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BackgroundThe fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus.ResultsWe have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli.ConclusionsMany aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

BackgroundFructose is an abundant sugar in plants as it is a breakdown product of both major sucrose-cleaving enzymes. To enter metabolism, fructose is phosphorylated by a fructokinase (FRK). Known FRKs are members of a diverse family of carbohydrate/purine kinases known as the phosphofructokinase B (pfkB) family. The complete complement of active fructokinases has not been reported for any plant species.ResultsProtein sequence analysis of the 22 Arabidopsis thaliana pfkB members identified eight highly related predicted proteins, including one with previously demonstrated FRK activity. For one, At1g50390, the predicted open reading frame is half the size of active FRKs, and only incompletely spliced RNAs were identified, which led to a premature stop codon, both indicating that this gene does not produce active FRK. The remaining seven proteins were expressed in E. coli and phosphorylated fructose specifically in vitro leading us to propose a unifying nomenclature (FRK1-7). Substrate inhibition was observed for fructose in all FRKs except FRK1. Fructose binding was on the same order of magnitude for FRK1-6, between 260 and 480 μM. FRK7 was an outlier with a fructose Km of 12 μM. ATP binding was similar for all FRKs and ranged between 52 and 280 μM. YFP-tagged AtFRKs were cytosolic, except plastidic FRK3. T-DNA alleles with non-detectable wild-type RNAs in five of the seven active FRK genes produced no overt phenotype. We extended our sequence comparisons to include putative FRKs encoded in other plant sequenced genomes. We observed that different subgroups expanded subsequent to speciation.ConclusionsArabidopsis thaliana as well as all other plant species analyzed contain multiple copies of genes encoding FRK activity. Sequence comparisons among multiple species identified a minimal set of three distinct FRKs present on all species investigated including a plastid-localized form. The selective expansion of specific isozymes results in differences in FRK gene number among species. AtFRKs exhibit substrate inhibition, typical of their mammalian counterparts with the single AtFRK1 lacking this property, suggesting it may have a distinct in vivo role. Results presented here provide a starting point for the engineering of specific FRKs to affect biomass production.

BackgroundConiophora olivacea is a basidiomycete fungus belonging to the order Boletales that produces brown-rot decay on dead wood of conifers. The Boletales order comprises a diverse group of species including saprotrophs and ectomycorrhizal fungi that show important differences in genome size.ResultsIn this study we report the 39.07-megabase (Mb) draft genome assembly and annotation of C. olivacea. A total of 14,928 genes were annotated, including 470 putatively secreted proteins enriched in functions involved in lignocellulose degradation. Using similarity clustering and protein structure prediction we identified a new family of 10 putative lytic polysaccharide monooxygenase genes. This family is conserved in basidiomycota and lacks of previous functional annotation. Further analyses showed that C. olivacea has a low repetitive genome, with 2.91% of repeats and a restrained content of transposable elements (TEs). The annotation of TEs in four related Boletales yielded important differences in repeat content, ranging from 3.94 to 41.17% of the genome size. The distribution of insertion ages of LTR-retrotransposons showed that differential expansions of these repetitive elements have shaped the genome architecture of Boletales over the last 60 million years.ConclusionsConiophora olivacea has a small, compact genome that shows macrosynteny with Coniophora puteana. The functional annotation revealed the enzymatic signature of a canonical brown-rot. The annotation and comparative genomics of transposable elements uncovered their particular contraction in the Coniophora genera, highlighting their role in the differential genome expansions found in Boletales species.

Subtelomeres (ST) are chromosome regions that separate telomeres from euchromatin and play relevant roles in various biological processes of the cell. While their functions are conserved, ST structure and genetic compositions are unique to each species. This study aims to identify and characterize the subtelomeric regions of the 13 Toxoplasma gondii chromosomes of the Me49 strain. Here, STs were defined at chromosome ends based on poor gene density. The length of STs ranges from 8.1 to 232.4 kbp, with a gene density of 0.049 genes/kbp, lower than the Me49 genome (0.15 kbp). Chromatin organization showed that H3K9me3, H2A.X, and H3.3 are highly enriched near telomeres and the 5′ end of silenced genes, decaying in intensity towards euchromatin. H3K4me3 and H2A.Z/H2B.Z are shown to be enriched in the 5′ end of the ST genes. Satellite DNA was detected in almost all STs, mainly the sat350 family and a novel satellite named sat240. Beyond the STs, only short dispersed fragments of sat240 and sat350 were found. Within STs, there were 12 functional annotated genes, 59 with unknown functions (Hypothetical proteins), 15 from multigene FamB, and 13 from multigene family FamC. Some genes presented low interstrain synteny associated with the presence of satellite DNA. Orthologues of FamB and FamC were also detected in Neospora caninum and Hammondia hammondi. A re-analysis of previous transcriptomic data indicated that ST gene expression is strongly linked to the adaptation to different situations such as extracellular passage (evolve and resequencing study) and changes in metabolism (lack of acetyl-CoA cofactor). In conclusion, the ST region of the T. gondii chromosomes was defined, the STs genes were determined, and it was possible to associate them with high interstrain plasticity and a role in the adaptability of T. gondii to environmental changes. [ABSTRACT FROM AUTHOR]

