Inter-species horizontal transfer of biosynthetic gene clusters: an evolutionary driver for chemical diversity in bacterial communities.

The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.

SummaryTo quantify the major environmental drivers of stream bacterial population dynamics, we modelled temporal differences in stream bacterial communities to quantify community shifts, including those relating to cyclical seasonal variation and more sporadic bloom events. We applied Illumina MiSeq 16S rRNA bacterial gene sequencing of 892 stream biofilm samples, collected monthly for 36‐months from six streams. The streams were located a maximum of 118 km apart and drained three different catchment types (forest, urban and rural land uses). We identified repeatable seasonal patterns among bacterial taxa, allowing their separation into three ecological groupings, those following linear, bloom/trough and repeated, seasonal trends. Various physicochemical parameters (light, water and air temperature, pH, dissolved oxygen, nutrients) were linked to temporal community changes. Our models indicate that bloom events and seasonal episodes modify biofilm bacterial populations, suggesting that distinct microbial taxa thrive during these events including non‐cyanobacterial community members. These models could aid in determining how temporal environmental changes affect community assembly and guide the selection of appropriate statistical models to capture future community responses to environmental change.

Bacterial secondary metabolites are crucial bioactive compounds with significant therapeutic potential, playing key roles in ecological processes and the discovery of novel antimicrobial agents and natural products. Cenotes, as extreme environments, harbour untapped microbial diversity and hold an interesting potential as sources of novel secondary metabolites. While research has focused on the fauna and flora of cenotes, the study of their microbial communities and their biosynthetic capabilities remains limited. Advances in metagenomics and genome sequencing have greatly improved the capacity to explore these communities and their metabolites. In this study, we analysed the microbial diversity and biotechnological potential of micro-organisms inhabiting sediments from a coastal cenote. Metagenomic analyses revealed a rich diversity of bacterial and archaeal communities, containing several novel biosynthetic gene clusters (BGCs) linked to secondary metabolite production. Notably, polyketide synthase BGCs, including those encoding ladderanes and aryl-polyenes, were identified. Bioinformatics analyses of these pathways suggest the presence of compounds with potential industrial and pharmaceutical applications. These findings highlight the biotechnological value of cenotes as reservoirs of secondary metabolites. The study and conservation of these ecosystems are essential to facilitate the discovery of new bioactive compounds that could benefit various industries.

Background: Microbial secondary metabolites (SM), especially those produced by soil microorganisms, have been a valuable source of antibiotics, antitumor agents, pigments, growth-promoting substances, etc., with tremendous market potential. These molecules are encoded by biosynthetic gene clusters (BGCs) within the bacterial genome. Their synthesis confers survival advantages by facilitating chemical defense, interspecies communication, and adaptation to well-defined ecological niches. Caves, particularly Movile Cave (Romania) - a sulfidic autotrophic-based ecosystem - can be considered extreme environments suited to investigate and discover novel bioactive microbial molecules. Here, low nutrient availability can lead to resource competition and, consequently, antimicrobial production to deter nearby microbial competitors. Aim: Our study focused on highlighting the biosynthetic gene clusters (BGCs) and biosynthetic potential of the sediment-associated microbiome in Movile Cave. Methods: Over 100 high-quality metagenome-assembled genomes (MAGs) were retrieved by whole-community shotgun-sequencing of 7 sediment samples collected in different Movile Cave’s galleries (Chiciudean et al. 2022). Detected MAGs were then analyzed for the presence of BGCs by antiSMASH (v. 6.1.1) (Blin et al. 2021) whereas antibiotic resistance genes were predicted by ResFinder (v. 4.1) (Florensa et al. 2022). The statistical analysis of BGCs data was performed by Past (v. 4.03). Results: We detected 637 BGCs across 106 high-quality MAGs that were affiliated to 22 phyla. The diversity of predicted BGCs varies across sediment samples with no apparent correlation to the number of analyzed MAGs per sample. The MAGs recovered from the sulfidic water-sediment interface (sample code PMV4) had the lowest alpha BGCs diversity among all sampled locations and it was clearly distinct in BGCs composition and abundance (β-diversity) from dry gallery samples (PMV7 and PMV8). The most abundant BGCs predicted in Movile Cave metagenomic dataset encode for terpenes, non-ribosomal peptides (NRPs) and ribosomally synthesised and post-translationally modified peptides (RIPPs). Acidobacteriota and Chloroflexota- affiliated MAGs were most enriched in BCG containing 20 and 23 BGCs per MAG, respectively, in contrast with the candidate phylum Ca. Patescibacteria-related MAGs that showed no SM biosynthetic capabilities. Two antimicrobial resistance (AR) genes (ole(C), oqxB) encoding resistance to antibiotics (i.e. oleandomycin, chloramphenicol, ciprofloxacin, trimethoprim) and disinfectants were identified in MAGs affiliated with the class Actinomycetia and Gammaproteobacteria. Based on the analyzed data, the biosynthetic potential of Movile Cave is significant compared with other microbiomes (Donia et al. 2014) and has a pronounced degree of novelty whereas the resistome (that is the genetic potential for antibiotic resistance) is reduced. Considering the uncommon futures of Movile Cave environment, future in-depth analysis of the identified BGCs might lead to the discovery of novel bioactive compounds. Additionally, the seclusion of this environment may provide an exciting opportunity for surveying the occurrence and environmental drivers of natural AR traits.

