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This study explores the organization, conservation, and diversity of biosynthetic gene clusters (BGCs) among Bacillus sp. strain BH32, a plant-beneficial bacterial endophyte, and its closest non-type Bacillus cereus group strains. BGC profiles were predicted for each of the 17 selected strains using antiSMASH, resulting in the detection of a total of 198 BGCs. We quantitatively compared the BGCs and analyzed their conservation, distribution, and evolutionary relationships. The study identified both conserved and singleton BGCs across the studied Bacillus strains, with minimal variation, and discovered two major BGC synteny blocks composed of homologous BGCs conserved within the B. cereus group. The identified BGC synteny blocks provide insight into the evolutionary relationships and diversity of BGCs within this complex group.

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The rapid expansion of multi-omics data has transformed biological research, offering unprecedented opportunities to explore complex genomic relationships across diverse organisms. However, the vast volume and heterogeneity of these datasets presents significant challenges for analyses. Here we introduce SocialGene, a comprehensive software suite designed to collect, analyze, and organize multi-omics data into structured knowledge graphs, with the ability to handle small projects to repository-scale analyses. Originally developed to enhance genome mining for natural product drug discovery, SocialGene has been effective across various applications, including functional genomics, evolutionary studies, and systems biology. SocialGene's concerted Python and Nextflow libraries streamline data ingestion, manipulation, aggregation, and analysis, culminating in a custom Neo4j database. The software not only facilitates the exploration of genomic synteny but also provides a foundational knowledge graph supporting the integration of additional diverse datasets and the development of advanced search engines and analyses. This manuscript introduces some of SocialGene's capabilities through brief case studies including targeted genome mining for drug discovery, accelerated searches for similar and distantly related biosynthetic gene clusters in biobank-available organisms, integration of chemical and analytical data, and more. SocialGene is free, open-source, MIT-licensed, designed for adaptability and extension, and available from github.com/socialgene.

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With the current spread of clinically relevant multidrug-resistant (MDR) pathogens, insufficient unearthing of new anti-infectives, and the high cost required for approval of new antimicrobial agents, a strong need for getting these agents via more economic and other alternative routes has emerged. With the discovery of the biosynthetic pathways of various antibiotics pointing out the role of each gene/protein in their antibiotic-producing strains, it became apparent that the biosynthetic gene clusters can be manipulated to produce modified antibiotics. This new approach is known as the combinatorial biosynthesis of new antibiotics which can be employed for obtaining novel derivatives of these valuable antibiotics using genetically modified antibiotic-producing strains (pathway engineering). In this review and based on the available biosynthetic gene clusters of the major aminoglycoside antibiotics (AGAs), the possible alterations or modifications that could be done by co-expression of certain gene(s) previously known to be involved in unique biosynthetic steps have been discussed. In this review defined novel examples of modified AGA using this approach were described and the information provided will act as a platform of researchers to get and develop new antibiotics by the antibiotic-producing bacterial strains such as Streptomyces, Micromonospora,…etc. This way, novel antibiotics with new biological activities could be isolated and used in the treatment of infectious diseases conferring resistance to existing antibiotics.

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The mitochondrial genome of Dematophora necatrix is 121,350 base pairs in length with a G + C content of 30.19%. Phylogenetic analysis showed that D. necatrix grouped with other members of the Xylariaceae, with which its mitogenome also shares a broadly similar architecture and gene content. The D. necatrix mitogenome contains 14 protein-coding and 26 tRNA-encoding genes, as well as one copy each of the rnl, rns, rps3 and nat1 genes. However, as much as 80% of this genome is intronic or non-coding. This is likely due to expansions and rearrangements caused by the large number of group I introns and the homing endonucleases and reverse-transcriptases they encode. Our study thus provides a valuable foundation from which to explore the mitochondrion's role in the biology of D. necatrix, and also serves as a resource for investigating the pathogen's population biology and general ecology.

