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BMC Biology May 2024Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan...
BACKGROUND
Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan predators, raptorial Haptorian ciliates are particularly fascinating as they possess offensive extrusomes known as toxicysts, which are rapidly discharged upon prey contact. However, our understanding of the genetic processes and specific toxins involved in toxicyst formation and discharge is still limited.
RESULTS
In this study, we investigated the predation strategies and subcellular structures of seven Haptoria ciliate species and obtained their genome sequences using single-cell sequencing technology. Comparative genomic analysis revealed distinct gene duplications related to membrane transport proteins and hydrolytic enzymes in Haptoria, which play a crucial role in the production and discharge of toxicysts. Transcriptomic analysis further confirmed the abundant expression of genes related to membrane transporters and cellular toxins in Haptoria compared to Trichostomatia. Notably, polyketide synthases (PKS) and L-amino acid oxidases (LAAO) were identified as potentially toxin genes that underwent extensive duplication events in Haptoria.
CONCLUSIONS
Our results shed light on the evolutionary and genomic adaptations of Haptorian ciliates for their predation strategies in evolution and provide insights into their toxic mechanisms.
Topics: Ciliophora; Genomics; Genome, Protozoan; Transcriptome
PubMed: 38715037
DOI: 10.1186/s12915-024-01904-2 -
Microbiology Spectrum Jun 2024The cyanosiphophage Mic1 specifically infects the bloom-forming FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded...
UNLABELLED
The cyanosiphophage Mic1 specifically infects the bloom-forming FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded DNA genome consists of 98 putative open reading frames, 63% of which are of unknown function. Here, we investigated the transcriptome dynamics of Mic1 and its host using RNA sequencing. In the early, middle, and late phases of the 10 h lytic cycle, the Mic1 genes are sequentially expressed and could be further temporally grouped into two distinct clusters in each phase. Notably, six early genes, including that encodes a TnpB-like transposase, immediately reach the highest transcriptional level in half an hour, representing a pioneer cluster that rapidly regulates and redirects host metabolism toward the phage. An in-depth analysis of the host transcriptomic profile in response to Mic1 infection revealed significant upregulation of a polyketide synthase pathway and a type III-B CRISPR system, accompanied by moderate downregulation of the photosynthesis and key metabolism pathways. The constant increase of phage transcripts and relatively low replacement rate over the host transcripts indicated that Mic1 utilizes a unique strategy to gradually take over a small portion of host metabolism pathways after infection. In addition, genomic analysis of a less-infective Mic1 and a Mic1-resistant host strain further confirmed their dynamic interplay and coevolution via the frequent horizontal gene transfer. These findings provide insights into the mutual benefit and symbiosis of the highly polymorphic cyanobacteria and cyanophages.
IMPORTANCE
The highly polymorphic is one of the predominant bloom-forming cyanobacteria in eutrophic freshwater bodies and is infected by diverse and abundant cyanophages. The presence of a large number of defense systems in genome suggests a dynamic interplay and coevolution with the cyanophages. In this study, we investigated the temporal gene expression pattern of Mic1 after infection and the corresponding transcriptional responses of its host. Moreover, the identification of a less-infective Mic1 and a Mic1-resistant host strain provided the evolved genes in the phage-host coevolution during the multiple-generation cultivation in the laboratory. Our findings enrich the knowledge on the interplay and coevolution of and its cyanophages and lay the foundation for the future application of cyanophage as a potential eco-friendly and bio-safe agent in controlling the succession of harmful cyanobacterial blooms.
Topics: Microcystis; Bacteriophages; China; Transcriptome; Lakes; Genome, Viral; Evolution, Molecular
PubMed: 38695606
DOI: 10.1128/spectrum.00298-24 -
Acta Biochimica Et Biophysica Sinica Jun 2024Biological control of pests and pathogens has attracted much attention due to its green, safe and effective characteristics. However, it faces the dilemma of... (Review)
Review
Biological control of pests and pathogens has attracted much attention due to its green, safe and effective characteristics. However, it faces the dilemma of insignificant effects in large-scale applications. Therefore, an in-depth exploration of the metabolic potential of biocontrol fungi based on big omics data is crucial for a comprehensive and systematic understanding of the specific modes of action operated by various biocontrol fungi. This article analyzes the preferences for extracellular carbon and nitrogen source degradation, secondary metabolites (nonribosomal peptides, polyketide synthases) and their product characteristics and the conversion relationship between extracellular primary metabolism and intracellular secondary metabolism for eight different filamentous fungi with characteristics appropriate for the biological control of bacterial pathogens and phytopathogenic nematodes. Further clarification is provided that , encoding a large number of hydrolase enzymes capable of degrading pathogen protection barrier, can be directly applied in the field as a predatory biocontrol fungus, whereas , as an antibiosis-active biocontrol control fungus, can form dominant strains on preferred substrates and produce a large number of secondary metabolites to achieve antibacterial effects. By clarifying the levels of biological control achievable by different biocontrol fungi, we provide a theoretical foundation for their application to cropping habitats.