The human gut microbiota contains biosynthetic gene clusters (BGCs) that encode bioactive secondary metabolites, which play pivotal roles in microbe-microbe and host-microbe interactions and serve as a rich source of pharmaceutical lead compounds. Understanding the horizontal transfer of BGCs can reveal insights into microbial adaptation, resource utilization, and evolutionary mechanisms, thereby advancing biotechnological applications. Despite its importance, horizontal transfer of BGCs within the gut microbiota remains poorly understood. In this study, we introduce a novel tool, the Horizontally Transferred Biosynthetic Gene Clusters Finder (HTBGC-Finder), designed to systematically identify potential horizontally transferred BGCs (HTBGCs) within the extensive human gut microbiota. Using HTBGC-Finder, we identified 81 potential HTBGCs, underscoring the prevalence and significance of horizontal gene transfer in shaping the genetic landscape of the gut microbiome. Remarkably, ribosomally synthesized and post-translationally modified peptides (RiPPs) constituted the majority of these HTBGCs (76 out of 81, 93.83%), exhibiting a significantly higher transfer rate compared to non-RiPPs (Chi-squared test, p < 0.001). Upon detailed examination of BGCs, cyclic-lactone-autoinducer (CLA) and RiPP recognition element (RRE)-containing BGCs were predominant, representing nearly three-quarters of the total (45, or 55.56%, and 14, or 17.28%, respectively). Notably, CLA BGCs also demonstrated a higher transfer rate than non-CLA BGCs (Chi-squared test, p < 0.001). Taxonomy profiling revealed that horizontal BGC transfer occurred exclusively in the phyla Bacteroidota (synonym Bacteroidetes) and Bacillota (synonym Firmicutes), with 50 and 31 instances, respectively. Furthermore, cross-phylum transfer events were observed, highlighting the complex interactions between the gut microbiota and host health. These findings offer valuable insights into the horizontal transfer dynamics of BGCs within the gut microbiome and their potential implications for host-microbiota interactions.