ETHYLENE-INSENSITIVE3 (EIN3) or EIN3-Like (EIL) proteins, play critical roles in integrating ethylene signaling and physiological regulation in plants by modulating the expression of various downstream genes, such as ethylene-response factors (ERFs). However, little is known about the characteristics of EIN3/EILs in the gymnosperm Ginkgo biloba. In the present study, a genome-wide comparative analysis of Ginkgo EIN3/EIL gene family was performed with those from an array of species, including bryophytes (Physcomitrella patens), gymnosperms (Cycas panzhihuaensis), and angiosperms (Arabidopsis thaliana, Gossypium raimondii, Gossypium hirsutum, Oryza sativa, and Brachypodium distachyon). Within the constructed phylogenetic tree for the 53 EIN3/EILs identified, 5 GbEILs from G. biloba, 2 PpEILs from P. patens, and 3 CpEILs from C. panzhihuaensis were assigned to one cluster, suggesting that their derivation occurred after the split of their ancestors and angiosperms. Although considerable divergence accumulated in amino acid sequences along with the evolutionary process, the specific EIN3_DNA-binding domains were evolutionarily conserved among the 53 EIN3/EILs. Collinearity analysis indicated that whole-genome or segmental duplication and subsequent purifying selection might have prompted the generation and evolution of EIN3/EIL multigene families. Based on the expression patterns of five GbEILs at the four developmental stages of Ginkgo ovules, one GbEIL gene (Gb_03292) was further investigated for its role in mediating ethylene signaling. The functional activity of Gb_03292 was closely related to ethylene signaling, as it complemented the triple response via ectopic expression in ein3eil1 double mutant Arabidopsis. Additionally, GbEIL likely modulates the expression of a Ginkgo ERF (Gb_15517) by directly binding to its promoter. These results demonstrated that the GbEIL gene could have participated in mediating ethylene signal transduction during ovule development in G. biloba. The present study also provides insights into the conservation of ethylene signaling across the gymnosperm G. biloba and angiosperm species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The 4-coumarate coenzyme A ligase (4CL) plays a critical role in the phenylpropane metabolic pathway and is a key enzyme in plant growth metabolism and stress responses. Using bioinformatics methods, 50 Me4CL gene were identified within the cassava genome u, and a comprehensive analysis of the cassava 4CL gene family was conducted. The results showed that these 50 4CL proteins are divided into four subfamilies, with members within the same subfamily sharing similar or identical gene structures. Co-linearity analysis revealed that cassava and rubber trees have the highest number of homologous genes, indicating a close homologous relationship between them. Analysis of 20 cis-acting elements in the promoter region of Me4CL32 revealed the presence of hormone-responsive elements such as gibberellin, auxin, abscisic acid, and as well as elements related to meristematic tissue regulation. results Quantitative real-time PCR (qRT-PCR) results showed that Me4CL32 gene expression changes in response to abiotic stressors (drought, salt, cold, heat) and hormonal stimuli(GA3 and ABA), indicating that Me4CL32 can respond to both environmental pressures and hormone signals. RNA-seq transcriptome and single-cell transcriptome analyses were used to examine the expression patterns of Me4CLs. Additionally, subcellular localization studies demonstrated that the Me4CL32 protein is confined to the chloroplasts of cassava leaves.Investigating the functionality of this gene family aids in comprehending plant growth, development, and stress resistance mechanisms. Furthermore, it furnishes a theoretical basis for future research on developing resilient cassava germplasm and the enhancing cassava's environmental tolerance., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Historically, small molecules biosynthesised by bacteria have been an excellent source for antibacterial drugs. Today, however, the rediscovery of known compounds is a significant hurdle to developing new antimicrobials. Here we use a genome mining and synthetic biology approach to discover the ambocidins: calcium-dependent lipodepsipeptides that are active against drug-resistant Gram-positive pathogens. By cloning a silent biosynthetic gene cluster (the amb cluster) from Streptomyces ambofaciens ATCC 2387 and integrating this into the chromosome of Streptomyces avermitilis we induce expression of ambocidin A and B: two new N ϵ -hydroxyarginine-containing cyclic lipodepsipeptides active against drug-resistant Gram-positive pathogens. Using a panel of Streptomyces host strains, we show that the choice of heterologous host is critical for producing the biologically active compounds, and that inappropriate host choice leads to aberrant production inactive derivatives. We show that N ϵ -hydroxyarginine is the product of a heme-dependent oxygenase and that it enhances biological activity. Ambocidin A inhibits cell wall biosynthesis by binding to Lipid II at a different site than vancomycin. Furthermore, unlike daptomycin, ambocidin A retains potent antimicrobial activity in the presence of lung surfactant, giving it the potential to treat bacterial pneumonia. Our work expands the family of calcium-dependent lipopeptide antibiotics with a new member exhibiting a distinct mechanism of action., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Bacillus pumilus plays an essential role in agricultural applications as a beneficial microbe and for sustainable agriculture production. However, the underlying mechanisms of B. pumilus strains remain unclear as to how they are beneficial for plants as stress tolerant and growth promoters. Bacillus pumilus was isolated from the rhizosphere soil of Artemisia vulgaris. NGS (next-generation sequencing) was performed for the strain to gain new insights into the molecular mechanisms underlying plant-microbial interactions. NGS revealed 3,910 genes, 3294 genes with protein-coding, and 11 functional genomic regions related to diverse agronomic traits including stress tolerance. We identified the two possible phytohormone biosynthesis approaches from metabolic regions1(terpense→diterpense→betacarotene→xanthoxin→ABA)2(terpense→diterpense→geranyl diphosphate →C20 →GA). Several gene clusters related to the biosynthesis of phytohormones, stress tolerance, and agricultural diversification were predicted. The genome provides insights into the possible mechanisms of this bacterium for stress tolerance and its future applications. The genomic organization of B. pumilus revealed several hallmarks of its plant growth promotion and pathogen suppression activities. Our results provide detailed genomic information for the strain and reveal its potential stress tolerance mechanisms, laying the foundation for developing effective stress tolerance strategies against abiotic stress., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

This study showcases 121 new genomes of spore-forming Bacillales from strains collected globally from a variety of habitats, assembled using Oxford Nanopore long-read and MGI short-read sequences. Bacilli are renowned for their capacity to produce diverse secondary metabolites with use in agriculture, biotechnology, and medicine. These secondary metabolites are encoded within biosynthetic gene clusters (smBGCs). smBGCs have significant research interest due to their potential as sources of new bioactivate compounds. Our dataset includes 62 complete genomes, 2 at chromosome level, and 57 at contig level, covering a genomic size range from 3.50 Mb to 7.15 Mb. Phylotaxonomic analysis revealed that these genomes span 16 genera, with 69 of them belonging to Bacillus. A total of 1,176 predicted BGCs were identified by in silico genome mining. We anticipate that the open-access data presented here will expand the reported genomic information of spore-forming Bacillales and facilitate a deeper understanding of the genetic basis of Bacillales' potential for secondary metabolite production., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Leaderless bacteriocins are a unique class of bacteriocins that possess antimicrobial activity after translation and have few cases of documented resistance. Aureocin A53 and lacticin Q are considered two of the most well-studied leaderless bacteriocins. Here, we used in silico genome mining to search for novel aureocin A53-like leaderless bacteriocins in GenBank and MGnify. We identified 757 core peptides across 430 genomes with 75 species found currently without characterized leaderless bacteriocin production. These include putative novel species containing bacteriocin gene clusters (BGCs) from the genera Streptomyces (sp. NBC_00237) and Agrococcus (sp. SL85). To date, all characterized leaderless bacteriocins have been found within the phylum Bacillota, but this study identified 97 core peptides within the phylum Actinomycetota. Members of this phylum are traditionally associated with the production of antibiotics, such is the case with the genus Streptomyces . Actinomycetota is an underexplored phylum in terms of bacteriocin production with no characterized leaderless bacteriocin production to date. The two novel leaderless bacteriocins arcanocin and arachnicin from Actinomycetota members Arcanobacterium sp. and Arachnia sp., respectively, were chemically synthesized and antimicrobial activity was verified. These peptides were encoded in human gut (PRJNA485056) and oral (PRJEB43277) microbiomes, respectively. This research highlights the biosynthetic potential of Actinomycetota in terms of leaderless bacteriocin production and describes the first antimicrobial peptides encoded in the genera Arcanobacterium and Arachnia .IMPORTANCEBacteriocins are gathering attention as alternatives to current antibiotics given the increasing incidence of antimicrobial resistance. Leaderless bacteriocins are considered a commercially attractive subclass of bacteriocins due to the ability to synthesize active peptide and low levels of documented resistance. Therefore, in this work, we mined publicly available data to determine how widespread and diverse leaderless bacteriocins are within the domain of bacteria. Actinomycetota, known for its antibiotic producers but lacking described and characterized bacteriocins, proved to be a rich source of leaderless bacteriocins-97 in total. Two such peptides, arcanocin and arachnicin, were chemically synthesized and have antimicrobial activity. These bacteriocins may provide a novel source of novel antimicrobials that could aid in the development of future alternative antimicrobials and highlight that the Actinomycetota are an underexplored resource of bacteriocin peptides., Competing Interests: The authors declare no conflict of interest.

Background: Gene duplication is a dominant mechanism for the evolution of genomes and plays a key role in genome expansion. Gene duplication via retroposition produces RNA-mediated intron-less copies called retrocopies, that may gain regulatory sequence and biological function to generate retrogenes. Retrocopies dynamics have been reported in several model insect species, but there is still a huge knowledge gap about retrocopies dynamics in most insects, and their role in adaptation., Results: In this study, we reported retrocopy dynamics in 40 species of insect pests of plants belonging to six insect orders. We identified a total of 9,930 retrocopies, which is so far the largest set of retrocopies identified in insects. The identified retrocopies were further grouped into 2,599 Retrogenes, 4,578 Chimeras, 1,241 Intact retrocopies, and 1,512 Pseudogene. We also analyzed all the identified retrogenes that were annotated into 506 gene families. The highest number of retrogenes annotated belong to the heat shock proteins superfamily and are present across all the 40 species from the six orders. We found a significant expansion of the heat shock protein superfamily in the studied species. Almost all the retrogenes, including those belonging to heat shock proteins, are under purifying selection. In summary, we report the retrocopies and retrogenes dynamics in a large set of insect pests of plants and the expansion of the heat shock protein family due to retroposition., Conclusion: This study unveils retrocopy dynamics in the insect pests of plants and highlights the evolution of new genes due to retroposition, and their role in important gene families' expansion., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

