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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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Myxobacteria are predatory bacteria with antimicrobial activity, utilizing complex mechanisms to kill their prey and assimilate their macromolecules. Having large genomes encoding hundreds of secondary metabolites, hydrolytic enzymes and antimicrobial peptides, these organisms are widely studied for their antibiotic potential. MyxoPortal is a comprehensive genomic database hosting 262 genomes of myxobacterial strains. Datasets included provide genome annotations with gene locations, functions, amino acids and nucleotide sequences, allowing analysis of evolutionary and taxonomical relationships between strains and genes. Biosynthetic gene clusters are identified by AntiSMASH, and dbAMP-generated antimicrobial peptide sequences are included as a resource for novel antimicrobial discoveries, while curated datasets of CRISPR/Cas genes, regulatory protein sequences, and phage associated genes give useful insights into each strain's biological properties. MyxoPortal is an intuitive open-source database that brings together application-oriented genomic features that can be used in taxonomy, evolution, predation and antimicrobial research. MyxoPortal can be accessed at http://dicsoft1.physics.iisc.ac.in/MyxoPortal/. Database URL:  http://dicsoft1.physics.iisc.ac.in/MyxoPortal/. Graphical Abstract., (© The Author(s) 2024. Published by Oxford University Press.)

Two myxobacterial strains (KH5-1 T 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 KH5-1 T and NO1 were affiliated with the genus Corallococcus, with the closest neighbours being Corallococcus exercitus AB043A T (99.77% and 99.84%, respectively). Genome comparisons using orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridisation similarity (dDDH) with strains KH5-1 T and NO1 and their phylogenetically close relatives in Corallococcus spp. were below the thresholds. The major cellular fatty acids of strains KH5-1 T and NO1 were iso-C 15:0 (31.9%, 30.0%), summed feature 3 (comprising C 16:1 ω7c and/or C 1 6:1 ω6c) (20.2%, 17.7%), and iso-C 17:0 (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 KH5-1 T and NO1 represent a new species in the genus Corallococcus, for which the proposed name is Corallococcus caeni sp. nov. The type strain is KH5-1 T (= NCIMB 15510 T  = JCM 36609 T )., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and destruct genetic invaders. Systematic collation and characterization of endogenous CRISPR-Cas systems are conducive to our understanding and potential utilization of this natural genetic machinery. In this study, we screened 39 complete and 692 incomplete genomes of myxobacteria using a combined strategy to dispose of the abridged genome information and revealed at least 19 CRISPR-Cas subtypes, which were distributed with a taxonomic difference and often lost stochastically in intraspecies strains. The cas genes in each subtype were evolutionarily clustered but deeply separated, while most of the CRISPRs were divided into four types based on the motif characteristics of repeat sequences. The spacers recorded in myxobacterial CRISPRs were in high G+C content, matching lots of phages, tiny amounts of plasmids, and, surprisingly, massive organismic genomes. We experimentally demonstrated the immune and self-target immune activities of three endogenous systems in Myxococcus xanthus DK1622 against artificial genetic invaders and revealed the microhomology-mediated end-joining mechanism for the immunity-induced DNA repair but not homology-directed repair. The panoramic view and immune activities imply potential omnipotent immune functions and applications of the endogenous CRISPR-Cas machinery., Importance: Serving as an adaptive immune system, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) empower prokaryotes to fend off the intrusion of external genetic materials. Myxobacteria are a collective of swarming Gram-stain-negative predatory bacteria distinguished by intricate multicellular social behavior. An in-depth analysis of their intrinsic CRISPR-Cas systems is beneficial for our understanding of the survival strategies employed by host cells within their environmental niches. Moreover, the experimental findings presented in this study not only suggest the robust immune functions of CRISPR-Cas in myxobacteria but also their potential applications., Competing Interests: The authors declare no conflict of interest.