Marine actinobacterium Streptomyces xinghaiensis NRRL B-24674T has been characterized as a novel species, but thus far, its biosynthetic potential remains unexplored. In this study, the high-quality genome sequence of S. xinghaiensis NRRL B-24674T was obtained, and the production of anticomplement agents, xiamycin analogs, and siderophores was investigated by genome mining. Anticomplement compounds are valuable for combating numerous diseases caused by the abnormal activation of the human complement system. The biosynthetic gene cluster (BGC) nrps1 resembles that of complestatins, which are potent microbial-derived anticomplement agents. The identification of the nrps1 BGC revealed a core peptide that differed from that in complestatin; thus, we studied the anticomplement activity of this strain. The culture broth of S. xinghaiensis NRRL B-24674T displayed good anticomplement activity. Subsequently, the disruption of the genes in the nrps1 BGC resulted in the loss of anticomplement activity, confirming the involvement of this BGC in the biosynthesis of anticomplement agents. In addition, the mining of the BGC tep5, which resembles that of the antiviral pentacyclic indolosesquiterpene xiamycin, resulted in the discovery of nine xiamycin analogs, including three novel compounds. In addition to the BGCs responsible for desferrioxamine B, neomycin, ectoine, and carotenoid, 18 BGCs present in the genome are predicted to be novel. The results of this study unveil the potential of S. xinghaiensis as a producer of novel anticomplement agents and provide a basis for further exploration of the biosynthetic potential of S. xinghaiensis NRRL B-24674T for the discovery of novel bioactive compounds by genome mining.

Water and nitrogen sources have always been the primary limiting factors for vegetation growth in arid and semi-arid regions and play an important role in the physiological ecology of vegetation. In this work, we studied the effects of water deficit and nitrogen addition on the physiological traits and rhizosphere bacterial microbial community of Haloxylon ammodendron seedlings in sterilized and non-sterilized soil habitats. A pot experiment was conducted to control the water and nitrogen sources of H. ammodendron seedlings. The water deficit treatment was divided into two groups based on gradient: a normal water group (CK, 70% field water holding capacity) and water deficit group (D, 30% field water holding capacity). The nitrogen addition treatment was divided into a no addition group (CK, 2.8 mg·kg−1) and addition group (N, 22.4 mg·kg−1). At the end of the growing season, the biochemical indexes of H. ammodendron seedlings were measured, and the rhizosphere soil was subjected to 16S rDNA-high-throughput sequencing to determine the rhizosphere bacterial community composition of H. ammodendron seedlings under different treatments. The results showed that the root-to-crown ratio of H. ammodendron seedlings increased significantly (p &lt; 0.05) under the water deficit treatment compared to the control and nitrogen addition treatments, indicating that H. ammodendron seedlings preferred to allocate biological carbon to the lower part of the ground. In contrast, plant height and root length were significantly lower (p &lt; 0.05) under water deficit treatment compared to the control, and no significant change was observed under water deficit and nitrogen addition compared to the control, indicating that water deficit inhibited the growth of H. ammodendron seedlings and nitrogen addition mitigated the effect of water deficit on the growth of H. ammodendron seedlings. Under sterilized soil conditions, both water deficit and nitrogen addition significantly increased the abundance and diversity of bacterial communities in H. ammodendron seedlings (p &lt; 0.05). Conversely, under non-sterilized conditions, both inhibited the diversity of microbial bacterial communities, and the microbial characteristic species under different controls were different. Therefore, in the short-term experiment, H. ammodendron seedlings were affected by water deficit and allocated greater quantities of biomass to the underground part, especially in the non-sterile microbial environment; different initial soil conditions resulted in divergent responses of rhizosphere bacterial communities to water deficit and nitrogen addition. Under different initial soil conditions, the same water deficit and nitrogen addition treatment will lead to the development of distinct differences in rhizosphere bacterial community composition.

Microbial communities are closely related to the overall health and quality of soil, but studies on microbial ecology in apple pear orchard soils are limited. In the current study, 28 soil samples were collected from three apple pear orchards, and the composition and structure of fungal and bacterial communities were investigated by high-throughput sequencing. The molecular ecological network showed that the keystone taxa of bacterial communities were Actinobacteria, Proteobacteria, Gemmatimonadetes, Acidobacteria, Nitrospirae, and Chloroflexi, and the keystone taxon of fungal communities was Ascomycota. Mantel tests showed that soil texture and pH were important factors shaping soil bacterial and fungal communities, and soil water soluble organic carbon (WSOC) and nitrate nitrogen (NO3−-N) were also closely related to soil bacterial communities. Canonical correspondence analysis (CCA) and variation partition analysis (VPA) revealed that geographic distance, soil texture, pH, and other soil properties could explain 10.55%, 13.5%, and 19.03% of the overall variation in bacterial communities, and 11.61%, 13.03%, and 20.26% of the overall variation in fungal communities, respectively. The keystone taxa of bacterial and fungal communities in apple pear orchard soils and their strong correlation with soil properties could provide useful clues toward sustainable management of orchards.