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Cyanobacteria are globally occurring photosynthetic bacteria notable for their contribution to primary production and production of toxins which have detrimental ecosystem impacts. Furthermore, cyanobacteria can form mutualistic symbiotic relationships with a diverse set of eukaryotes, including land plants, aquatic plankton and fungi. Nevertheless, not all cyanobacteria are found in symbiotic associations suggesting symbiotic cyanobacteria have evolved specializations that facilitate host-interactions. Photosynthetic capabilities, nitrogen fixation, and the production of complex biochemicals are key functions provided by host-associated cyanobacterial symbionts. To explore if additional specializations are associated with such lifestyles in cyanobacteria, we have conducted comparative phylogenomics of molecular functions and of biosynthetic gene clusters (BGCs) in 984 cyanobacterial genomes. Cyanobacteria with host-associated and symbiotic lifestyles were concentrated in the family Nostocaceae, where eight monophyletic clades correspond to specific host taxa. In agreement with previous studies, symbionts are likely to provide fixed nitrogen to their eukaryotic partners, through multiple different nitrogen fixation pathways. Additionally, our analyses identified chitin metabolising pathways in cyanobacteria associated with specific host groups, while obligate symbionts had fewer BGCs. The conservation of molecular functions and BGCs between closely related symbiotic and free-living cyanobacteria suggests the potential for additional cyanobacteria to form symbiotic relationships than is currently known.

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Metallophores are small molecule chelators that many microbes use to obtain trace metals from their environment. Through genome mining, where genomes are scanned for metallophore biosynthesis genes, one can not only identify which organisms are likely to produce a metallophore, but also predict the metallophore structure, thus preventing undesired reisolation of known compounds and accelerating characterization. Furthermore, the presence of accessory genes for the transport, utilization, and regulation can suggest the biological function and fate of a metallophore. Modern, user-friendly tools have made powerful genomic analyses accessible to scientists with no bioinformatics experience, but these tools are often not utilized to their full potential. This chapter provides an introduction to metallophore genomics and demonstrates how to use the free, publicly available antiSMASH platform to infer metallophore function and structure.

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Bacillus thuringiensis Berliner is recognized as a predominant bioinsecticide but its antifungal potential has been relatively underexplored. A novel B. thuringiensis strain NBAIR BtAr was isolated and morphologically characterized using light and scanning electron microscopy, revealing presence of bipyramidal, cuboidal, and spherical parasporal crystals. The crude form of lipopeptides was extracted from NBAIR BtAr and assessed for its antagonistic activity in vitro, and demonstrated 100 % inhibition of Sclerotium rolfsii Sacc. at a minimum inhibitory concentration of 50 µL of the crude lipopeptide extract per mL of potato dextrose agar. To identify the antagonistic genes responsible, we performed whole genome sequencing of NBAIR BtAr, revealing the presence of circular chromosome of 5,379,913 bp and 175,362 bp plasmid with 36.06 % guanine-cytosine content and 5814 protein-coding sequences. Average nucleotide identity and whole genome phylogenetic analysis delineated the NBAIR BtAr strain as konkukian serovar. Gene ontology analysis revealed associations of 1474, 1323, and 1833 genes with biological processes, molecular function, and cellular components, respectively. Antibiotics & secondary metabolite analysis shell analysis of the whole genome yielded secondary metabolites biosynthetic gene clusters with 100 %, 85 %, 40 %, and 35 % similarity for petrobactin, bacillibactin, fengycin, and paenilamicin, respectively. Also, novel biosynthetic gene clusters, along with antimicrobial genes, including zwittermicin A, chitinase, and phenazines, were identified. Moreover, the presence of eight bacteriophage sequences, 18 genomic islands, insertion sequences, and one CRISPR region indicated prior occurrences of genetic exchange and thus improved competitive fitness of the strain. Overall, the whole genome sequence of NBAIR BtAr is presented, with its taxonomic classification and critical genetic attributes that contribute to its strong antagonistic activity against S. rolfsii.

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Advances in genome engineering and associated technologies have reinvigorated natural products research. Here we highlight the latest developments in the field across the discover-design-build-test-learn cycle of bioengineering, from recent progress in computational tools for AI-supported genome mining, enzyme and pathway engineering, and compound identification to novel host systems and new techniques for improving production levels, and place these trends in the context of responsible research and innovation, emphasizing the importance of anticipatory analysis at the early stages of process development.