TBC1D3 is a primate-specific gene family that has expanded in the human lineage and has been implicated in neuronal progenitor proliferation and expansion of the frontal cortex. The gene family and its expression have been challenging to investigate because it is embedded in high-identity and highly variable segmental duplications. We sequenced and assembled the gene family using long-read sequencing data from 34 humans and 11 nonhuman primate species. Our analysis shows that this particular gene family has independently duplicated in at least five primate lineages, and the duplicated loci are enriched at sites of large-scale chromosomal rearrangements on Chromosome 17. We find that all human copy-number variation maps to two distinct clusters located at Chromosome 17q12 and that humans are highly structurally variable at this locus, differing by as many as 20 copies and ∼1 Mbp in length depending on haplotypes. We also show evidence of positive selection, as well as a significant change in the predicted human TBC1D3 protein sequence. Last, we find that, despite multiple duplications, human TBC1D3 expression is limited to a subset of copies and, most notably, from a single paralog group: TBC1D3-CDKL These observations may help explain why a gene potentially important in cortical development can be so variable in the human population., (© 2024 Guitart et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Calcium-dependent protein kinases (CDPKs) phosphorylate downstream target proteins in response to signals transmitted by free calcium ions (Ca 2+ , one of the second messengers) and thus play important regulatory roles in many biological processes, such as plant growth, development, and stress response., Results: A bioinformatic analysis, as well as thorough evolutionary and expression investigations, were conducted to confirm previous reports of functional evidence for plant CDPKs. Using the Phytozome database's BLAST search engine and the HMM search tool in TBtools software, we discovered that CDPKs are well conserved from green algae to flowering angiosperms in various gene family sizes. Additional investigations of the obtained CDPKs revealed high conservation of domain and motif numbers, gene architectures, and patterns. However, this conservation differed among plant species. Phylogenetic analysis demonstrated that the CDPK gene family diverged from a common ancient gene. Similarly, investigations into plant interspecies evolutionary relationships revealed common ancestral plant species, suggesting speciation of plants and evolution based on plant adaptation and diversification. A search for the driving force of CDPK gene family expansion revealed that dispersed duplication events, among other duplication events, contributed largely to CDPK gene family expansion. Gene localization analysis in P. trichocarpa  demonstrated that most CDPK genes are localized within several cell organelles and bind other kinases and proteins to perform their biological functions efficiently. Using RNA-seq data and qPCR analyses, we postulated that PtCDPKs play functional roles in abiotic stress responses by regulating cold, heat, drought and salt stress to varying extents., Conclusion: The CDPK genes are well conserved in plants and are critical entities in abiotic stress regulation, and further exploration and manipulation of these genes in the future may provide solutions to some of the challenges in agriculture, forestry and food security., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Sugars Will Eventually be Exported Transporter (SWEET) gene family is a unique type of sugar transporter that plays a vital role in metabolic regulation, growth, development, and stress response in multiple species. This study aimed to systematically identify the SWEET gene family members and detect the regulation of gene expression and their potential roles of the SWEET gene family in Brassica juncea., Results: A total of 66 BjSWEET (Brassica juncea Sugar Will Eventually be Exported Transporter) genes distributed across 17 chromosomes were identified. The gene structure and motifs were relatively conserved, with all members containing the MtN3/saliva domain. Phylogenetic analysis revealed that the SWEET gene family can be classified into four subfamilies (Clades I, II, III, and IV). Collinearity analysis revealed that there were 118 pairs of segment duplicates, indicating that some BjSWEET genes were obtained via segmental duplication. The promoter regions of the BjSWEET genes contained many plant hormone-related response elements, stress-related response elements, growth and development elements, and light-responsive regulatory elements. Furthermore, analysis of the expression profiles revealed that the expression levels of the BjSWEET genes differed among the eight different tissues. qRT‒PCR analysis of six selected BjSWEET genes revealed that the expression levels of BjSWEET17.2, BjSWEET17.4, BjSWEET12.2, and BjSWEET12.3 were significantly upregulated under drought treatment, suggesting that these genes may respond to drought stress in B. juncea., Conclusion: This study systematically identified and analyzed the SWEET gene family members in B. juncea for the first time, laying the foundation for further research on the molecular mechanisms of drought resistance in B. juncea and providing theoretical guidance for the application of these genes in other species., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The evolution of operons has puzzled evolutionary biologists since their discovery, and many theories exist to explain their emergence, spreading, and evolutionary conservation. In this work, we suggest that DNA replication introduces a selective force for the clustering of functionally related genes on chromosomes, which we interpret as a preliminary and necessary step in operon formation. Our reasoning starts from the observation that DNA replication produces copy number variations of genomic regions, and we propose that such changes perturb metabolism. The formalization of this effect by exploiting concepts from metabolic control analysis suggests that the minimization of such perturbations during evolution could be achieved through the formation of gene clusters and operons. We support our theoretical derivations with simulations based on a realistic metabolic network, and we confirm that present-day genomes have a degree of compaction of functionally related genes, which is significantly correlated to the proposed perturbations introduced by replication. The formation of clusters of functionally related genes in microbial genomes has puzzled microbiologists since their first discovery. Here, we suggest that replication, and the copy number variations due to the replisome passage, might play a role in the process through a perturbation in metabolite homeostasis. We provide theoretical support to this hypothesis, and we found that both simulations and genomic analysis support our hypothesis., Importance: The formation of clusters of functionally related genes in microbial genomes has puzzled microbiologists since their discovery. Here, we suggest that replication, and the copy number variations due to the replisome passage, might play a role in the process through a perturbation in metabolite homeostasis. We provide theoretical support to this hypothesis, and we found that both simulations and genomic analysis support our hypothesis., Competing Interests: The authors declare no conflict of interest.

Cytokinin Response Factors (CRFs) play a crucial role in plant growth and development, hormone signaling, and responses to biotic and abiotic stresses. However, there have been no reports on CRF genes in rice until now. We analyzed the CRF families in four rice subspecies: cultivated rice Oryza sativa Japonica Group, Oryza sativa Indica Group, and Oryza sativa (circum-Aus1 var. N22), as well as wild rice Oryza rufipogon. We identified 7, 6, 6, and 7 CRF in their genomes, respectively, distributed across different chromosomes. The protein motifs and gene structures of CRF in these four types of rice show high conservation. Cis-regulatory element analysis revealed that the promoter regions of the CRF contain numerous hormone and stress-related elements. The number of CRF in these four types of rice is not influenced by gene duplication. The expression pattern showed that OsCRF exhibit significant tissue-specific expression. The qRT-PCR results showed that OsCRF strongly responded to low-temperature stress and can be induced by melatonin and cytokinin to increase expression levels. In addition, the nuclear localisation of OsCRF4/5 was confirmed to be as predicted. The results above will provide a foundation for further and deeper investigation of CRFs., Competing Interests: Declarations Competing interests The authors declare no competing interests. Conflict of interest There are no conflicts of interest to declare., (© 2024. The Author(s).)

Plants are chemical engineers par excellence. Collectively they make a vast array of structurally diverse specialized metabolites. The raw materials for building new pathways (genes encoding biosynthetic enzymes) are commonly recruited directly or indirectly from primary metabolism. Little is known about how new metabolic pathways and networks evolve in plants, or what key nodes contribute to branches that lead to the biosynthesis of diverse chemicals. Here we review the molecular mechanisms underlying the generation of biosynthetic branchpoints. We also consider examples in which new metabolites are formed through the joining of precursor molecules arising from different biosynthetic routes, a scenario that greatly increases both the diversity and complexity of specialized metabolism. Given the emerging importance of metabolic gene clustering in helping to identify new enzymes and pathways, we further cover the significance of biosynthetic gene clusters in relation to metabolic networks and dedicated biosynthetic pathways. In conclusion, an improved understanding of the branchpoints between metabolic pathways will be key in order to be able to predict and illustrate the complex structure of metabolic networks and to better understand the plasticity of plant metabolism. This article is part of the theme issue 'The evolution of plant metabolism'.

