but genomic introgression possibly blurs the species boundary between H. discus discus and H. madaka.

Genome Mining of the Marine-Derived Fungus Trichoderma erinaceum F1-1 Unearths Bergamotene-Type Sesquiterpenoids.

Terpenoids are a vast group of natural products known for their remarkable biological properties and structural diversity. UbiA terpene synthases are increasingly recognized for producing various terpenoids. In this study, we identified a biosynthetic gene cluster (bgt) encoding a UbiA terpene synthase BgtA in the genome of the marine-derived fungus Trichoderma erinaceum F1-1. The gene bgtA was validated to encode the biosynthesis of (-)-α-trans-bergamotene (1). Heterologous expression of the bgt gene cluster in the characterized host Aspergillus nidulans LO8030 activated the biosynthetic pathway, leading to the isolation of eight previously undocumented bergamotene-derived sesquiterpenoids (2-9). Their structures, including the absolute configurations, were elucidated by a combination of spectroscopic analysis, ECD spectra, chemical hydrolysis, single-crystal X-ray diffraction, and biosynthetic considerations. We further demonstrated that the production of these structurally intricate sesquiterpenoids in heterologous expression is attributable to the concerted action of the UbiA terpene synthase BgtA, the cytochrome P450 BgtC, and endogenous enzymes. This study underscores the immense biosynthetic potential of fungal UbiA terpene synthase gene clusters and shows genome mining is a promising strategy for the discovery of novel terpenoids from fungi.

Genomic Characterization of Bacillus sp. THPS1: A Hot Spring-Derived Species with Functional Features and Biotechnological Potential

Bacillus sp. THPS1 is a novel strain isolated from a high-temperature hot spring in Thailand, exhibiting distinctive genomic features that enable adaptation to an extreme environment. This study aimed to characterize the genomic and functional attributes of Bacillus sp. THPS1 to understand its adaptation strategies and evaluate its potential for biotechnological applications. The draft genome is 5.38 Mbp with a GC content of 35.67%, encoding 5606 genes, including those linked to stress response and sporulation, which are essential for survival in high-temperature conditions. Phylogenetic analysis and average nucleotide identity (ANI) values confirmed its classification as a distinct species within the Bacillus genus. Pangenome analysis involving 19 others closely related thermophilic Bacillus species identified 1888 singleton genes associated with heat resistance, sporulation, and specialized metabolism, suggesting adaptation to nutrient-deficient, high-temperature environments. Genomic analysis revealed 12 biosynthetic gene clusters (BGCs), including those for polyketides and non-ribosomal peptides, highlighting its potential for synthesizing secondary metabolites that may facilitate its adaptation. Additionally, the presence of three Siphoviridae phage regions and 96 mobile genetic elements (MGEs) suggests significant genomic plasticity, whereas the existence of five CRISPR arrays implies an advanced defense mechanism against phage infections, contributing to genomic stability. The distinctive genomic features and functional capacities of Bacillus sp. THPS1 make it a promising candidate for biotechnological applications, particularly in the production of heat-stable enzymes and the development of resilient bioformulations.

Enhancing Solanine Production and Antifungal Activity by Streptomyces abikoensis XH-17 through Combined Ribosome Engineering and Post-Translational Modification

Solanine, a strong poison alkaloid typically produced by staple food crops, such as sprouted potatoes, poses a potential risk to food safety, therefore, controlling solanine levels is one of the important targets for human health. Streptomyces species are ubiquitous across diverse environments and are acclaimed for their extensive production of bioactive compounds, including antibiotics, anticancer agents, and immunosuppressants. Despite their potential, many biosynthetic gene clusters (BGCs) in Streptomyces remain silent under standard laboratory conditions. The present study introduces a combination strategy that integrates ribosome engineering and post-translational modification (PTM) to activate the silent BGCs in Streptomyces XH-17, which was identified as S. abikoensis by whole gene cluster sequencing and phylogenetic analysis. The antimicrobial activity of the engineered strain increased by 3.74-fold, 1.40-fold, and 4.08-fold against Alternaria alternata, Fusarium oxysporum, and F. proliferatum, respectively. The main compounds responsible for antifungal activity were identified as α-chaconine and α-solanine by high-resolution mass spectrometry (HRMS), and their BGCs were silent in the wild type (WT). Compared to WT, the two compounds’ production increased by 5.78-fold and 4.68-fold in engineered strain XH-17rpoB+Svp, respectively, and the biosynthetic pathways of α-chaconine and α-solanine were inferred by bioinformatics analysis. This is the first to identify solanine and presumed its biosynthetic pathway from Streptomyces. Our research provides new insights into the control of solanine to protect food safety from Streptomyces contamination. The results from the current investigation provide an effective pipeline to activate silent gene clusters in Streptomyces and offer new insight into discovering new bioactive compounds within this genus.

Could the transcription factor AtnN coordinating the aspercryptin secondary metabolite gene cluster in Aspergillus nidulans be a global regulator?