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To investigate their roles in extracellular electron transfer (EET), the porin-cytochrome (pcc) gene clusters Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 of the Gram-negative bacterium Geobacter metallireducens were deleted. Failure to delete all pcc gene clusters at the same time suggested their essential roles in extracellular reduction of Fe(III)-citrate by G. metallireducens. Deletion of Gmet0825-0828 had no impact on bacterial reduction of Fe(III)-citrate but diminished bacterial reduction of ferrihydrite and abolished anode reduction and direct interspecies electron transfer (DIET) to Methanosarcina barkeri and Geobacter sulfurreducens. Although it had no impact on the bacterial reduction of Fe(III)-citrate, deletion of Gmet0908-0910 delayed ferrihydrite reduction, abolished anode reduction, and diminished DIET. Deletion of Gmet0911-0913 had little impact on DIET but diminished bacterial reductions of Fe(III)-citrate, ferrihydrite, and anodes. Most importantly, deletions of both Gmet0825-0828 and Gmet0908-0910 restored bacterial reduction of ferrihydrite and anodes and DIET. Enhanced expression of Gmet0911-0913 in this double mutant when grown in coculture with G. sulfurreducens ΔhybLΔfdnG suggested that this cluster might compensate for impaired EET functions of deleting Gmet0825-0828 and Gmet0908-0910. Thus, these pcc gene clusters played essential, distinct, overlapping, and compensatory roles in EET of G. metallireducens that are difficult to characterize as deletion of some clusters affected expression of others. The robustness of these pcc gene clusters enabled G. metallireducens to mediate EET to different acceptors for anaerobic growth even when two of its three pcc gene clusters were inactivated by mutation. The results from this investigation provide new insights into the roles of pcc gene clusters in bacterial EET. IMPORTANCE: The Gram-negative bacterium Geobacter metallireducens is of environmental and biotechnological significance. Crucial to the unique physiology of G. metallireducens is its extracellular electron transfer (EET) capability. This investigation sheds new light on the robust roles of the three porin-cytochrome (pcc) gene clusters, which are directly involved in EET across the bacterial outer membrane, in the EET of G. metallireducens. In addition to their essential roles, these gene clusters also play distinct, overlapping, and compensatory roles in the EET of G. metallireducens. The distinct roles of the pcc gene clusters enable G. metallireducens to mediate EET to a diverse group of electron acceptors for anaerobic respirations. The overlapping and compensatory roles of the pcc gene clusters enable G. metallireducens to maintain and restore its EET capability for anaerobic growth when one or two of its three pcc gene clusters are deleted from the genome.

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Multidrug-resistant pathogens pose an earnest risk to human health. Therefore, new antibiotics need to be developed quickly. Most of the antibiotics we use today are derived from secondary metabolites, which are produced by plants. Genome mining tools allow us to detect biosynthetic gene clusters (BGCs) responsible for the production of secondary metabolites. Focusing on the most promising BGCs-coding antibiotics with unique pathways is currently a challenge. In silico approach like genome mining are used to visualise the action of these bioactive chemicals. Camelina sativa is a well-known medicinal plant and it would be interesting to study its secondary metabolites. In this work, we found seven bioactive compounds in this plant using the genome mining approach. Further, the clusters of genes involved in the biosynthesis of these compounds were analysed with their metabolic pathways. This work illuminates new ground on the evolution of BGCs for the nutritional improvement of C. sativa.

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Entomopathogenic fungi belonging to the Order Hypocreales are renowned for their ability to infect and kill insect hosts, while their endophytic mode of life and the beneficial rhizosphere effects on plant hosts have only been recently recognized. Understanding the molecular mechanisms underlying their different lifestyles could optimize their potential as both biocontrol and biofertilizer agents, as well as the wider appreciation of niche plasticity in fungal ecology. This study describes the comprehensive whole genome sequencing and analysis of one of the most effective entomopathogenic and endophytic EPF strains, Metarhizium brunneum V275 (commercially known as Lalguard Met52), achieved through Nanopore and Illumina reads. Comparative genomics for exploring intraspecies variability and analyses of key gene sets were conducted with a second effective EPF strain, M. brunneum ARSEF 4556. The search for strain- or species-specific genes was extended to M. brunneum strain ARSEF 3297 and other species of genus Metarhizium, to identify molecular mechanisms and putative key genome adaptations associated with mode of life differences. Genome size differed significantly, with M. brunneum V275 having the largest genome amongst M. brunneum strains sequenced to date. Genome analyses revealed an abundance of plant-degrading enzymes, plant colonization-associated genes, and intriguing intraspecies variations regarding their predicted secondary metabolic compounds and the number and localization of Transposable Elements. The potential significance of the differences found between closely related endophytic and entomopathogenic fungi, regarding plant growth-promoting and entomopathogenic abilities, are discussed, enhancing our understanding of their diverse functionalities and putative applications in agriculture and ecology.