Archangium gephyra KYC5002 produces tubulysins during the death phase. In this study, we aimed to determine whether dead cells produce tubulysins. Cells were cultured for three days until the verge of the death phase, disrupted via ultrasonication, incubated for 2 h, and examined for tubulysin production. Non-disrupted cells produced 0.14 mg/L of tubulysin A and 0.11 mg/L of tubulysin B. Notably, tubulysin A production was increased by 4.4-fold to 0.62 mg/L and that of tubulysin B was increased by 6.7-fold to 0.74 mg/L in the disrupted cells. The same increase in tubulysin production was observed when the cells were killed by adding hydrogen peroxide. However, when the enzymes were inactivated via heat treatment of the cultures at 65 °C for 30 min, no significant increase in tubulysin production due to cell death was observed. Reverse transcription-quantitative polymerase chain reaction analysis of tubB mRNA revealed that the expression levels of tubulysin biosynthetic enzyme genes increased during the death phase compared to those during the vegetative growth phase. Our findings suggest that A. gephyra produces biosynthetic enzymes and subsequently uses them for tubulysin production in the cell death phase or during cell lysis by predators., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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&#95;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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Myxobacteria have comparatively large genomes that contain many biosynthetic genes with the potential to produce secondary metabolites. Based on genome mining, we discovered a new biosynthetic gene cluster of class III lanthipeptide in the genome of the myxobacterium Melittangium boletus. The biosynthetic gene cluster contained a precursor peptide-coding gene bolA, and a class III lanthipeptide synthetase-coding gene bolKC. The expression vector containing bolA and bolKC was constructed using synthetic DNA with codon-optimized sequences based on the commercially available vector pET29b. Co-expression of the two genes in the host Escherichia coli BL21(DE3) yielded a new class III lanthipeptide named boletupeptin. The structure of boletupeptin was proposed to have one unit of labionin, as determined by mass spectrometry experiments after reductive cleavage. This is the first report of a class III lanthipeptide from a myxobacterial origin., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In the past century, microbial natural products have proven themselves to be substantial and fruitful sources of anti-infectives. In addition to the well-studied Actinobacteria, understudied bacterial taxa like the Gram-negative myxobacteria have increasingly gained attention in the ongoing search for novel and biologically active natural products. In the course of a regional sampling campaign to source novel myxobacteria, we recently uncovered new myxobacterial strains MCy12716 and MCy12733 belonging to the Myxococcaceae clade. Early bioactivity screens of the bacterial extracts revealed the presence of bioactive natural products that were identified as angiolam A and several novel derivatives. Sequencing of the corresponding producer strains allowed the identification of the angiolam biosynthetic gene cluster, which was verified by targeted gene inactivation. Based on bioinformatic analysis of the biosynthetic gene cluster, a concise biosynthesis model was devised to explain angiolam biosynthesis. Importantly, novel angiolam derivatives uncovered in this study named angiolams B, C, and D were found to display promising antiparasitic activities against the malaria pathogen Plasmodium falciparum in the 0.3-0.8 µM range.IMPORTANCEThe COVID-19 pandemic and continuously emerging antimicrobial resistance (AMR) have recently raised awareness about limited treatment options against infectious diseases. However, the shortage of treatment options against protozoal parasitic infections, like malaria, is much more severe, especially for the treatment of so-called neglected tropical diseases. The detection of anti-parasitic bioactivities of angiolams produced by MCy12716 and MCy12733 displays the hidden potential of scarcely studied natural products to have promising biological activities in understudied indications. Furthermore, the improved biological activities of novel angiolam derivatives against Plasmodium falciparum and the evaluation of its biosynthesis display the opportunities of the angiolam scaffold on route to treat protozoal parasitic infections as well as possible ways to increase the production of derivatives with improved bioactivities., Competing Interests: The authors declare no conflict of interest.

The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from Haliangium ochraceum that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide-based antiphage signaling system (CBASS). Cyclic tri-adenosine monophosphate (AMP)-induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase. Subsequently, activated PCaspase cleaves a multitude of proteins, which results in a strong interference phenotype in vivo in Escherichia coli. Taken together, our findings reveal how a CRISPR-Cas-based detection of a target RNA triggers a cascade of caspase-associated proteolytic activities.