Products of dormant secondary metabolite gene clusters of fungal genomes can be exploited for medical purposes as bioactive agents. These clusters can be switched on under oxidative stress and may endow fungi with a versatile chemical armory in a competitive niche. In Aspergillus nidulans, the aspercryptin gene cluster, including the synthase [atnA (AN7884)] and its transcription factor (atnN), was activated under menadione sodium bisulfite (MSB) treatment. In this study, we generated and phenotypically examined the gene deletion and overexpression mutants of atnN and studied the secondary metabolite production of the mutants. Overexpression of atnN significantly reduced the colony growth of surface cultures compared to the control. The ΔatnN gene deletion strain showed higher sensitivity to tert-butyl hydroperoxide (tBOOH), while the atnNOE strain was more resistant to MSB, Congo Red, and sorbitol. Interestingly, deletion of atnN decreased cleistothecia formation of A. nidulans. Manipulation of atnN affected the synthesis of several secondary metabolites, for example, the siderophore production of A. nidulans. The extracellular triacetylfusarinine C (TAFC) production decreased, while the intracellular ferricrocin (FC) concentration of the cultures increased in the atnNOE mutant cultivating A. nidulans in a complex medium containing 1% mycological peptone and 2% maltose. In Czapek-Dox Broth medium, increased asperthecin production was observed in the ΔatnN mutant. The mycotoxin sterigmatocystin synthesis elevated in the ΔatnN mutant, while reduced in the atnNOE mutant on minimal medium. Our study supports previous observations that secondary metabolite production is coordinated in a complex way, and the linkage of stress response, sexual reproduction, and secondary metabolite production can be governed by several transcription factors.

Oxygen-dependent alternative mRNA splicing and a cone-specific motor protein revealed by single-cell RNA sequencing in hypoxic retinas

Restricted oxygen supply in the aging eye may lead to hypoxic conditions in the outer retina and contribute not only to physiological aging but also to nonhereditary degenerative retinal diseases. To understand the hypoxic response of specific retinal cell types, we performed single-cell RNA sequencing of retinas isolated from mice exposed to hypoxia. Significantly upregulated expression of marker genes in hypoxic clusters confirmed a general transcriptional response to hypoxia. By focusing on the hypoxic response in photoreceptors, we identified and confirmed a kinesin motor protein (Kif4) that was specifically and strongly induced in hypoxic cones. In contrast, RNA-binding proteins Rbm3 and Cirbp were differentially expressed across clusters but demonstrated isoform switching in hypoxia. The resulting short variants of these gene transcripts are connected to epitranscriptomic regulation, a notion supported by the differential expression of writers, readers and erasers of m6A RNA methylations in the hypoxic retina. Our data indicate that retinal cells adapt to hypoxic conditions by adjusting their transcriptome at various levels including gene expression, alternative splicing and the epitranscriptome. Adaptational processes may be cell-type specific as exemplified by the cone-specific upregulation of Kif4 or general like alternative splicing of RNA binding proteins.

Lrp Family Regulator SCAB_Lrp2 Responds to the Precursor Tryptophan and Represses the Thaxtomin Biosynthesis in Streptomyces scabies

ABSTRACTStreptomyces scabies is a well‐researched plant pathogen belonging to the genus Streptomyces. Its virulence is linked to the production of the secondary metabolite thaxtomin A, which is tightly regulated at the transcriptional level. The leucine‐responsive regulatory protein (Lrp) family is conserved in prokaryotes and is involved in various crucial biological processes. However, the regulatory interaction between Lrp protein and pathogenic Streptomyces species remains poorly understood. This study aims to explore the role of SCAB_Lrp2 in regulating thaxtomin biosynthesis and pathogenicity, and to analyse the shared and unique features of Lrp homologues in S. scabies. We observed that SCAB_Lrp2 (SCAB_75421) showed significant homology with SCAB_Lrp, a recognised activator of thaxtomin A production in S. scabies. Our results revealed a regulatory interaction between SCAB_Lrp2 and SCAB_Lrp in terms of their targets, although SCAB_Lrp2 does not respond to the amino acid‐effectors of SCAB_Lrp. In contrast to SCAB_Lrp, deletion of SCAB_Lrp2 resulted in a notable increase in thaxtomin A production with the emergence of a hypervirulent phenotype in S. scabies. Further analysis revealed that SCAB_Lrp2 represses the transcription of the thaxtomin biosynthetic gene cluster by directly regulating the cluster‐situated regulator (CSR) gene txtR. Moreover, the precursor of thaxtomin, tryptophan, acts as an effector of SCAB_Lrp2, strengthening the repressive effect on thaxtomin biosynthesis through txtR. These findings provide new insights into the functional conservation and diversity of Lrp homologues involved in the biosynthesis of thaxtomin phytotoxins in pathogenic Streptomyces species.

Genomic analysis and overexpressing the acetyl-CoA carboxylase of oleaginous Saccharomyces cerevisiae CU-TPD4 for enhanced lipid production from agricultural residues

Single-cell oil produced using oleaginous yeasts is emerging as a promising source for biofuels and oleochemicals. Saccharomyces cerevisiae is the model yeast and is widely utilized in several industrial applications. This study presents a genome analysis of the oleaginous S. cerevisiae strain CU-TPD4, which has been reported to accumulate a high amount of lipids. The analysis revealed a genome size of 17.5 million bases and 7,619 predicted genes were identified. To understand the differences between oleaginous and non-oleaginous S. cerevisiae, a comparative analysis was conducted. The results identified 5,169 shared gene clusters, 36 unique clusters in S. cerevisiae S288C and 22 unique clusters in S. cerevisiae CU-TPD4. Notably, mutations in proteins associated in lipid biosynthesis that affect phosphorylation, such as ACC1, ALE1, and FAA4, were observed, influencing lipid accumulation. Additionally, overexpression of the ACC1 gene under different promoters revealed an approximately 16% improved lipid production under the ENO2 promoter compared to the wild-type strain. Subsequently, lipid production process using rice straw hydrolysate was evaluated. The strain produced a highest total lipid of 1.60 g/L, corresponding to a lipid content of 34.12%. Interestingly, the FA composition of the lipids obtained from the rice straw hydrolysate was comprised of more than 75% monounsaturated FAs. This study provides the first draft genome of oleaginous S. cerevisiae CU-TPD4 and insights into genetic improvements to facilitate lipid production.