Integrating perspectives in actinomycete research: an ActinoBase review of 2020-21

Last year ActinoBase, a Wiki-style initiative supported by the UK Microbiology Society, published a review highlighting the research of particular interest to the actinomycete community. Here, we present the second ActinoBase review showcasing selected reports published in 2020 and early 2021, integrating perspectives in the actinomycete field. Actinomycetes are well-known for their unsurpassed ability to produce specialised metabolites, of which many are used as therapeutic agents with antibacterial, antifungal, or immunosuppressive activities. Much research is carried out to understand the purpose of these metabolites in the environment, either within communities or in host interactions. Moreover, many efforts have been placed in developing computational tools to handle big data, simplify experimental design, and find new biosynthetic gene cluster prioritisation strategies. Alongside, synthetic biology has provided advances in tools to elucidate the biosynthesis of these metabolites. Additionally, there are still mysteries to be uncovered in understanding the fundamentals of filamentous actinomycetes' developmental cycle and regulation of their metabolism. This review focuses on research using integrative methodologies and approaches to understand the bigger picture of actinomycete biology, covering four research areas: i) technology and methodology; ii) specialised metabolites; iii) development and regulation; and iv) ecology and host interactions

Phylogenomic analysis of natural products biosynthetic gene clusters allows discovery of arseno-organic metabolites in model streptomycetes

We are indebted with Marnix Medema, Paul Straight and Sean Rovito, for useful discussions and critical reading of the manuscript, as well as with Alicia Chagolla and Yolanda Rodriguez of the MS Service of Unidad Irapuato, Cinvestav, and Araceli Fernandez for technical support in high-performance computing. This work was funded by Conacyt Mexico (grants No. 179290 and 177568) and FINNOVA Mexico (grant No. 214716) to FBG. PCM was funded by Conacyt scholarship (No. 28830) and a Cinvestav posdoctoral fellowship. JF and JFK acknowledge funding from the College of Physical Sciences, University of Aberdeen, UK.Peer reviewedPublisher PD

ActDES- a Curated Actinobacterial Database for Evolutionary Studies

Actinobacteria is a large and diverse phylum of bacteria that contains medically and ecologically relevant organisms. Many members are valuable sources of bioactive natural products and chemical precursors that are exploited in the clinic and made using the enzyme pathways encoded in their complex genomes. Whilst the number of sequenced genomes has increased rapidly in the last 20 years, the large size, complexity and high G+C content of many actinobacterial genomes means that the sequences remain incomplete and consist of large numbers of contigs with poor annotation, which hinders large-scale comparative genomic and evolutionary studies. To enable greater understanding and exploitation of actinobacterial genomes, specialized genomic databases must be linked to high-quality genome sequences. Here, we provide a curated database of 612 high-quality actinobacterial genomes from 80 genera, chosen to represent a broad phylogenetic group with equivalent genome re-annotation. Utilizing this database will provide researchers with a framework for evolutionary and metabolic studies, to enable a foundation for genome and metabolic engineering, to facilitate discovery of novel bioactive therapeutics and studies on gene family evolution. This article contains data hosted by Microreact

Whole genome sequencing reveals widespread distribution of typhoidal toxin genes and VirB/D4 plasmids in bovine-associated nontyphoidal Salmonella

Aedes aegypti is the primary urban mosquito vector of viruses causing dengue, Zika and chikungunya fevers –for which vaccines and efective pharmaceuticals are still lacking. Current strategies to suppress arbovirus outbreaks include removal of larval-breeding sites and insecticide treatment of larval and adult populations. Insecticidal control of Ae. aegypti is challenging, due to a recent rapid global increase in knockdown-resistance (kdr) to pyrethroid insecticides. Widespread, heavy use of pyrethroid spacesprays has created an immense selection pressure for kdr, which is primarily under the control of the voltage-gated sodium channel gene (vgsc). To date, eleven replacements in vgsc have been discovered, published and shown to be associated with pyrethroid resistance to varying degrees. In Mexico, F1,534C and V1,016I have co-evolved in the last 16 years across Ae. aegypti populations. Recently, a novel replacement V410L was identifed in Brazil and its efect on vgsc was confrmed by electrophysiology. Herein, we screened V410L in 25 Ae. aegypti historical collections from Mexico, the frst heterozygote appeared in 2002 and frequencies have increased in the last 16 years alongside V1,016I and F1,534C. Knowledge of the specifc vgsc replacements and their interaction to confer resistance is essential to predict and to develop strategies for resistance management