2-Deoxystreptamine (2DOS) is the unique chemically stable aminocyclitol scaffold of clinically important aminoglycoside antibiotics such as neomycin, kanamycin, and gentamicin, which are produced by Actinomycetes. The 2DOS core can be decorated with various deoxyaminosugars to make structurally diverse pseudo-oligosaccharides. After the discovery of biosynthetic gene clusters for 2DOS-containing aminoglycoside antibiotics, the function of each biosynthetic enzyme has been extensively elucidated. The common biosynthetic intermediates 2DOS, paromamine and ribostamycin are constructed by conserved enzymes encoded in the gene clusters. The biosynthetic intermediates are then converted to characteristic architectures by unique enzymes encoded in each biosynthetic gene cluster. In this Personal Account, we summarize both common biosynthetic pathways and the pathways used for structural diversification.

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Excessive use of nitrogen fertilizer affects the sustainable development of the Korla fragrant pear orchard. Semi-decomposed sheep manure is favored because of its advantages of being pollution-free, containing more microorganisms, and being friendly to soil. However, the effects of nitrogen fertilizer combined with sheep manure on soil nutrient cycling and microbial community in pear orchards are still unclear. This study involved a two-year field experiment to investigate fertilization’s effects on the 0–20 cm soil layer of 10–12-year-old Korla fragrant pear trees at maturity. The purpose of this study was to explore the effect of reducing nitrogen fertilizer combined with sheep manure on soil fertility and microbial community in Korla fragrant pear orchard. The treatments of no nitrogen fertilizer (N0), conventional fertilization (N), 20% reduction in nitrogen based on conventional fertilization (N2), a combination of 20% nitrogen reduction with sheep manure F1 (22,500 kg·hm−2), and 20% nitrogen reduction with sheep manure F2 (33,750 kg·hm−2) formed the experimental treatment of nitrogen reduction with sheep manure, denoted as N2F1 and N2F2. The results showed that nitrogen application increased soil physicochemical indicators but decreased soil pH and bacterial community richness and diversity. After nitrogen reduction, soil total nitrogen (TN), alkaline hydrolysis nitrogen (AN), available phosphorus (AP), microbial biomass nitrogen (SMBN), bacterial community richness, fungal community evenness, and diversity were inhibited, but bacterial community diversity was increased. Nitrogen reduction combined with sheep manure treatment increased the content of nitrate nitrogen (NO3−–N), ammonium nitrogen (NH4+–N), soil organic matter (SOM), pH, microbial biomass carbon (SMBC), and SMBN and increased the evenness and diversity of the bacterial community but inhibited the richness of the bacterial community. Among them, N2F2 treatment had the best effect on SMBC and SMBN. Soil pH, NO3−–N, and SOM were the primary environmental variables influencing bacterial and fungal community levels. The application of nitrogen significantly influenced pear orchard yields, but the yield of pears treated showed no significant variation with nitrogen reduction and nitrogen reduction combined with sheep manure based on complete nitrogen application. In summary, 20% nitrogen reduction (300 kg·hm−2) combined with 22,500–33,750 kg·hm−2 sheep manure better promotes the stability and health of soil microbial communities, and the use of organic fertilizer represents the most efficient approach to quickly enhancing soil fertility and the variation of microbial communities. These findings are highly relevant when improving land productivity, ensuring food security, and promoting environmental sustainability in fruit tree farming.