Plant chemical diversity is largely owing to a number of enzymes which catalyse reactions involved in the assembly, and in the subsequent chemical modifications, of the core structures of major classes of plant specialized metabolites. One such reaction is acylation. With this in mind, to study the deep evolutionary history of BAHD and the serine-carboxypeptidase-like (SCPL) acyltransferase genes, we assembled phylogenomic synteny networks based on a large-scale inference analysis of orthologues across whole-genome sequences of 126 species spanning Stramenopiles and Archaeplastida, including Arabidopsis thaliana , tomato ( Solanum lycopersicum ) and maize ( Zea mays ). As such, this study combined the study of genomic location with changes in gene sequences. Our analyses revealed that serine-carboxypeptidase (SCP)/serine-carboxypeptidase-like (SCPL) genes had a deeper evolutionary origin than BAHD genes, which expanded massively on the transition to land and with the development of the vascular system. The two gene families additionally display quite distinct patterns of copy number variation across phylogenies as well as differences in cross-phylogenetic syntenic network components. In unlocking the above observations, our analyses demonstrate the possibilities afforded by modern phylogenomic (syntenic) networks, but also highlight their current limitations, as demonstrated by the inability of phylogenetic methods to separate authentic SCPL acyltransferases from standard SCP peptide hydrolases.This article is part of the theme issue 'The evolution of plant metabolism'.

Background: Microbes produce diverse bioactive natural products with applications in fields such as medicine and agriculture. In their genomes, these natural products are encoded by physically clustered genes known as biosynthetic gene clusters (BGCs). Genome and metagenome sequencing advances have enabled high-throughput identification of BGCs as a promising avenue for natural product discovery. BGC mining from (meta)genomes using in silico tools has allowed access to a vast diversity of potentially novel natural products. However, a fundamental limitation has been the ability to assemble complete BGCs, especially from complex metagenomes. With their fragmented assemblies, short-read technologies struggle to recover complete BGCs, such as the long and repetitive nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Recent advances in long-read sequencing, such as the High Fidelity (HiFi) technology from PacBio, have reduced this limitation and can help retrieve both accurate and complete BGCs from metagenomes, warranting improvement in the existing BGC identification approach for better utilization of HiFi data., Results: Here, we present HiFiBGC, a command-line-based workflow to identify BGCs in PacBio HiFi metagenomes. HiFiBGC leverages an ensemble of assemblies from three HiFi-tailored metagenome assemblers and the reads not represented in these assemblies. Based on our analyses of four HiFi metagenomic datasets from four different environments, we show that HiFiBGC identifies, on average, 78% more BGCs than the top-performing single-assembler-based method. This increase is due to HiFiBGC's ensemble assembly approach, which improves recovery by 25%, as well as from the inclusion of mostly fragmented BGCs identified in the unmapped reads., Conclusions: HiFiBGC is a computational workflow for identifying BGCs in long-read HiFi metagenomes, implemented majorly using Python programming language and workflow manager Snakemake. HiFiBGC is available on GitHub at https://github.com/ay-amityadav/HiFiBGC under the MIT license. The code related to the figures and analyses presented in the manuscript is available at https://github.com/ay-amityadav/HiFiBGC_analyses ., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The fundamental leucine zipper (bZIP) genes play a crucial role as transcriptional coactivators in plants. Pseudoroegneria libanotica served as the maternal contributor to several species within the tribe Triticeae (Poaceae), exhibiting commendable resilience to various stressors. Consequently, bZIP genes of Pseudoroegneria libanotica response to abiotic stresses was important for further understanding of stress tolerance studies in Triticeae. A total of 108 PsebZIPs were identified in this study, and they were divided into 10 subgroups. PsebZIPs were found to be extensively involved in the biological activities within P. libanotica. The RNA-seq results revealed differential responses of PsebZIPs to drought stress in both aerial and root tissues. PsebZIP44 and PsebZIP46 were regarded as potential genes capable of responding to abiotic stress, and their varying degrees of responsiveness to drought, salinity, heavy metals, heat, and oxidative stress have been demonstrated in yeast. Homologous proteins with intact bZIP gene structures of PsebZIP44 or PsebZIP46 have not been identified in wheat. The current investigation not only provides a fundamental basis for further probing into the biological functions of PsebZIPs within P. libanotica but also reveals previously undiscovered potential genes with the ability to respond to abiotic stress. They could contribute to improving the genetic constitution of Triticeae crops, including wheat., Competing Interests: Declarations Ethics approval and consent to participate This study did not involve any human or animal research participant data. The plant sample tested in this study was not an endangered species, and the collection of samples did not cause any environmental problems. All experimental procedures were approved by the Academic Committee of Sichuan Agricultural University. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Plesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors., Results: In this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides' capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides., Conclusions: Overall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Pollen coat proteins (PCPs) are cysteine-rich small-molecule proteins, which exhibit high levels of polymorphism and are expressed in gametocytes. Previous investigations have revealed that PCP genes are involved in pollen wall synthesis, pollen-stigma recognition, and pollen development and germination. However, gene expression and function of PCP family in pollen development is not well understood in Wucai (Brassica campestris L.). In this study, genome-wide identification and expression analysis of the BcPCP gene family members were conducted, including their physical and chemical properties, chromosome localization, phylogenetic relationships, gene structure, and tertiary structure. A total of 20 BcPCP genes were identified and classified into three subfamilies showing high homology to Arabidopsis thaliana. Expression pattern analysis indicated that the BcPCP gene family exhibits higher expression levels in reproductive organs, suggesting their potential involvement in the reproductive development. Notably, BraA02g002400.3 C, potentially associated with male sterility, was identified through multiple transcriptomic and proteomic datasets. Subsequently, sequence analysis revealed its homology with the Arabidopsis GRP20 gene, and thus it was named BcGRP20. Functional analysis of this gene showed that overexpression of BcGRP20 gene in the Arabidopsis grp20 mutant could restore anther fertility. Overall, our findings indicate that BcGRP20 plays a critical role in pollen development and may be the causative gene for male sterility in Wucai. This study provides candidate genes for further functional identification of BcPCP genes in Wucai, which are crucial for anther development., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Microbial communities are shaped by cell-cell interactions. Although archaea are often found in associations with other microorganisms, the mechanisms structuring these communities are poorly understood. Here, we report on the structure and function of haloarchaeal contractile injection systems (CISs). Using a combination of functional assays and time-lapse imaging, we show that Halogeometricum borinquense exhibits antagonism toward Haloferax volcanii by inducing cell lysis and inhibiting proliferation. This antagonism is contact-dependent and requires a functional CIS, which is encoded by a gene cluster that is associated with toxin-immunity pairs. Cryo-focused ion beam milling and imaging by cryo-electron tomography revealed that these CISs are bound to the cytoplasmic membrane, resembling the bacterial type six secretion systems (T6SSs). We show that related T6SS gene clusters are conserved and expressed in other haloarchaeal strains, which exhibit antagonistic behavior. Our data provide a mechanistic framework for understanding how archaea may shape microbial communities and affect the food webs they inhabit.

The Jumonji C (JmjC) structural domain-containing gene family plays essential roles in stress responses. However, descriptions of this family in Brassica rapa ssp. pekinensis (Chinese cabbage) are still scarce. In this study, we identified 29 members of the BrJMJ gene family, with cis-acting elements related to light, low temperature, anaerobic conditions, and phytohormone responses. Most BrJMJs were highly expressed in the siliques and flowers, suggesting that histone demethylation may play a crucial role in reproductive organ development. The expression of BrJMJ1, BrJMJ2, BrJMJ5, BrJMJ13, BrJMJ21 and BrJMJ24 gradually increased with higher Cd concentration under Cd stress, while BrJMJ4 and BrJMJ29 could be induced by osmotic, salt, cold, and heat stress. These results demonstrate that BrJMJs are responsive to abiotic stress and support future analysis of their biological functions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Many complex terpenoids, predominantly isolated from plants and fungi, show drug-like physicochemical properties. Recent advances in genome mining revealed actinobacteria as an almost untouched treasure trove of terpene biosynthetic gene clusters (BGCs). In this study, we characterized a terpene BGC with an unusual architecture. The selected BGC includes, among others, genes encoding a terpene cyclase fused to a truncated reductase domain and a cytochrome P450 monooxygenase (P450) that is split over three gene fragments. Functional characterization of the BGC in a heterologous host led to the identification of several new members of the trans -eunicellane family of diterpenoids, the euthailols, that feature unique oxidation patterns. A combination of bioinformatic analyses, structural modeling studies, and heterologous expression revealed a dual function of the pathway-encoded hypothetical protein that acts as an isomerase and an oxygenase. Moreover, in the absence of other tailoring enzymes, a P450 hydroxylates the eunicellane scaffold at a position that is not modified in other eunicellanes. Surprisingly, both the modifications installed by the hypothetical protein and one of the P450s exhibit partial redundancy. Bioactivity assays revealed that some of the euthailols show growth inhibitory properties against Gram-negative ESKAPE pathogens. The characterization of the euthailol BGC in this study provides unprecedented insights into the partial functional redundancy of tailoring enzymes in complex diterpenoid biosynthesis and highlights hypothetical proteins as an important and largely overlooked family of tailoring enzymes involved in the maturation of complex terpenoids.

