Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Macroevolutionary Dynamics in Micro-organisms: Generalists Give Rise to Specialists Across Biomes in the Ubiquitous Bacterial Phylum Myxococcota.

Prokaryotes dominate the Tree of Life, but our understanding of the macroevolutionary processes generating this diversity is still limited. Habitat transitions are thought to be a key driver of prokaryote diversity. However, relatively little is known about how prokaryotes successfully transition and persist across environments, and how these processes might vary between biomes and lineages. Here, we investigate biome transitions and specialization in natural populations of a focal bacterial phylum, the Myxococcota, sampled across a range of replicated soils and freshwater and marine sediments in Cornwall (UK). By targeted deep sequencing of the protein-coding gene rpoB, we found >2,000 unique Myxococcota lineages, with the majority (77%) classified as biome specialists and with only <5% of lineages distributed across the salt barrier. Discrete character evolution models revealed that specialists in one biome rarely transitioned into specialists in another biome. Instead, evolved generalism mediated transitions between biome specialists. State-dependent diversification models found variation in speciation rates across the tree, but this variation was independent of biome association or specialization. Our findings were robust to phylogenetic uncertainty, different levels of species delineation, and different assumed amounts of unsampled diversity resulting in an incomplete phylogeny. Overall, our results are consistent with a "jack-of-all-trades" tradeoff where generalists suffer a cost in any individual environment, resulting in rapid evolution of niche specialists and shed light on how bacteria could transition between biomes.

Recent advances in discovery and biosynthesis of natural products from myxobacteria: an overview from 2017 to 2023.

Covering: 2017.01 to 2023.11Natural products biosynthesized by myxobacteria are appealing due to their sophisticated chemical skeletons, remarkable biological activities, and intriguing biosynthetic enzymology. This review aims to systematically summarize the advances in the discovery methods, new structures, and bioactivities of myxobacterial NPs reported in the period of 2017-2023. In addition, the peculiar biosynthetic pathways of several structural families are also highlighted.

Corallococcus caeni sp. nov., a novel myxobacterium isolated from activated sludge.

Two myxobacterial strains (KH5-1T and NO1) were isolated from the activated sludge tanks treating municipal sewage wastewater in Japan. These strains were recognised as myxobacteria based on their phenotypic characteristics of swarming colonies and fruiting bodies. Phylogenetic analyses using the 16S rRNA gene revealed that strains KH5-1T and NO1 were affiliated with the genus Corallococcus, with the closest neighbours being Corallococcus exercitus AB043AT (99.77% and 99.84%, respectively). Genome comparisons using orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridisation similarity (dDDH) with strains KH5-1T and NO1 and their phylogenetically close relatives in Corallococcus spp. were below the thresholds. The major cellular fatty acids of strains KH5-1T and NO1 were iso-C15:0 (31.9%, 30.0%), summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c) (20.2%, 17.7%), and iso-C17:0 (12.1%, 14.8%), and the major respiratory quinone was found to be menaquinone (MK)-8. Based on the phenotypic, chemotaxonomic, and phylogenetic evidence, strains KH5-1T and NO1 represent a new species in the genus Corallococcus, for which the proposed name is Corallococcus caeni sp. nov. The type strain is KH5-1T (= NCIMB 15510T = JCM 36609T).

Three Fe(III)-reducing and nitrogen-fixing bacteria, Anaeromyxobacter terrae sp. nov., Anaeromyxobacter oryzisoli sp. nov. and Anaeromyxobacter soli sp. nov., isolated from paddy soil.

Three microaerophilic bacterial strains, designated SG22T, SG63T and SG29T were isolated from paddy soils in PR China. Cells of these strains were Gram-staining-negative and long rod-shaped. SG22T, SG63T and SG29T showed the highest 16S rRNA gene sequence similarities with the members of the genus Anaeromyxobacter. The results of phylogenetic and phylogenomic analysis also indicated that these strains clustered with members of the genus Anaeromyxobacter. The main respiratory menaquinone of SG22T, SG63T and SG29T was MK-8 and the major fatty acids were iso-C15 : 0, iso-C17 : 0 and C16 : 0. SG22T, SG29T and SG63T not only possessed iron reduction ability but also harboured genes (nifHDK) encoding nitrogenase. The genomic DNA G+C contents of SG22T, SG63T and SG29T ranged from 73.3 to 73.5 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values between SG22T, SG63T and SG29T and the closely related species of the genus Anaeromyxobacter were lower than the cut-off values (dDDH 70 % and ANI 95-96 %) for prokaryotic species delineation. On the basis of these results, strains SG22T, SG63T and SG29T represent three novel species within the genus Anaeromyxobacter, for which the names Anaeromyxobacter terrae sp. nov., Anaeromyxobacter oryzisoli sp. nov. and Anaeromyxobacter soli sp. nov., are proposed. The type strains are SG22T (= GDMCC 1.3185T = JCM 35581T), SG63T (= GDMCC 1.2914T = JCM 35124T) and SG29T (= GDMCC 1.2911T = JCM 35123T).