Introduction - Natural products (NP) are bioactive compounds produced by plants, fungi and bacteria, and represent a vital source for many pharmaceutical drugs. Genes involved in NP biosynthesis are usually clustered in a genome[1], and known as Biosynthetic Gene Clusters (BGC). Previous work on identifying BGCs showed supervised learning to be effective for bacteria[2,3]. Fungi BGC genomic diversity makes their identification challenging[4]. We propose an alignment-free approach based on supervised learning to predict BGCs in fungi, capable of generalizing predictions for various BGC types, focusing on precision while requiring less data curation. Methodology - Our approach has 3 main steps: collect a curated dataset; design a classification framework; and evaluate predictions against annotated BGCs. Positive instances were curated from MIBiG (http://mibig.secondarymetabolites.org), and synthetic negative instances from OrthoDB (http://orthodb.org). We used protein k-mers and protein annotated domains as features. Classification models are based on Random Forest, C-Support Vector (SVC), LinearSVC, Nu-SVC, Logit classifiers and a LSTM network. We tested our models using annotated Aspergillus niger BGCs[5]. Preliminary Results &amp; Conclusion - Despite the task complexity, preliminary results for the positive class for all classifiers show trends with F-measure around 0.6, further improved if handling class imbalance. State-of-the-art methods seem to have limited performance possibly due to the genomic diversity aspect of these clusters. [1] Anne Osbourn. Secondary metabolic gene clusters: evolutionary toolkits for chemical innovation. Trends in Genetics, 26(10):449–457, 2010. [2] Priyesh Agrawal, Shradha Khater, Money Gupta, Neetu Sain, and Debasisa Mohanty. RiPPMiner: a bioinformatics resource for deciphering chemical structures of RiPPs based on prediction of cleavage and cross-links. Nucleic Acids Research, 45(W1):W80–W88, 2017. [3] Geoffrey D Hannigan, David Prihoda, Andrej Palicka, Jindrich Soukup, Ondrej Klempir, Lena Rampula, Jindrich Durcak, Michael Wurst, Jakub Kotowski, Dan Chang, et al. A deep learning genome mining strategy improves biosynthetic gene cluster prediction. bioRxiv, page 500694, 2018. [4] Inge Kjærbølling, Tammi C Vesth, Jens C Frisvad, Jane L Nybo, Sebastian Theobald, Alan Kuo, Paul Bowyer, Yudai Matsuda, Stephen Mondo, Ellen K Lyhne, et al. Linking secondary metabolites to gene clusters through genome sequencing of six diverse aspergillus species. Proceedings of the National Academy of Sciences, 115(4):E753–E761, 2018. [5] Diane O Inglis, Jonathan Binkley, Marek S Skrzypek, Martha B Arnaud, Gustavo C Cerqueira, Prachi Shah, Farrell Wymore, Jennifer R Wortman, and Gavin Sherlock. Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC microbiology, 13(1):91, 2013.

The discovery of novel bioactive compounds has significant implications for diverse biotechnological applications. However, microbial genomes remain largely underexplored regarding their potential to produce secondary metabolites. Here, we explore the biotechnological potential of microorganisms from the highly biodiverse Amazon region through in silico genome mining. A total of 40 bacterial genomes originating from water, soil, and animal and plant-associated microorganisms were selected from a public database and analyzed using AntiSMASH and PRISM, predicting, respectively, 402 and 195 biosynthetic gene clusters (BGCs) related to secondary metabolite production. The most frequent BGCs were associated with polyketides, nonribosomal peptides, and ribosomally synthesized and post-translationally modified peptides, diverse classes of secondary metabolites commonly associated with biotechnological applications. To evaluate similarity with previously characterized clusters, the predicted BGCs were compared to entries in the MIBiG repository using BiG-SCAPE, revealing 12 clusters with close relationships to known BGCs. The remaining clusters showed low similarity, indicating that many potentially novel biosynthetic pathways remain uncharacterized, highlighting the limitations of current reference databases and the need for experimental validation. The results highlight the underexplored biotechnological potential of Amazonian microorganisms and reinforce the importance of expanding microbial genome sequencing and mining efforts in this biodiversity hotspot. The findings have significant implications for the discovery of novel bioactive compounds with diverse applications.