Ribosomally synthesized lanthionine-containing peptides (lanthipeptides) have emerged as a promising source of antimicrobials against multidrug resistance pathogens. An effective way to discover and engineer lanthipeptides is through heterologous expression of their biosynthetic gene clusters (BGCs) in a host of choice. Here we report a plug-and-play pathway refactoring strategy for rapid evaluation of lanthipeptide BGCs in Bacillus subtilis based on the T7 expression system. As a proof of concept, we used this strategy to not only observe the successful production of a known lanthipeptide haloduracin β but also discover two new human-microbiota-derived lanthipeptides that previously failed to be produced in Escherichia coli . The resulting B. subtilis plug-and-play T7 expression system should enable the genome mining of new lanthipeptides in a high-throughput manner.

We report the successful cell-free reconstitution of two natural product biosynthetic pathways of divergent complexity and structural classes. We first constructed the teleocidin biosynthetic pathway using our BY-2 (tobacco) cell-free protein synthesis (CFPS) system. We discovered a direct interaction between TleA and MbtH, and showed that the BY-2 system is capable of producing more than 80 mg/L teleocidin B-3 with cofactor supplementation and ∼20 mg/L with no cofactors supplemented, demonstrating the high metabolic activity of the system. We then extended our methodology and report the first successful cell-free biosynthesis of UK-2 diol (precursor to the commercially valuable secondary metabolite UK-2A) from simple building blocks by refactoring a complex pathway of 10 proteins in the wheat germ CFPS system. We show that plant CFPS systems are suitable for reconstructing pathways and identifying the functions of uncharacterized genes linked to biosynthetic gene clusters and rate-limiting biosynthetic steps.

The efficiency of valuable metabolite production by engineered microorganisms underscores the importance of stable and controllable gene expression. While plasmid-based methods offer flexibility, integrating genes into host chromosomes can establish stability without selection pressure. However, achieving site-directed multicopy integration presents challenges, including site selection and stability. We introduced a stable multicopy integration method by using a novel dual-plasmid mini-Tn5 system to insert genes into Escherichia coli 's genome. The gene of interest was combined with a removable antibiotic resistance gene. After the selection of bacteria with inserted genes, the antibiotic resistance gene was removed. Optimizations yielded an integration efficiency of approximately 5.5 × 10 -3 per recipient cell in a single round. Six rounds of integration resulted in 19 and 5 copies of the egfp gene in the RecA + strain MG1655 and the RecA - strain XL1-Blue MRF', respectively. Additionally, we integrated a polyhydroxybutyrate (PHB) synthesis gene cluster into E. coli MG1655, yielding an 8-copy integration strain producing more PHB than strains with the cluster on a high-copy plasmid. The method was efficient in generating gene insertions in various E. coli strains, and the inserted genes were stable after extended culture. This stable, high-copy integration tool offers potential for diverse applications in synthetic biology.

A cryptic cluster of genes, known as the ebo cluster, has been found in a variety of genomic contexts among bacteria and algae. In Pseudomonas fluorescens NZI7, the ebo cluster (a.k.a. EDB cluster) is involved in the bacterial repellent mechanism against nematode grazing. In cyanobacteria, the cluster plays a role in the transport of the scytonemin monomer from the cytosol to the periplasm. Despite their broad distribution and interesting phenotypes, neither the pathway nor the functions of the enzymes are known. Here we show that EboD proteins from the ebo clusters in Nostoc punctiforme and Sporocytophaga myxococcoides catalyze the cyclization of mannose 6-phosphate to a novel cyclitol, 2-deoxy-4- epi - scyllo -inosose. The enzyme product is postulated to be a precursor of a signaling molecule or a transporter in the organisms. This study sheds the first light onto ebo/EDB pathways and established a functionally distinct enzyme that extends the diversity of sugar phosphate cyclases.