Corallococcus senghenyddensis sp. nov., a myxobacterium with potent antimicrobial activity.

AIM: Corallococcus species are diverse in the natural environment with 10 new Corallococcus species having been characterized in just the last 5 years. As well as being an abundant myxobacterial genus, they produce several secondary metabolites, including Corallopyronin, Corramycin, Coralmycin, and Corallorazine. We isolated a novel strain Corallococcus spp RDP092CA from soil in South Wales, UK, using Candida albicans as prey bait and characterized its predatory activities against pathogenic bacteria and yeast. METHODS AND RESULTS: The size of the RDP092CA genome was 8.5 Mb with a G + C content of 71.4%. Phylogenetically, RDP092CA is closely related to Corallococcus interemptor, C. coralloides, and C. exiguus. However, genome average nucleotide identity and digital DNA-DNA hybridization values are lower than 95% and 70% when compared to those type strains, implying that it belongs to a novel species. The RDP092CA genome harbours seven types of biosynthetic gene clusters (BGCs) and 152 predicted antimicrobial peptides. In predation assays, RDP092CA showed good predatory activity against Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii, and Staphylococcus aureus but not against Enterococcus faecalis. It also showed good antibiofilm activity against all five bacteria in biofilm assays. Antifungal activity against eight Candida spp. was variable, with particularly good activity against Meyerozyma guillermondii DSM 6381. Antimicrobial peptide RDP092CA_120 exhibited potent antibiofilm activity with >50% inhibition and >60% dispersion of biofilms at concentrations down to 1 µg/ml. CONCLUSIONS: We propose that strain RDP092CA represents a novel species with promising antimicrobial activities, Corallococcus senghenyddensis sp. nov. (=NBRC 116490T =CCOS 2109T), based on morphological, biochemical, and genomic features.

Heterologous biosynthesis of myxobacterial lanthipeptides melittapeptins.

The myxobacteria are an attractive bioresource for bioactive compounds since the large size genome contains many biosynthetic gene clusters of secondary metabolites. The genome of the myxobacterium Melittangium boletus contains three biosynthetic gene clusters for lanthipeptide production. One of the gene clusters includes genes coding lanthipeptide precursor (melA), class II lanthipeptide synthetase (melM), and transporter (melT). The amino acid sequence of melA indicated similarity with that of known lanthipeptides mersacidin and lichenicidin A1 by the alignment. To perform heterologous production of new lanthipeptides, the expression vector containing the essential genes (melA and melM) was constructed by utilizing codon-optimized synthetic genes. The co-expression of two genes in the host bacterial cells of Escherichia coli BL21 (DE3) afforded new lanthipeptides named melittapeptins A-C. The structures of melittapeptins A-C including lanthionine/methyllanthionine bridge pattern were proposed based on protease digestion and MS/MS experiments. The native strain of M. boletus did not produce melittapeptins A-C, so heterologous production using the biosynthetic gene cluster was effective in obtaining the lanthipeptides. Melittapeptins A-C showed specific and potent antibacterial activity to the Gram-positive bacterium Micrococcus luteus. To the best of our knowledge, this is the first report of antibacterial lanthipeptides derived from myxobacterial origin. KEY POINTS: • New lanthipeptides melittapeptins were heterologously produced in Escherichia coli. • Melittapeptins showed specific antibacterial activity against Micrococcus luteus. • Melittapeptins were the first antibacterial lanthipeptides of myxobacterial origin.

Discovery and Characterization of a Myxobacterial Lanthipeptide with Unique Biosynthetic Features and Anti-inflammatory Activity.