Expanding Primary Metabolism Helps Generate the Metabolic Robustness To Facilitate Antibiotic Biosynthesis in Streptomyces

Abstract The expansion of the genetic repertoire of an organism by gene duplication or horizontal gene transfer (HGT) can aid adaptation. Streptomyces bacteria are prolific producers of bioactive specialized metabolites that have adaptive functions in nature and have found extensive utility in human medicine. Whilst the biosynthesis of these specialized metabolites is directed by dedicated biosynthetic gene clusters, little attention has been focussed on how these organisms have evolved robustness into their genomes to facilitate the metabolic plasticity required to provide chemical precursors for biosynthesis during the complex metabolic transitions from vegetative growth to specialized metabolite production and sporulation. Here we examine genetic redundancy in Actinobacteria and show that specialised metabolite producing bacterial families exhibit gene family expansion in primary metabolism. Focussing on a gene duplication event we show that the two pyruvate kinases in the genome of S. coelicolor arose by an ancient duplication event and that each have evolved altered enzymatic kinetics, with Pyk1 having a 20-fold higher Kcat than Pyk2 (4703 sec-1 compared to 215 sec-1 respectively) yet both are constitutively expressed. The pyruvate kinase mutants were also found to be compromised in terms of fitness when compared to wild-type Streptomyces. These data suggest that expanding gene familes can help maintain cell functionality during metabolic perturbation such as nutrient limitation and/or specialized metabolite production. Importance The rise of antimicrobial resistant infections has prompted a resurgence in interest in understanding the production of specialized metabolites by Streptomyces such as antibiotics. The presence of multiple genes encoding the same enzymatic function is an aspect of Streptomyces biology that has received little attention, however understanding how the metabolic expansion influences these organisms can help enhance production of clinically useful molecules. Here we show that expanding the number of pyruvate kinases enables metabolic adaptation, increases strain fitness and represents an excellent target for metabolic engineering of industrial specialized metabolite producing bacteria and the activation of cryptic specialized metabolites

Biosynthetic gene profiling and genomic potential of the novel photosynthetic marine bacterium Roseibaca domitiana

Shifting the bioprospecting targets toward underexplored bacterial groups combined with genome mining studies contributes to avoiding the rediscovery of known compounds by revealing novel, promising biosynthetic gene clusters (BGCs). With the aim of determining the biosynthetic potential of a novel marine bacterium, strain V10T, isolated from the Domitian littoral in Italy, a comparative phylogenomic mining study was performed across related photosynthetic bacterial groups from an evolutionary perspective. Studies on polyphasic and taxogenomics showed that this bacterium constitutes a new species, designated Roseibaca domitiana sp. nov. To date, this genus has only one other validly described species, which was isolated from a hypersaline Antarctic lake. The genomic evolutionary study linked to BGC diversity revealed that there is a close relationship between the phylogenetic distance of the members of the photosynthetic genera Roseibaca, Roseinatronobacter, and Rhodobaca and their BGC profiles, whose conservation pattern allows discriminating between these genera. On the contrary, the rest of the species related to Roseibaca domitiana exhibited an individual species pattern unrelated to genome size or source of isolation. This study showed that photosynthetic strains possess a streamlined content of BGCs, of which 94.34% of the clusters with biotechnological interest (NRPS, PKS, RRE, and RiPP) are completely new. Among these stand out T1PKS, exclusive of R. domitiana V10T, and RRE, highly conserved only in R. domitiana V10T and R. ekhonensis, both categories of BGCs involved in the synthesis of plant growth-promoting compounds and antitumoral compounds, respectively. In all cases, with very low homology with already patented molecules. Our findings reveal the high biosynthetic potential of infrequently cultured bacterial groups, suggesting the need to redirect attention to microbial minorities as a novel and vast source of bioactive compounds still to be exploited

Genomic study and lipidomic bioassay of Leeuwenhoekiella parthenopeia: A novel rare biosphere marine bacterium that inhibits tumor cell viability