Glutamate decarboxylase (GAD) is involved in GABA metabolism and plays an essential regulatory role in plant growth, abiotic stresses, and hormone response. This study investigated the expression mechanism of the GAD family during longan early somatic embryogenesis (SE) and identified 6 GAD genes based on the longan genome. Homology analysis indicated that DlGAD genes had a closer relationship with dicotyledonous plants. The analysis of cis-acting elements in the promoter region suggests that the GAD genes were associated with various stress responses and hormones. RNA sequencing (RNA-Seq) and the qRT-PCR data indicated that most DlGAD genes were highly expressed in the incomplete compact pro-embryogenic cultures (ICpEC) and upregulated in longan embryogenic callus (EC) after treatments with 2,4-D, high temperature (35 °C), IAA, and ABA. Moreover, the RNA-Seq analysis also revealed that DlGADs exhibit different expression patterns in various tissues and organs. The subcellular localization results showed that DlGAD5 was localized in the cytoplasm, suggesting that it played a role in the cytoplasm. Transient overexpression of DlGAD5 enhanced the expression levels of DlGADs and increased the activity of glutamate decarboxylase in longan embryogenic callus (EC), while the content of glutamic acid decreased. Thus, the DlGAD gene can play an important role in the early somatic embryogenesis of longan by responding to hormones such as IAA and ABA. DlGAD5 can affect the growth and development of longan by stimulating the expression of the DlGAD gene family, thereby increasing the GAD activity in the early SE of longan, participating in hormone synthesis and signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The swimming crab, Portunus trituberculatus is one of crucial aquaculture crabs with significant differences in growth and economic performance between male and female swimming crabs. Consequently, the culture of female populations presents higher economic value. The doublesex and mab-3 related transcription factor (Dmrt) gene family are known to play crucial role in gonad differentiation and development. However, there is limited information about this gene family in Portunus trituberculatus. In this study, we identified seven members of the Dmrt gene family in P. trituberculatus based on the published transcriptome and genome data and designated as Ptdmrt-1, Ptdoublesex (Ptdsx), Ptidmrt-1, Ptdmrt-11E, Ptidmrt-2, Ptdmrt-99B, and Ptdmrt-3 based on the homology analysis results, respectively. These Ptdmrt genes distributed across 6 chromosomes and were predicted to encode 283 aa, 288 aa, 529 aa, 436 aa, 523 aa, 224 aa, and 435 aa protein precursors, respectively. The expression patterns of these dmrt genes were characterized by qRT-PCR and gonad transcriptome data. The results showed that five members (Ptdmrt-99B, Ptdsx, Ptdmrt-1, Ptdmrt-3, and Ptdmrt-11E) were differentially expressed between the testis and ovary. In addition, their expression patterns from zoea 2 to juvenile 1 were characterized by published transcriptome data and the results showed that they were lowly expressed and did not exhibit notable difference except for Ptdsx during early development. Overall, majority of Ptdmrt genes were involved in gonad differentiation in the swimming crab. Current findings provide a solid foundation for further exploration of the roles of these genes in gonad development and differentiation in P. trituberculatus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The multifunctional proteins of class 2 CRISPR systems such as Cas9, have been employed to activate cryptic biosynthetic gene clusters (BGCs) in Streptomyces, which represent a large and hidden reservoir of natural products. However, such approaches are not applicable to most Streptomyces strains with reasons to be comprehended. Inspired by the prevalence of the class 1 subtype especially the type I-E CRISPR system in Streptomyces, here we report the development of the type I-E CRISPR system into a series of transcriptional regulation tools. We further demonstrate the effectiveness of such activators in nine phylogenetically distant Streptomyces strains. Using these tools, we successfully activate 13 out of 21 BGCs and lead to the identification and characterization of one polyketide, one Ripp and three alkaloid products. Our work is expected to have a profound impact and to facilitate the discovery of numerous structurally diverse compounds from Streptomyces., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Phloem protein 2 (PP2), a dimeric lectin, is known for its involvement in plant responses to biotic and abiotic stresses. However, research on PP2 proteins in Moso bamboo is lacking., Results: In this study, comprehensive genome-wide analysis of the PP2-like gene family was conducted in Moso bamboo (Phyllostachys edulis), which has a significant economic and ecological value. Using HMMER3 search and InterPro domain analysis, 23 PP2-like genes (PhePP2-1 to PhePP2-23) were identified in the P. edulis genome. These genes were distributed across 12 chromosomal scaffolds, with proteins ranging from 216 to 556 amino acids in length. Phylogenetic analysis, including 163 PP2 proteins from eight plant species, revealed six distinct groups, with Group III and Group V being the largest. Gene structure and motif analyses indicated conserved domains across the PhePP2 proteins. In addition, Cis-element analysis of the promoter regions highlighted their potential regulatory roles in hormone, stress, and light responses. Expression pattern analysis using RNA-seq data showed differential expression of PhePP2 genes under drought, salt, salicylic acid, and abscisic acid treatments, indicating their involvement in stress response pathways. Furthermore, qPCR validation in different tissues and organs of Moso bamboo confirmed the expression profiles of the selected PhePP2 genes., Conclusions: This study provides a comprehensive understanding of the functional roles of PP2-like genes in Moso bamboo and insights into their potential applications in enhancing stress tolerance and growth in plants., Competing Interests: Declarations Ethics approval and consent to participate The bamboo shoots in our study were from the north campus of Leshan Normal University, China. These plant materials do not include any wild species at risk of extinction. No specific permits were required for sample collection in this study. We complied with relevant institutional, national, and international guidelines and legislation for plant study. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: The OVATE gene family is a new class of transcriptional repressors, which play an important regulatory role in plant growth and development. Many studies have proved that the OVATE gene family can regulate the development of plant tissues and organs and resist stress, but its quantity and functional role in Rosa roxburghii remain unknown., Results: In this study, 14 OVATE family members were identified by re-annotating the genome of Rosa roxburghii, and these members were unevenly distributed on 6 chromosomes. Evolutionary analysis indicated that these family members were classified into three groups. In their promoter regions, many hormone-related cis-acting elements such as ABA, GA, and MeJA were identified. Segmental duplication is an important driving force for the expansion of the OVATE family in Rosa roxburghii. Transcriptome sequencing and RT-qPCR analysis showed that OVATE gene family had a specific tissue expression pattern in Rosa roxburghii. For instance, the expression level of gene Rr602241 in leaves was more than 4 times that of other tissues. The gene Rr101515 was highly expressed in FR1 and FR4 stages of fruit tree development, and was highly homologous to the gene regulating fruit shape in tomatoes. These results suggest that members of the OVATE gene family may have diverse functions in different tissues. Furthermore, based on the transcriptome data of eight tissues, a transcriptional regulatory co-expression network of different transcription factors and 14 OVATE genes was constructed., Conclusion: In conclusion, our study provides the expression profiles of the OVATE family and reveals the potential functional roles of different members in the growth and development of Rosa roxburghii Tratt., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. The sampling of plant material was performed in compliance with institutional guidelines. The research conducted in this study required neither approval from an ethics committee, nor involved any human or animal subjects. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Echinocandin B (ECB) is a key precursor of the antifungal drug anidulafungin and its biosynthesis occurs via ani gene cluster in Aspergillus nidulans NRRL8112. Strain improvement for industrial ECB production has mainly relied on mutation breeding due to the lack of genetic tools., Results: Here, a CRISPR-base-editing tool was developed in A. nidulans NRRL8112 for simultaneous inactivation of the nkuA gene and two marker genes, pryoA and riboB, which enabled efficient genetic manipulation. Then, in-vivo plasmid assembly was harnessed for ani gene expression screening, identifying the rate-limiting enzyme AniA and a pathway-specific transcription factor AniJ. Stepwise titer enhancement was achieved by overexpressing aniA and/or aniJ, and ECB production reached 1.5 g/L during 5-L fed-batch fermentation, an increase of ~ 30-fold compared with the parent strain., Conclusion: This study, for the first time, revealed the regulatory mechanism of ECB biosynthesis and harnessed genetic engineering for the development of an efficient ECB-producing strain., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Legionella pneumophila is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. L. pneumophila requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the Legionella- containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify L. pneumophila fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to L. pneumophila replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in L. pneumophila fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit , for Legionella infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support L. pneumophila replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. Legionella pneumophila is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for L. pneumophila are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of L. pneumophila fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria., Competing Interests: The authors declare no conflict of interest.

Mangiferin, a C-glucosyl xanthone, is a biologically active glycoside naturally synthesized in mango. Glycosyltransferase can catalyze the biosynthesis of mangiferin. In this study, we identified 221 members of the UGT glycosyltransferase family in mango. The 221 MiUGT genes were grouped into 13 subfamilies through phylogenetic tree analysis with Arabidopsis, Chinese bayberry, and mango. All UGT family members in mango were unevenly distributed on 17 chromosomes and found that tandem duplication dominated the expansion of UGT family members in mango. Purification selection primarily influenced the evolution of the mango UGT family members. In addition, cis-element analysis of the mango UGT gene family revealed the presence of MYB binding sites, which are involved in flavonoid biosynthesis; which further supports the role of UGT family members in the synthesis of flavonoids. To verify these results, we analyzed the expression of UGT family members in mango leaves, stems, and different developmental stages of fruit peel. The RNA-seq and qRT-PCR results showed significant differences in the expression patterns of MiUGT genes in various tissues and developmental stages of mango. We identified MiUGT gene-specific expression at different stages of fruit development. These results lay a theoretical foundation for research on the relationship between members of the mango UGT family and the synthesis of flavonoids, mangiferin., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Telomere-linked RecQ helicase (tlh) genes have been reported in several fungi and a choanoflagellate in the regions adjacent to the terminal telomere repeats. In this study, we explored the Telomere-linked RecQ helicase (tlh) genes in four strains of Fusarium oxysporum, offering new insights into their genomic structure, functional motifs, and impact on chromosomal ends. We conducted a comprehensive analysis, comparing the tlh genes of F. oxysporum with those previously identified in other organisms and uncovering significant similarities. Through comparative genomics, we identified conserved protein motifs across these genes, including a TLH domain, C 2 H 2 , and RecQ helicase motifs. Our phylogenetic analysis positions the F. oxysporum tlh genes in a cluster with other known tlhs, suggesting a shared evolutionary origin. Mutation analysis revealed a relatively low level of deleterious mutations in tlh gene paralogs, with a notable proportion of full-size structures maintained across strains. Analysis of subtelomeric sequences indicates that a region with almost identical sequences flanks the majority of chromosome ends, termed tlh-containing region (TLHcr), across these strains. The presence of TLHcrs at chromosome ends, either as single entities or in arrays, underscores their potential role in telomere function and genome stability. Our findings provide a detailed examination of tlh genes in four strains of F. oxysporum, laying the groundwork for future studies on their biological significance and evolutionary history in fungal genomes., Competing Interests: Declarations Conflicts of interest The authors declare no conflict of interest. Ethics approval The authors declare no ethics approval required. Consent to participate Not applicable. Consent for publication Not applicable., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microcystis spp. threaten freshwater ecosystems through the proliferation of cyanobacterial harmful algal blooms (cyanoHABs) and production of the hepatotoxin, microcystin. While microcystin and its biosynthesis pathway, encoded by the mcy genes, have been well studied for over 50 years, a recent study found that Microcystis populations in western Lake Erie contain a transcriptionally active partial mcy operon, in which the A2 domain of mcyA and mcyB-C are present but the mcyD-J genes are absent. Here, we investigate the potential biosynthetic products and the evolutionary history of this partial operon. Our results reveal two candidate tetrapeptide constructs, with an X variable position, to be produced by strains with the partial operon. The partial operon appears necessary and sufficient for tetrapeptide biosynthesis and likely evolved from a single ancestor hundreds to tens of thousands of years ago. Bioactivity screens using Hep3B cells indicate a mild elevation of some markers of hepatotoxicity and inflammation, suggesting the need to further assess the effects of these novel secondary metabolites on freshwater ecosystems and public health. The need to assess these effects is even more pressing given the detection of tetrapeptides in both culture and western Lake Erie, which is a vital source of fresh water. Results from this study emphasize previous findings in which novel bacterial secondary metabolites may be derived from the molecular evolution of existing biosynthetic machinery under different environmental forcings.