The genomes of myxobacteria harbor a variety of biosynthetic gene clusters encoding numerous secondary metabolites, including ribosomally synthesized and post-translationally modified peptides (RiPPs) with diverse chemical structures and biological activities. However, the biosynthetic potential of RiPPs from myxobacteria remains barely explored. Herein, we report a novel myxobacteria lanthipeptide myxococin identified from Myxococcus fulvus. Myxococins represent the first example of lanthipeptides, of which the characteristic multiple thioether rings are installed by employing a Class II lanthipeptide synthetase MfuM and a Class I lanthipeptide cyclase MfuC in a cascaded way. Unprecedentedly, we biochemically characterized the first M61 family aminopeptidase MfuP involved in RiPP biosynthesis, demonstrating that MfuP showed the activity of an endopeptidase activity. MfuP is leader-independent but strictly selective for the multibridge structure of myxococin A and responsible for unwrapping two rings via amide bond hydrolysis, yielding myxococin B. Furthermore, the X-ray crystal structure of MfuP and structural analysis, including active-site mutations, are reported. Finally, myxococins are evaluated to exhibit anti-inflammatory activity in lipopolysaccharide-induced macrophages without detectable cytotoxicity.

The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition.

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Constructing a Myxobacterial Natural Product Database to Facilitate NMR-Based Metabolomics Bioprospecting of Myxobacteria.

Myxobacteria are fascinating prokaryotes featuring a potent capacity for producing a wealth of bioactive molecules with intricate chemical topology as well as intriguing enzymology, and thus it is critical to developing an efficient pipeline for bioprospecting. Herein, we construct the database MyxoDB, the first public compendium solely dedicated to myxobacteria, which enabled us to provide an overview of the structural diversity and taxonomic distribution of known myxobacterial natural products. Moreover, we demonstrated that the cutting-edge NMR-based metabolomics was effective to differentiate the biosynthetic priority of myxobacteria, whereby MyxoDB could greatly streamline the dereplication of multifarious known compounds and accordingly speed up the discovery of new compounds. This led to the rapid identification of a class of linear di-lipopeptides (archangimins) and a rare rearranged sterol (corasterol) that were endowed with unique chemical architectures and/or biosynthetic enzymology. We also showcased that NMR-based metabolomics, MyxoDB, and genomics can also work concertedly to accelerate the targeted discovery of a polyketidic compound pyxipyrrolone C. All in all, this study sets the stage for the discovery of many more novel natural products from underexplored myxobacterial resources.

Two reasons to kill: predation and kin discrimination in myxobacteria.

Myxobacteria are social microbial predators that use cell-cell contacts to identify bacterial or fungal prey and to differentiate kin relatives to initiate cellular responses. For prey killing, they assemble Tad-like and type III-like secretion systems at contact sites. For kin discrimination (KD), they assemble outer membrane exchange complexes composed of the TraA and TraB receptors at contacts sites. A type VI secretion system and Rhs proteins also mediate KD. Following cellular recognition, these systems deliver appropriate effectors into target cells. For prey, this leads to cell death and lysis for nutrient consumption by myxobacteria. In KD, a panel of effectors are delivered, and if adjacent cells are clonal cells, resistance ensues because they express a cognate panel of immunity factors; while nonkin lack complete immunity and are intoxicated. This review compares and contrasts recent findings from these systems in myxobacteria.

Synthesis of isochromanone containing natural products from myxobacteria.

This review details the biological activity, biosynthesis and synthesis of isochromanone metabolites isolated from myxobacteria. Strategies towards the synthesis of the isochomanone and oxazole fragments of these natural products are highlighted.

Structure Elucidation and Biosynthesis of Nannosterols A and B, Myxobacterial Sterols from Nannocystis sp. MNa10993.

Myxobacteria represent an underinvestigated source of chemically diverse and biologically active secondary metabolites. Here, we report the discovery, isolation, structure elucidation, and biological evaluation of two new bacterial sterols, termed nannosterols A and B (1, 2), from the terrestrial myxobacterium Nannocystis sp. (MNa10993). Nannosterols feature a cholestanol core with numerous modifications including a secondary alcohol at position C-15, a terminal vicinal diol side chain at C-24-C-25 (1, 2), and a hydroxy group at the angular methyl group at C-18 (2), which is unprecedented for bacterial sterols. Another rare chemical feature of bacterial triterpenoids is a ketone group at position C-7, which is also displayed by 1 and 2. The combined exploration based on myxobacterial high-resolution secondary metabolome data and genomic in silico investigations exposed the nannosterols as frequently produced sterols within the myxobacterial suborder of Nannocystineae. The discovery of the nannosterols provides insights into the biosynthesis of these new myxobacterial sterols, with implications in understanding the evolution of sterol production by prokaryotes.