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Members of the bacterial genus Pantoea produce a variety of antimicrobial products that are effective against plant, animal, and human pathogens. To date, little is known about the distribution and evolutionary history of these clusters. We surveyed the public databases for the 12 currently known antibiotic biosynthetic gene clusters found across Pantoea strains to determine their distribution. We show that some clusters, namely pantocin B, PNP-3, and PNP-4 are found strictly in Pantoea, while agglomerin, andrimid, AGA, dapdiamide, herbicolin, PNP-1, PNP-2, PNP-5, and pantocin A, are more broadly distributed in distantly related genera within Vibrionaceae, Pectobacteriaceae, Yersiniaceae, Morganellaceae, and Hafniaceae. We evaluated the evolutionary history of these gene clusters relative to a cpn60-based species tree, considering the flanking regions of each cluster, %GC, and presence of mobile genetic elements, and identified potential occurrences of horizontal gene transfer. Lastly, we also describe the biosynthetic gene cluster of pantocin B in the strain Pantoea agglomerans Eh318 more than 20 years after this antibiotic was first described.

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Advanced techniques can accelerate the pace of natural product discovery from microbes, which has been lagging behind the drug discovery era. Therefore, the present review article discusses the various interdisciplinary and cutting-edge techniques to present a concrete strategy that enables the high-throughput screening of novel natural compounds (NCs) from known microbes. Recent bioinformatics methods revealed that the microbial genome contains a huge untapped reservoir of silent biosynthetic gene clusters (BGC). This article describes several methods to identify the microbial strains with hidden mines of silent BGCs. Moreover, antiSMASH 5.0 is a free, accurate, and highly reliable bioinformatics tool discussed in detail to identify silent BGCs in the microbial genome. Further, the latest microbial culture technique, HiTES (high-throughput elicitor screening), has been detailed for the expression of silent BGCs using 500-1000 different growth conditions at a time. Following the expression of silent BGCs, the latest mass spectrometry methods are highlighted to identify the NCs. The recently emerged LAESI-IMS (laser ablation electrospray ionization-imaging mass spectrometry) technique, which enables the rapid identification of novel NCs directly from microtiter plates, is presented in detail. Finally, various trending 'dereplication' strategies are emphasized to increase the effectiveness of NC screening.

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Cyanobacteria, as oxygenic phototrophs, offer significant potential for sustainable biotechnology applications. Cyanobacterial natural products, with antimicrobial, anticancer, and plant growth-promoting properties, hold promise in pharmaceuticals, agriculture, and environmental remediation. By leveraging advanced technologies, cyanobacteria can significantly impact various industries, supporting the green biotechnology agenda. Recent advancements in integrated omics, orphan gene cluster activation, genetic manipulation, and chemo-enzymatic methods are expanding their biotechnological relevance. Omics technologies revolutionize cyanobacterial natural product research by facilitating biosynthetic gene cluster identification. Heterologous expression and pathway reconstitution enable complex natural product production, while high-titer strategies like metabolic engineering enhance yields. Interdisciplinary research and technological progress position cyanobacteria as valuable sources of bioactive compounds, driving sustainable biotechnological practices forward.

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Antibiotic resistance is a worldwide problem that imposes a devastating effect on developing countries and requires immediate interventions. Initially, most of the antibiotic drugs were identified by culturing soil microbes. However, this method is prone to discovering the same antibiotics repeatedly. The present study employed a shotgun metagenomics approach to investigate the taxonomic diversity, functional potential, and biosynthetic capacity of microbiomes from two natural agricultural farmlands located in Bekeka and Welmera Choke Kebelle in Ethiopia for the first time. Analysis of the small subunit rRNA revealed bacterial domain accounting for 83.33% and 87.24% in the two selected natural farmlands. Additionally, the analysis showed the dominance of Proteobacteria representing 27.27% and 28.79% followed by Actinobacteria making up 12.73% and 13.64% of the phyla composition. Furthermore, the analysis revealed the presence of unassigned bacteria in the studied samples. The metagenome functional analysis showed 176,961 and 104, 636 number of protein-coding sequences (pCDS) from the two samples found a match with 172,655 and 102, 275 numbers of InterPro entries, respectively. The Genome ontology annotation suggests the presence of 5517 and 3293 pCDS assigned to the "biosynthesis process". Numerous Kyoto Encyclopedia of Genes and Genomes modules (KEGG modules) involved in the biosynthesis of terpenoids and polyketides were identified. Furthermore, both known and novel Biosynthetic gene clusters, responsible for the production of secondary metabolites, such as polyketide synthases, non-ribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptides (Ripp), and Terpene, were discovered. Generally, from the results it can be concluded that the microbiomes in the selected sampling sites have a hidden functional potential for the biosynthesis of secondary metabolites. Overall, this study can serve as a strong preliminary step in the long journey of bringing new antibiotics to the market.