Myxobacteria (phylum Myxococcota) are abundant and virtually ubiquitous microbial predators. Facultatively multicellular organisms, they are able to form multicellular fruiting bodies and swarm across surfaces, cooperatively hunting for prey. Myxobacterial communities are able to kill a wide range of prey microbes, assimilating their biomass to fuel population growth. Their mechanism of predation is exobiotic - hydrolytic enzymes and toxic metabolites are secreted into the extracellular environment, killing and digesting prey cells from without. However, recent observations of single-cell predation and contact-dependent prey killing challenge the dogma of myxobacterial predation being obligately cooperative. Regardless of their predatory mechanisms, myxobacteria have a broad prey range, which includes Gram-negative bacteria, Gram-positive bacteria and fungi. Pangenome analyses have shown that their extremely large genomes are mainly composed of accessory genes, which are not shared by all members of their species. It seems that the diversity of accessory genes in different strains provides the breadth of activity required to prey upon such a smorgasbord of microbes, and also explains the considerable strain-to-strain variation in predatory efficiency against specific prey. After providing a short introduction to general features of myxobacterial biology which are relevant to predation, this review brings together a rapidly growing body of work into the molecular mechanisms and genetic basis of predation, presenting a summary of current knowledge, highlighting trends in research and suggesting strategies by which we can potentially exploit myxobacterial predation in the future., (Copyright © 2024. Published by Elsevier Inc.)

Myxobacteria are Gram-negative eubacteria and they thrive in a variety of habitats including soil rich in organic matter, rotting wood, animal dung and marine environment. Myxobacteria are a promising source of new compounds associated with diverse bioactive spectrum and unique mode of action. The genome information of myxobacteria has revealed many orphan biosynthetic pathways indicating that these bacteria can be the source of several novel natural products. In this review, we highlight the biology of myxobacteria with emphasis on their habitat, life cycle, isolation methods and enlist all the bioactive secondary metabolites purified till date and their mode of action., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Myxobacteria serve as a treasure trove of secondary metabolites. During our ongoing search for bioactive natural products, a novel subclass of disorazoles termed disorazole Z was discovered. Ten disorazole Z family members were purified from a large-scale fermentation of the myxobacterium Sorangium cellulosum So ce1875 and characterized by electrospray ionization-high-resolution mass spectrometry (ESI-HRMS), X-ray, nuclear magnetic resonance (NMR), and Mosher ester analysis. Disorazole Z compounds are characterized by the lack of one polyketide extension cycle, resulting in a shortened monomer in comparison to disorazole A, which finally forms a dimer in the bis-lactone core structure. In addition, an unprecedented modification of a geminal dimethyl group takes place to form a carboxylic acid methyl ester. The main component disorazole Z1 shows comparable activity in effectively killing cancer cells to disorazole A1 via binding to tubulin, which we show induces microtubule depolymerization, endoplasmic reticulum delocalization, and eventually apoptosis. The disorazole Z biosynthetic gene cluster (BGC) was identified and characterized from the alternative producer S. cellulosum So ce427 and compared to the known disorazole A BGC, followed by heterologous expression in the host Myxococcus xanthus DK1622. Pathway engineering by promoter substitution and gene deletion paves the way for detailed biosynthesis studies and efficient heterologous production of disorazole Z congeners. IMPORTANCE Microbial secondary metabolites are a prolific reservoir for the discovery of bioactive compounds, which prove to be privileged scaffolds for the development of new drugs such as antibacterial and small-molecule anticancer drugs. Consequently, the continuous discovery of novel bioactive natural products is of great importance for pharmaceutical research. Myxobacteria, especially Sorangium spp., which are known for their large genomes with yet-underexploited biosynthetic potential, are proficient producers of such secondary metabolites. From the fermentation broth of Sorangium cellulosum strain So ce1875, we isolated and characterized a family of natural products named disorazole Z, which showed potent anticancer activity. Further, we report on the biosynthesis and heterologous production of disorazole Z. These results can be stepping stones toward pharmaceutical development of the disorazole family of anticancer natural products for (pre)clinical studies., Competing Interests: The authors declare no conflict of interest.