Heterologous Biosynthesis of Myxobacterial Antibiotic Miuraenamide A.

The hard-to-culture slightly halophilic myxobacterium "Paraliomyxa miuraensis" SMH-27-4 produces antifungal cyclodepsipeptide miuraenamide A (1). Herein, the region (85.9 kbp) containing the biosynthetic gene cluster (BGC) coding the assembly of 1 was identified and heterologously expressed in Myxococcus xanthus. A biosynthetic pathway proposed using in silico analysis was verified through the gene disruption of the heterologous transformant. In addition to the core polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) genes, tyrosine halogenase and O-methyltransferase genes participated in the biosynthesis of 1 as their gene-disrupted mutants produced a new congener, debromomiuraenamide A (4), and a previously isolated congener, miuraenamide E (3), respectively. Multigene disruption provided a heterologous mutant that produced 1 with the highest yield among the prepared mutants. When fed on 3-bromo-L-tyrosine, this mutant produced more 1 in the yield of 1.21 mg/L, which was 20 times higher than that produced by the initially prepared heterologous transformant. Although this yield was comparable to that of the original producer SMH-27-4 (1 mg/L), the culture time was 4.5 times shorter than that of SMH-27-4, indicating a five-fold efficiency in productivity. The results indicate the great potential of the miuraenamide BGC for the future contribution to drug development through logical gene manipulation.

Differential response to prey quorum signals indicates predatory specialization of myxobacteria and ability to predate Pseudomonas aeruginosa.

Multiomic analysis of transcriptional and metabolic responses from the predatory myxobacteria Myxococcus xanthus and Cystobacter ferrugineus exposed to prey signalling molecules of the acylhomoserine lactone and quinolone quorum signalling classes provided insight into predatory specialization. Acylhomoserine lactone quorum signals elicited a general response from both myxobacteria. We suggest that this is likely due to the generalist predator lifestyles of myxobacteria and ubiquity of acylhomoserine lactone signals. We also provide data that indicates the core homoserine lactone moiety included in all acylhomoserine lactone scaffolds to be sufficient to induce this general response. Comparing both myxobacteria, unique transcriptional and metabolic responses were observed from Cystobacter ferrugineus exposed to the quinolone signal 2-heptylquinolin-4(1H)-one (HHQ) natively produced by Pseudomonas aeruginosa. We suggest that this unique response and ability to metabolize quinolone signals contribute to the superior predation of P. aeruginosa observed from C. ferrugineus. These results further demonstrate myxobacterial eavesdropping on prey signalling molecules and provide insight into how responses to exogenous signals might correlate with prey range of myxobacteria.

Coenzyme B12 -dependent and independent photoregulation of carotenogenesis across Myxococcales.

Light-induced carotenogenesis in Myxococcus xanthus is controlled by the B12 -based CarH repressor and photoreceptor, and by a separate intricate pathway involving singlet oxygen, the B12 -independent CarH paralogue CarA and various other proteins, some eukaryotic-like. Whether other myxobacteria conserve these pathways and undergo photoregulated carotenogenesis is unknown. Here, comparative analyses across 27 Myxococcales genomes identified carotenogenic genes, albeit arranged differently, with carH often in their genomic vicinity, in all three Myxococcales suborders. However, CarA and its associated factors were found exclusively in suborder Cystobacterineae, with carA-carH invariably in tandem in a syntenic carotenogenic operon, except for Cystobacter/Melittangium, which lack CarA but retain all other factors. We experimentally show B12 -mediated photoregulated carotenogenesis in representative myxobacteria, and a remarkably plastic CarH operator design and DNA binding across Myxococcales. Unlike the two characterized CarH from other phyla, which are tetrameric, Cystobacter CarH (the first myxobacterial homologue amenable to analysis in vitro) is a dimer that combines direct CarH-like B12 -based photoregulation with CarA-like DNA binding and inhibition by an antirepressor. This study provides new molecular insights into B12 -dependent photoreceptors. It further establishes the B12 -dependent pathway for photoregulated carotenogenesis as broadly prevalent across myxobacteria and its evolution, exclusively in one suborder, into a parallel complex B12 -independent circuit.

Sandacrabins - Structurally Unique Antiviral RNA Polymerase Inhibitors from a Rare Myxobacterium.