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Bacillus atrophaeus HAB-5 is a plant growth-promoting rhizobacterium (PGPR) that exhibits several biotechnological traits, such as enhancing plant growth, colonizing the rhizosphere, and engaging in biocontrol activities. In this study, we conducted whole-genome sequencing of B. atrophaeus HAB-5 using the single-molecule real-time (SMRT) sequencing platform by Pacific Biosciences (PacBio; United States), which has a circular chromosome with a total length of 4,083,597 bp and a G + C content of 44.21%. The comparative genomic analysis of B. atrophaeus HAB-5 with other strains, Bacillus amyloliquefaciens DSM7, B. atrophaeus SRCM101359, Bacillus velezensis FZB42, B. velezensis HAB-2, and Bacillus subtilis 168, revealed that these strains share 2,465 CDSs, while 599 CDSs are exclusive to the B. atrophaeus HAB-5 strain. Many gene clusters in the B. atrophaeus HAB-5 genome are associated with the production of antimicrobial lipopeptides and polypeptides. These gene clusters comprise distinct enzymes that encode three NRPs, two Transat-Pks, one terpene, one lanthipeptide, one T3PKS, one Ripp, and one thiopeptide. In addition to the likely IAA-producing genes (trpA, trpB, trpC, trpD, trpE, trpS, ywkB, miaA, and nadE), there are probable genes that produce volatile chemicals (acoA, acoB, acoR, acuB, and acuC). Moreover, HAB-5 contained genes linked to iron transportation (fbpA, fetB, feuC, feuB, feuA, and fecD), sulfur metabolism (cysC, sat, cysK, cysS, and sulP), phosphorus solubilization (ispH, pstA, pstC, pstS, pstB, gltP, and phoH), and nitrogen fixation (nif3-like, gltP, gltX, glnR, glnA, nadR, nirB, nirD, nasD, narl, narH, narJ, and nark). In conclusion, this study provides a comprehensive genomic analysis of B. atrophaeus HAB-5, pinpointing the genes and genomic regions linked to the antimicrobial properties of the strain. These findings advance our knowledge of the genetic basis of the antimicrobial properties of B. atrophaeus and imply that HAB-5 may employ a variety of commercial biopesticides and biofertilizers as a substitute strategy to increase agricultural output and manage a variety of plant diseases.

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Microbial genomes produced by standard single-cell amplification methods are largely incomplete. Here, we show that primary template-directed amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard multiple displacement amplification-based approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters.

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Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggest their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. To address the origins of this complexity and understand its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery.IMPORTANCEPolycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically important bacteria, including Streptomyces and Lysobacter spp. Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggest new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize the practical limitations of genomics-based discovery by exposing hidden complexity.

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Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes form a major line of defense in plants, acting in both pathogen recognition and resistance machinery activation. NLRs are reported to form large gene clusters in limber pine (Pinus flexilis), but it is unknown how widespread this genomic architecture may be among the extant species of conifers (Pinophyta). We used comparative genomic analyses to assess patterns in the abundance, diversity, and genomic distribution of NLR genes. Chromosome-level whole genome assemblies and high-density linkage maps in the Pinaceae, Cupressaceae, Taxaceae, and other gymnosperms were scanned for NLR genes using existing and customized pipelines. The discovered genes were mapped across chromosomes and linkage groups and analyzed phylogenetically for evolutionary history. Conifer genomes are characterized by dense clusters of NLR genes, highly localized on one chromosome. These clusters are rich in TNL-encoding genes, which seem to have formed through multiple tandem duplication events. In contrast to angiosperms and nonconiferous gymnosperms, genomic clustering of NLR genes is ubiquitous in conifers. NLR-dense genomic regions are likely to influence a large part of the plant's resistance, informing our understanding of adaptation to biotic stress and the development of genetic resources through breeding.

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