ABSTRACTThe aerodigestive tract (ADT) is the primary portal through which pathogens and other invading microbes enter the body. As the direct interface with the environment, we hypothesize that the ADT microbiota possess biosynthetic gene clusters (BGCs) for antibiotics and other specialized metabolites to compete with both endogenous and exogenous microbes. From 1,214 bacterial genomes, representing 136 genera and 387 species that colonize the ADT, we identified 3,895 BGCs. To determine the distribution of BGCs and bacteria in different ADT sites, we aligned 1,424 metagenomes, from nine different ADT sites, onto the predicted BGCs. We show that alpha diversity varies across the ADT and that each site is associated with distinct bacterial communities and BGCs. We identify specific BGC families enriched in the buccal mucosa, external naris, gingiva, and tongue dorsum despite these sites harboring closely related bacteria. We reveal BGC enrichment patterns indicative of the ecology at each site. For instance, aryl polyene and resorcinol BGCs are enriched in the gingiva and tongue, which are colonized by many anaerobes. In addition, we find that streptococci colonizing the tongue and cheek possess different ribosomally synthesized and posttranslationally modified peptide BGCs. Finally, we highlight bacterial genera with BGCs but are underexplored for specialized metabolism and demonstrate the bioactivity of Actinomyces against other bacteria, including human pathogens. Together, our results demonstrate that specialized metabolism in the ADT is extensive and that by exploring these microbiomes further, we will better understand the ecology and biogeography of this system and identify new bioactive natural products.IMPORTANCE Bacteria produce specialized metabolites to compete with other microbes. Though the biological activities of many specialized metabolites have been determined, our understanding of their ecology is limited, particularly within the human microbiome. As the aerodigestive tract (ADT) faces the external environment, bacteria colonizing this tract must compete both among themselves and with invading microbes, including human pathogens. We analyzed the genomes of ADT bacteria to identify biosynthetic gene clusters (BGCs) for specialized metabolites. We found that the majority of ADT BGCs are uncharacterized and the metabolites they encode are unknown. We mapped the distribution of BGCs across the ADT and determined that each site is associated with its own distinct bacterial community and BGCs. By further characterizing these BGCs, we will inform our understanding of ecology and biogeography across the ADT, and we may uncover new specialized metabolites, including antibiotics.

The amphibian skin microbiome has been the focus of recent studies aiming to better understand the role of these microbial symbionts in host defense against disease. However, host-associated microbial communities are complex and dynamic, and changes in their composition and structure can influence their function. Understanding temporal variation of bacterial communities on amphibian skin is critical for establishing baselines from which to improve the development of mitigation techniques based on probiotic therapy and provides long-term host protection in a changing environment. Here, we investigated whether microbial communities on amphibian skin change over time at a single site. To examine this, we collected skin swabs from two pond-breeding species of treefrogs, Agalychnis callidryas and Dendropsophus ebraccatus, over 4 years at a single lowland tropical pond in Panamá. Relative abundance of operational taxonomic units (OTUs) based on 16S rRNA gene amplicon sequencing was used to determine bacterial community diversity on the skin of both treefrog species. We found significant variation in bacterial community structure across long and short-term time scales. Skin bacterial communities differed across years on both species and between seasons and sampling days only in D. ebraccatus. Importantly, bacterial community structures across days were as variable as year level comparisons. The differences in bacterial community were driven primarily by differences in relative abundance of key OTUs and explained by rainfall at the time of sampling. These findings suggest that skin-associated microbiomes are highly variable across time, and that for tropical lowland sites, rainfall is a good predictor of variability. However, more research is necessary to elucidate the significance of temporal variation in bacterial skin communities and their maintenance for amphibian conservation efforts.