Structure elucidation and total synthesis of five unprecedented terpenoid-alkaloids, the sandacrabins, are reported, alongside with the first description of their producing organism Sandaracinus defensii MSr10575, which expands the Sandaracineae family by only its second member. The genome sequence of S. defensii as presented in this study was utilized to identify enzymes responsible for sandacrabin formation, whereby dimethylbenzimidazol, deriving from cobalamin biosynthesis, was identified as key intermediate. Biological activity profiling revealed that all sandacrabins except congener A exhibit potent antiviral activity against the human pathogenic coronavirus HCoV229E in the three digit nanomolar range. Investigation of the underlying mode of action discloses that the sandacrabins inhibit the SARS-CoV-2 RNA-dependent RNA polymerase complex, highlighting them as structurally distinct non-nucleoside RNA synthesis inhibitors. The observed segregation between cell toxicity at higher concentrations and viral inhibition opens the possibility for their medicinal chemistry optimization towards selective inhibitors.

Isolation of Nannocystis species from Iran and exploring their natural products.

Several different techniques were employed for the isolation of Nannocystis from various sources. A polyphasic approach was used for identification. Twelve strains of N. pusilla, N. exedens, and N. konarekensis with distinctive distribution between climates were identified. The bioactivity was examined against a panel of eight bacteria, two yeasts, and one fungus; cytotoxicity was tested on the L929 fibroblast cell line. Eleven strains mainly inhibit Gram-positive bacteria, and only one isolate was cytotoxic. The extract analyses by HPLC and LC-MS were compared to Myxobase, and eight different compounds were detected; a correlation was observed between compounds and producing species. 70% of strains had the potential to produce structurally diverse compounds. Nannochelins and althiomycin were the most abundant metabolites. The discovery of a new species of Nannocystis and the high potentiality of strains to produce secondary metabolites encourage further sampling and in-depth analysis of extracts to find new active metabolites.

Expanding the Ajudazol Cytotoxin Scaffold: Insights from Genome Mining, Biosynthetic Investigations, and Novel Derivatives.

Myxobacteria have proven to be a rich source of natural products, but their biosynthetic potential seems to be underexplored given the high number of biosynthetic gene clusters present in their genomes. In this study, a truncated ajudazol biosynthetic gene cluster in Cystobacter sp. SBCb004 was identified using mutagenesis and metabolomics analyses and a set of novel ajudazols (named ajudazols C-J, 3-10, respectively) were detected and subsequently isolated. Their structures were elucidated using comprehensive HR-MS and NMR spectroscopy. Unlike the known ajudazols A (1) and B (2), which utilize acetyl-CoA as the biosynthetic starter unit, these novel ajudazols were proposed to incorporate 3,3-dimethylacrylyl CoA as the starter. Ajudazols C-J (3-10, respectively) are characterized by varying degrees of hydroxylation, desaturation, and different glycosylation patterns. Two P450-dependent enzymes and one glycosyltransferase are shown to be responsible for the hydroxylation at C-8, the desaturation at C-15 and C-33, and the transfer of a d-ß-glucopyranose, respectively, based on mutagenesis results. One of the cytochrome P450-dependent enzymes and the glycosyltransferase were found to be encoded by genes located outside the biosynthetic gene cluster. Ajudazols C-H (3-8, respectively) exhibit cytotoxicity against various cancer cell lines.

Phenylalanine 314 is essential for the activity of maltogenic amylase from Corallococcus sp. EGB.

Maltogenic amylase CoMA from Corallococcus sp. strain EGB catalyzes the hydrolysis and transglycosylation of maltooligosaccharides and soluble starch into maltose, the sole hydrolysate. This process yields pure maltose with potentially wide applications. Here, we identified and evaluated the role of phenylalanine 314 (F314), a key amino acid located near the active center, in the catalytic activities of the CoMA. Site-directed mutagenesis analysis showed that the activity of a F314L mutant on potato starch substrate decreased to 26% of that of wild-type protein. Compared with the wild-type, F314L exhibited similar substrate specificity, hydrolysis pattern, pH, and temperature requirements. Circular dichroism spectrum data showed that the F314L mutation did not affect the structure of the folded protein. In addition, kinetic analysis demonstrated that F314L exhibited an increased Km value with lower substrate affinity. Homology modeling showed that the benzene ring structure of F314L was involved in π-π conjugation, which might potentially affect the affinity of CoMA toward starch. Taken together, these data demonstrated that F314 is essential for the hydrolytic activity of the CoMA from Corallococcus sp. strain EGB.