Topics: Fungi; Secondary Metabolism; Carbon; Biological Control Agents; Pest Control, Biological; Nitrogen; Animals; Metabolomics
PubMed: 38686460
DOI: 10.3724/abbs.2024056 -
Microbial Biotechnology May 2024Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal...
Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 μg/mL - 4.36-fold higher than WT value (48.9 μg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 μg/mL. Overexpression of fatty acid β-oxidation pathway genes in the strain further increased B1a titer to 452.8 μg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 μg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.
Topics: Polyketide Synthases; Metabolic Engineering; Acyl Coenzyme A; Streptomyces; Metabolic Networks and Pathways; Ivermectin; Bacterial Proteins
PubMed: 38683675
DOI: 10.1111/1751-7915.14470 -
Toxicon : Official Journal of the... May 2024Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein...
Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin (mcy) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosamine-phosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time-of-flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine-Homoarginine(MC-LHar) and [Aspartic acid]MC-Leucine-Homoarginine ([Asp]MC-LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of L-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.
Topics: Microcystins; Homoarginine; Biosynthetic Pathways; Multigene Family; Cyanobacteria; Tandem Mass Spectrometry
PubMed: 38670499
DOI: 10.1016/j.toxicon.2024.107733 -
ACS Synthetic Biology May 2024spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have...
spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in M1152Δ that could produce aklavinone, 9--aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and M1152Δ expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant hosts.
Topics: Polyketide Synthases; Anthracyclines; Streptomyces coelicolor; Streptomyces; Biosynthetic Pathways; Hydroxylation; Anti-Bacterial Agents
PubMed: 38662967
DOI: 10.1021/acssynbio.4c00043 -
Fungal Genetics and Biology : FG & B Jun 2024The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production...
The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.
Topics: Stachybotrys; Multigene Family; Polyketide Synthases; Mycotoxins; Phylogeny; Biosynthetic Pathways; Genetic Engineering; Secondary Metabolism; Fungal Proteins
PubMed: 38636782
DOI: 10.1016/j.fgb.2024.103892 -
Microbial Genomics Apr 2024are prolific producers of secondary metabolites from which many clinically useful compounds have been derived. They inhabit diverse habitats but have rarely been...
are prolific producers of secondary metabolites from which many clinically useful compounds have been derived. They inhabit diverse habitats but have rarely been reported in vertebrates. Here, we aim to determine to what extent the ecological source (bat host species and cave sites) influence the genomic and biosynthetic diversity of bacteria. We analysed draft genomes of 132 isolates sampled from 11 species of insectivorous bats from six cave sites in Arizona and New Mexico, USA. We delineated 55 species based on the genome-wide average nucleotide identity and core genome phylogenetic tree. isolates that colonize the same bat species or inhabit the same site exhibit greater overall genomic similarity than they do with from other bat species or sites. However, when considering biosynthetic gene clusters (BGCs) alone, BGC distribution is not structured by the ecological or geographical source of the that carry them. Each genome carried between 19-65 BGCs (median=42.5) and varied even among members of the same species. Nine major classes of BGCs were detected in ten of the 11 bat species and in all sites: terpene, non-ribosomal peptide synthetase, polyketide synthase, siderophore, RiPP-like, butyrolactone, lanthipeptide, ectoine, melanin. Finally, genomes carry multiple hybrid BGCs consisting of signature domains from two to seven distinct BGC classes. Taken together, our results bring critical insights to understanding -bat ecology and BGC diversity that may contribute to bat health and in augmenting current efforts in natural product discovery, especially from underexplored or overlooked environments.