The fraction of low-abundance microbiota in the marine environment is a promising target for discovering new bioactive molecules with pharmaceutical applications. Phenomena in the ocean such as diel vertical migration (DVM) and seasonal dynamic events influence the pattern of diversity of marine bacteria, conditioning the probability of isolation of uncultured bacteria. In this study, we report a new marine bacterium belonging to the rare biosphere, Leeuwenhoekiella parthenopeia sp. nov. Mr9T, which was isolated employing seasonal and diel sampling approaches. Its complete characterization, ecology, biosynthetic gene profiling of the whole genus Leeuwenhoekiella, and bioactivity of its extract on human cells are reported. The phylogenomic and microbial diversity studies demonstrated that this bacterium is a new and rare species, barely representing 0.0029% of the bacterial community in Mediterranean Sea metagenomes. The biosynthetic profiling of species of the genus Leeuwenhoekiella showed nine functionally related gene cluster families (GCF), none were associated with pathways responsible to produce known compounds or registered patents, therefore revealing its potential to synthesize novel bioactive compounds. In vitro screenings of L. parthenopeia Mr9T showed that the total lipid content (lipidome) of the cell membrane reduces the prostatic and brain tumor cell viability with a lower effect on normal cells. The lipidome consisted of sulfobacin A, WB 3559A, WB 3559B, docosenamide, topostin B-567, and unknown compounds. Therefore, the bioactivity could be attributed to any of these individual compounds or due to their synergistic effect. Beyond the rarity and biosynthetic potential of this bacterium, the importance and novelty of this study is the employment of sampling strategies based on ecological factors to reach the hidden microbiota, as well as the use of bacterial membrane constituents as potential novel therapeutics. Our findings open new perspectives on cultivation and the relationship between bacterial biological membrane components and their bioactivity in eukaryotic cells, encouraging similar studies in other members of the rare biosphere

MIBiG 3.0 : a community-driven effort to annotate experimentally validated biosynthetic gene clusters

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/

Unveiling the genomic potential of Pseudomonas type strains for discovering new natural products

12 páginas, 4 figurasMicrobes host a huge variety of biosynthetic gene clusters that produce an immeasurable array of secondary metabolites with many different biological activities such as antimicrobial, anticarcinogenic and antiviral. Despite the complex task of isolating and characterizing novel natural products, microbial genomic strategies can be useful for carrying out these types of studies. However, although genomic-based research on secondary metabolism is on the increase, there is still a lack of reports focusing specifically on the genus Pseudomonas. In this work, we aimed (i) to unveil the main biosynthetic systems related to secondary metabolism in Pseudomonas type strains, (ii) to study the evolutionary processes that drive the diversification of their coding regions and (iii) to select Pseudomonas strains showing promising results in the search for useful natural products. We performed a comparative genomic study on 194 Pseudomonas species, paying special attention to the evolution and distribution of different classes of biosynthetic gene clusters and the coding features of antimicrobial peptides. Using EvoMining, a bioinformatic approach for studying evolutionary processes related to secondary metabolism, we sought to decipher the protein expansion of enzymes related to the lipid metabolism, which may have evolved toward the biosynthesis of novel secondary metabolites in Pseudomonas. The types of metabolites encoded in Pseudomonas type strains were predominantly non-ribosomal peptide synthetases, bacteriocins, N-acetylglutaminylglutamine amides and ß-lactones. Also, the evolution of genes related to secondary metabolites was found to coincide with Pseudomonas species diversification. Interestingly, only a few Pseudomonas species encode polyketide synthases, which are related to the lipid metabolism broadly distributed among bacteria. Thus, our EvoMining-based search may help to discover new types of secondary metabolite gene clusters in which lipid-related enzymes are involved. This work provides information about uncharacterized metabolites produced by Pseudomonas type strains, whose gene clusters have evolved in a species-specific way. Our results provide novel insight into the secondary metabolism of Pseudomonas and will serve as a basis for the prioritization of the isolated strains. This article contains data hosted by Microreact.Z.S.S. and E.P.A. received grants from the Regional Government of Castile and Leon. Also, this work was supported by the Regional Government of Castile and Leon (Escalera de Excelencia CLU-2018-04) and co-funded by the Operational Program of the European Regional Development Fund for Castile and Leon 2014–2020.Peer reviewe