The GRAS gene is an important specific transcription factor in plants, which has multiple functions such as signal transduction, cell morphogenesis and stress response. Although it is widely distributed in plants and has been characterized in several species, however, information about the GRAS family in Taraxacum kok-saghyz Rodin remains unknown. Here, TkGRAS family members were identified and analyzed for molecular characterization, tissue expression patterns and induced expression patterns. A total of 64 GRAS family members were identified at the genome-wide level, which could be categorized into 14 subfamilies by phylogenetic analysis. Most TkGRASs were intronless and had essentially the same gene structure in the same subfamily. Meanwhile, there were multiple response elements found in the promoters of TkGRASs. Tissue expression patterns and induced expression patterns showed that TkGRASs were expressed in different tissues and induced by abiotic stresses. Notably, the expression level of TkGRAS20 was up-regulated under different stresses, suggesting that this gene plays a pivotal role in the stress response. TkGRAS20 showed transcriptional activity in yeast cells and localized in the nucleus and plasma membrane. In conclusion, our study provided valuable insights into the genetic mechanisms underlying stress tolerance in TKS, and several key genes may be used for genetic breeding to improve stress tolerance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Rice kernel smut, caused by Tilletia horrida, is becoming an increasingly serious disease in hybrid rice planting, leading to production losses and quality decline of male-sterile rice varieties. Successful infection requires an efficient energy source that the pathogen obtains from rice plants, such as carbohydrates. Glycoside hydrolases (GHs), one of the largest sub-families in the cell wall-degrading enzyme family, play a key role in the infection progress of pathogens. To investigate their roles in facilitating infection, in this study, we identified and characterized genes encoding GH family proteins of T. horrida and further explored the functions and structures of these genes., Materials and Methods: Through genome-wide sequencing and bioinformatics analyses, 52 GH genes were identified from T. horrida, named ThGhd_1 to ThGhd_52. The subcellular location, conserved motifs, and structures of ThGhds were identified by bioinformatics analyses., Results: Phylogenetic analysis revealed that ThGhds with similar domains clustered together, although some proteins clustered in different branches, which might reflect functional diversity. Protein-protein interaction network analysis revealed that ThGhds interact with partner proteins involved in reactive oxygen species signaling, protein kinase activity, and plant hormone signal transduction pathways. RNA-sequencing analysis showed that the expression of ThGhd genes responded differently at different infection time points, with dynamic changes detected during the T. horrida infection process, indicating that these genes are involved in interactions with rice and have potential roles in pathogenic mechanisms., Conclusions: The results of this study provide valuable resources for the structure elucidation of GH family proteins of T. horrida and can help to further elucidate their roles in pathogenesis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Terpenoids are the largest class of natural products, found across all domains of life. One of the most abundant bacterial terpenoids is the volatile odorant 2-methylisoborneol (2-MIB), partially responsible for the earthy smell of soil and musty taste of contaminated water. Many bacterial 2-MIB biosynthetic gene clusters were thought to encode a conserved transcription factor, named EshA in the model soil bacterium Streptomyces griseus. Here, we revise the function of EshA, now referred to as Sg Enc, and show that it is a Family 2B encapsulin shell protein. Using cryo-electron microscopy, we find that Sg Enc forms an icosahedral protein shell and encapsulates 2-methylisoborneol synthase (2-MIBS) as a cargo protein. Sg Enc contains a cyclic adenosine monophosphate (cAMP) binding domain (CBD)-fold insertion and a unique metal-binding domain, both displayed on the shell exterior. We show that Sg Enc CBDs do not bind cAMP. We find that 2-MIBS cargo loading is mediated by an N-terminal disordered cargo-loading domain and that 2-MIBS activity and Sg Enc shell structure are not modulated by cAMP. Our work redefines the function of EshA and establishes Family 2B encapsulins as cargo-loaded protein nanocompartments involved in secondary metabolite biosynthesis., (© 2024. The Author(s).)

Background: B3 genes encode transcription factors that play key roles in plant growth and development. However, the specific B3 genes involved in the seed development of Akebia trifoliata remain unexplored., Results: A total of 72 AktB3 genes were identified and classified into five subfamilies (ARF, LAV, RAV, HSI, and REM) based on phylogenetic analysis. These 72 AktB3 genes were unevenly distributed across 16 chromosomes. Collinear analysis indicated that segmental duplication has played a significant role in the evolution of AktB3 genes, and underwent purification selection. Expression profiling across seed development stages revealed that seven AktB3 genes, particularly from the LAV subfamily (AktABI3, AktFUS3, AktLEC2), were up-regulated at 70 days after flowering (DAF). Notably, the expression of oleosin exhibited a strong positive correlation with LAV subfamily genes, highlighting their potential roles as hub genes in lipid metabolism and seed development. Yeast two-hybrid (Y2H) and yeast one-hybrid (Y1H) experiments confirmed that AktFUS3-1, AktFUS3-2, and AktLEC2 form protein complexes and individually bind to the AktOLE1 promoter, thereby regulating downstream gene expression. These results provide direct evidence of the cooperative role these transcription factors play in controlling lipid metabolism, particularly related to oleosin proteins. Additionally, miRNA sequencing across three seed developmental stages identified 591 miRNAs and 1,673 target gene pairs. A total of 23 AktB3 genes were predicted to be targets of 20 miRNAs, with 11 miRNAs specifically targeting the ARF subfamily genes. Particularly, miR160-x, miR160-z, and miR167-z were predicted to target ARF subfamily genes, potentially influencing seed development. Moreover, the miRNA-B3 regulatory modules, especially involving ARF genes and miR160/167, require further study to clarify their roles in seed development., Conclusions: These findings contribute valuable resources for future functional studies of the molecular regulatory networks governing seed development in A. trifoliata., (© 2024. The Author(s).)

Conserved protein-coding sequences are critical for maintaining protein function across species. Odorant receptors (ORs), a large poorly understood multigene family responsible for odor detection, lack comprehensive classification methods that reflect their functional diversity. In this study, we propose a new approach called conserved motif-based classification (CMC) for classifying ORs based on amino acid sequence similarities within conserved motifs. Specifically, we focused on three well-conserved motifs: MAYDRYVAIC in TM3, KAFSTCASH in TM6, and PMLNPFIY in TM7. Using an unsupervised clustering technique, we classified human ORs (hORs) into two main clusters with six sub-clusters. CMC partly reflects previously identified subfamilies, revealing altered residue positions among the sub-clusters. These altered positions interacted with specific residues within or adjacent to the transmembrane domain, suggesting functional implications. Furthermore, we found that the CMC correlated with both ligand responses and ectopic expression patterns, highlighting its relevance to OR function. This conserved motif-based classification will help in understanding the functions and features that are not understood by classification based solely on entire amino acid sequence similarity., (© 2024. The Author(s).)