Topics: Animals; Chiroptera; Phylogeny; Genomics; Arizona; Bacteria
PubMed: 38625724
DOI: 10.1099/mgen.0.001238 -
Journal of Biotechnology Jun 2024The 2-pyrone moiety is present in a wide range of structurally diverse natural products with various biological activities. The plant biosynthetic routes towards these...
The 2-pyrone moiety is present in a wide range of structurally diverse natural products with various biological activities. The plant biosynthetic routes towards these compounds mainly depend on the activity of either type III polyketide synthase-like 2-pyrone synthases or hydroxylating 2-oxoglutarate dependent dioxygenases. In the present study, the substrate specificity of these enzymes is investigated by a systematic screening using both natural and artificial substrates with the aims of efficiently forming (new) products and understanding the underlying catalytic mechanisms. In this framework, we focused on the in vitro functional characterization of a 2-pyrone synthase Gh2PS2 from Gerbera x hybrida and two dioxygenases AtF6'H1 and AtF6'H2 from Arabidopsis thaliana using a set of twenty aromatic and aliphatic CoA esters as substrates. UHPLC-ESI-HRMS based analyses of reaction intermediates and products revealed a broad substrate specificity of the enzymes, enabling the facile "green" synthesis of this important class of natural products and derivatives in a one-step/one-pot reaction in aqueous environment without the need for halogenated or metal reagents and protective groups. Using protein modeling and substrate docking we identified amino acid residues that seem to be important for the observed product scope.
Topics: Pyrones; Esters; Arabidopsis; Substrate Specificity; Coenzyme A; Molecular Docking Simulation; Biological Products; Dioxygenases
PubMed: 38616039
DOI: 10.1016/j.jbiotec.2024.04.006 -
BMC Genomics Apr 2024Pseudoalteromonas viridis strain BBR56 was isolated from seawater at Dutungan Island, South Sulawesi, Indonesia. Bacterial DNA was isolated using Promega Genomic DNA...
Pseudoalteromonas viridis strain BBR56 was isolated from seawater at Dutungan Island, South Sulawesi, Indonesia. Bacterial DNA was isolated using Promega Genomic DNA TM050. DNA purity and quantity were assessed using NanoDrop spectrophotometers and Qubit fluorometers. The DNA library and sequencing were prepared using Oxford Nanopore Technology GridION MinKNOW 20.06.9 with long read, direct, and comprehensive analysis. High accuracy base calling was assessed with Guppy version 4.0.11. Filtlong and NanoPlot were used for filtering and visualizing the FASTQ data. Flye (2.8.1) was used for de novo assembly analysis. Variant calls and consensus sequences were created using Medaka. The annotation of the genome was elaborated by DFAST. The assembled genome and annotation were tested using Busco and CheckM. Herein, we found that the highest similarity of the BBR56 isolate was 98.37% with the 16 S rRNA gene sequence of P. viridis G-1387. The genome size was 5.5 Mb and included chromosome 1 (4.2 Mbp) and chromosome 2 (1.3 Mbp), which encoded 61 pseudogenes, 4 noncoding RNAs, 113 tRNAs, 31 rRNAs, 4,505 coding DNA sequences, 4 clustered regularly interspaced short palindromic repeats, 4,444 coding genes, and a GC content of 49.5%. The sequence of the whole genome of P. viridis BBR56 was uploaded to GenBank under the accession numbers CP072425-CP072426, biosample number SAMN18435505, and bioproject number PRJNA716373. The sequence read archive (SRR14179986) was successfully obtained from NCBI for BBR56 raw sequencing reads. Digital DNA-DNA hybridization results showed that the genome of BBR56 had the potential to be a new species because no other bacterial genomes were similar to the sample. Biosynthetic gene clusters (BGCs) were assessed using BAGEL4 and the antiSMASH bacterial version. The genome harbored diverse BGCs, including genes that encoded polyketide synthase, nonribosomal peptide synthase, RiPP-like, NRP-metallophore, hydrogen cyanide, betalactone, thioamide-NRP, Lant class I, sactipeptide, and prodigiosin. Thus, BBR56 has considerable potential for further exploration regarding the use of its secondary metabolite products in the human and fisheries sectors.
Topics: Humans; Pseudoalteromonas; Pseudogenes; Gene Library; DNA, Bacterial
PubMed: 38615000
DOI: 10.1186/s12864-024-10266-6