The rising incidence of Autism Spectrum Disorder (ASD) has become a major concern, affecting children's psychological well-being and placing a significant strain on healthcare systems. Despite its impact, the etiological mechanisms underpinning ASD remain elusive. This study leveraged dorsolateral prefrontal cortex gene data from 452 individuals of European descent, sourced from the CommonMindConsortium, and examined ASD-related gene expression data from the Gene Expression Omnibus (GEO) database (GSE18123), along with Genome-Wide Association Studies (GWAS) data from the Lundbeck Foundation Integrated Psychiatric Research and Psychiatric Genomics Consortium. Expression quantitative trait loci data were sourced from the GTExv8 database. We employed Transcriptome-Wide Association Studies (TWAS) and Weighted Gene Co-expression Network Analysis (WGCNA) to pinpoint genes within ASD-associated susceptibility gene families (ARHGAP, MAP). Four genes-ARHGAP27, MAPT, ARHGAP19, and MAP1B-were scrutinized, and their biological implications were elucidated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Protein-Protein Interaction (PPI) analysis and conditional analysis within the TWAS framework helped identify pivotal genes (ARHGAP27, MAPT). A subsequent verification phase involving Mendelian Randomization (MR) evaluated the potential causal links between the identified genes and ASD. The findings revealed no causal association between ARHGAP19, MAP1B, and ASD. In contrast, significant causal relationships were established for ARHGAP27 and MAPT, suggesting that ARHGAP27 may elevate ASD risk as a susceptibility gene, whereas MAPT appears to reduce the risk as a protective gene., Competing Interests: Enter: The authors have declared that no competing interests exist., (Copyright: © 2024 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The species Bacillus velezensis is known for its biosynthetic potential and metabolic versatility in producing several secondary metabolites and promoting plant growth. In this study, we isolated two B. velezensis strains, WGTg-8 and WGTm-299, from the termite gut and termitarium, which exhibited antimicrobial activity against multiple clinical and phytopathogens. The whole genomes of these strains were sequenced using the Illumina platform and annotated. The genome mining of the draft genome sequences revealed 48 biological gene clusters (BGCs) responsible for synthesizing various secondary metabolites. The construction of the similarity network of the BGCs and the comparative analysis with the genetically related organisms are aided in the identification of metabolites produced by these strains. We identified biosynthetic gene clusters (BGCs) coding for macrolactin H, bacilysin, bacillibactin, amylocyclin, comX4, and LCI, found in both strains with 100% similarity. The difficidin, bacillaene, thusin_alpha, and cericidin BGCs are exclusively found in strain WGTg-8, while the colicin BGC is exclusively present in the WGTm-299 strain. The fengycin and surfactin gene clusters are present in both strains with 80% similarity. Furthermore, 28 putative NRPS BGCs, NRPS-T1PKS hybrid clusters, a T1PKS, and a bacteriocin BGC were identified with very low similarity (≤ 25%) or no similarity with known antibiotics. In addition, we found several genes coding for plant growth-promoting properties, including nitrogen metabolism, hormone synthesis, sulfur metabolism, phosphate metabolism, and a few other properties., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Iron (Fe) and Zinc (Zn) are essential micronutrients for plant growth and development. ZIP (ZRT, IRT-like protein) transporters, known for their role in the regulation of Zinc and Iron uptake, are pivotal in facilitating the absorption, transport, and maintenance of Fe/Zn homeostasis in plants. Nicotiana tabacum has been widely used as a model plant for gene function analysis; however, the tobacco ZIP genes have not been identified systematically. In this study, we have identified a comprehensive set of 32 NtZIP genes, which were phylogenetically categorized into three distinct clades. The gene structures, characterized by their exon/intron organization, and the protein motifs are relatively conserved, particularly among genes within the same clade. These NtZIP genes exhibit an uneven distribution across 12 chromosomes. The gene localization analysis revealed the presence of 11 pairs of homeologous locus genes and 7 pairs of tandem duplication genes within the genome. To further explore the functionality of these genes, real-time quantitative reverse transcription PCR was employed to assess their expression levels in roots subjected to metal deficiency. The results indicated that certain NtZIP genes are specifically upregulated in response to either Fe or Zn deficiency. Additionally, the presence of specific cis-elements within their promoter regions, such as the E-box associated with Fe deficiency response and the ZDRE box linked to Zn deficiency response, was identified. This study lays a foundational groundwork for future research into the biological functions of NtZIP genes in tobacco in micronutrient regulation and homeostasis., (© The Author(s) 2024. Published by Oxford University Press.)

Heat stress has become one of the abiotic stresses that pose an increasing threat to maize production due to global warming. The Hsp20 gene family confers tolerance to various abiotic stresses in plants. However, very few Hsp20s have been identified in relation to maize thermotolerance. In this study, we conducted a comprehensive study of Hsp20s involved in thermotolerance in maize. A total of 33 maize Hsp20 genes (ZmHsp20s) were identified through scanning for a conserved α-crystalline domain (ACD), and they were categorized into 14 subfamilies based on phylogenetic analysis. These genes are distributed across all maize chromosomes and nine of them are in regions previously identified as heat-tolerance quantitative trait loci (hrQTL). These hrQTL-associated ZmHsp20s show variation in tissue-specific expression profiles under normal conditions, and seven of them possess 1-5 heat stress elements in their promoters. The integration of RNA-seq data with real-time RT-PCR analysis indicated that ZmHsp23.4, ZmHsp22.8B and ZmHsp18 were dramatically induced under heat stress. Additionally, these genes exhibited co-expression patterns with key ZmHsfs, which are crucial in the heat tolerance pathway. When a null mutant carrying a frame-shifted ZmHsp18 gene was subjected to heat stress, its survival rate decreased significantly, indicating a critical role of ZmHsp18 in maize thermotolerance. Our study lays the groundwork for further research into the roles of ZmHsp20s in enhancing maize's thermotolerance., (© 2024. The Author(s).)

Streptomyces species can form beneficial relationships with hosts as endophytes, including the phytopathogen-inhibiting strain, Streptomyces griseoaurantiacus MH191, isolated from wheat plants. Using genomic characterization and untargeted metabolomics, we explored the capacity of strain MH191 to inhibit a range of fungal phytopathogens through the production of secondary metabolites. Complete genome assembly of strain MH191 predicted 24 biosynthetic gene clusters. Secondary metabolite production was assessed following culture on six different media, with the detection of 205 putative compounds. Members of the manumycin family, undecylprodigiosin, and desferrioxamine were identified as the predominant metabolites. Antifungal activity was validated for undecylprodigiosin and manumycin. These compounds were produced from different BGCs, which showed similarity to asukamycin, undecylprodigiosin, and FW0622 gene clusters, respectively. The growth of strain MH191 on different media illustrated the metabolic regulation of these gene clusters and the strain's extended chemical potential, with the asukamycin gene cluster alone, producing a variety of antifungal metabolites. The study highlights the extended chemical capability of strain MH191, which could be exploited as a biological control agent for designing future crop protection solutions.

Fusarium meridionale is one of the pathogens causing maize ear rot, it produce bioactive secondary metabolites may threaten humans food safty, however, the production mechanism of the secondary metabolites and their interaction with maize ear remains poorly understood. To facilitate related studies, we sequenced and assembled the genome of F. meridionale strain JX18-4. The size of F. meridionale JX18-4 genome is 37.11 Mbp, include four nuclear chromosome contigs that consists of 11920 coding genes and one mitochondrial contig. 95.64% gene synteny collinearity was found between the assembly and the reference genomes F. graminearum strain PH-1. Compared to the sequences of seconary matabolism gene clusters sequences reported previously, the stain JX18-4 was predicted potential producing 8 clusters, including nivalenol, zearalenone, aurofusarin, fusarielin, fusaristatin A, fusarin, fusarubin and butenolide. This study aims to reveal the molecular mechanism of secondary metabolites producing, and the genomic information of JX18-4 will provide resources for the study of biological control mechanisms and plant-microbe interactions.