Polyp bail-out constitutes both a stress response and an asexual reproductive strategy that potentially facilitates dispersal of some scleractinian corals, including several dominant reef-building taxa in the family Pocilloporidae. Recent studies have proposed that microorganisms may be involved in onset and progression of polyp bail-out. However, changes in the coral microbiome during polyp bail-out have not been investigated. In this study, we induced polyp bail-out in Pocillopora corals using hypersaline and hyperthermal methods. Bacterial community dynamics during bail-out induction were examined using the V5-V6 region of the 16S-rRNA gene. From 70 16S-rRNA gene libraries constructed from coral tissues, 1,980 OTUs were identified. Gammaproteobacteria and Alphaproteobacteria consistently constituted the dominant bacterial taxa in all coral tissue samples. Onset of polyp bail-out was characterized by increased relative abundance of Alphaproteobacteria and decreased abundance of Gammaproteobacteria in both induction experiments, with the shift being more prominent in response to elevated temperature than to elevated salinity. Four OTUs, affiliated with Thalassospira , Marisediminitalea , Rhodobacteraceae , and Myxococcales , showed concurrent abundance increases at the onset of polyp bail-out in both experiments, suggesting potential microbial causes of this coral stress response. IMPORTANCE Polyp bail-out represents both a stress response and an asexual reproductive strategy with significant implications for reshaping tropical coral reefs in response to global climate change. Although earlier studies have suggested that coral-associated microbiomes likely contribute to initiation of polyp bail-out in scleractinian corals, there have been no studies of coral microbiome shifts during polyp bail-out. In this study, we present the first investigation of changes in bacterial symbionts during two experiments in which polyp bail-out was induced by different environmental stressors. These results provide a background of coral microbiome dynamics during polyp bail-out development. Increases in abundance of Thalassospira , Marisediminitalea , Rhodobacteraceae , and Myxococcales that occurred in both experiments suggest that these bacteria are potential microbial causes of polyp bail-out, shedding light on the proximal triggering mechanism of this coral stress response., Competing Interests: The authors declare no conflict of interest.

Myxobacteria have potential application value in developing new antibiotics and environmental protection. In this study, in order to establish a more suitable method for diversity studies of myxobacteria, the effects of primers, polymerase chain reaction (PCR) approaches and sample preservation methods on the results were compared by Illumina high-throughput sequencing. The results showed that the relative abundance and operational taxonomic unit (OTU) ratio of myxobacteria amplified by the universal primers accounted for 0.91-1.85% and 2.82-4.10% of total bacteria, indicating that myxobacteria were the dominant bacteria both in population and species numbers. The relative abundance and OTU number and ratio of myxobacteria amplified by the myxobacteria semi-specific primers were significantly higher than those amplified by the universal primers, of which the primer pair W2/802R specifically amplified myxobacteria of suborder Cystobacterineae, while the primer pair W5/802R mainly amplified myxobacteria of suborder Sorangineae and also amplified more species of suborder Nannocystineae at the same time. Among three PCR approaches, the relative abundance and OTU ratio of myxobacteria amplified by the touch-down PCR were the highest. More myxobacterial OTUs were detected in most dried samples. In conclusion, the combination of the myxobacteria semi-specific primer pairs W2/802R and W5/802R, touch-down PCR, and dry preservation of samples were more conducive to diversity studies of myxobacteria., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest, and manuscript is approved by all authors for publication. This article does not contain any studies with human participants or animals performed by any of the authors., (Copyright © 2023. Published by Elsevier B.V.)

As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora. These enzymes include three specialized β-1,3-glucanases (AcGlu13.1, -13.2 and -13.3) that act as a cooperative consortium to target β-1,3-glucans of Phytophthora. However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain β-1,3-glucans. Heterologous expression of AcGlu13.1, -13.2 or -13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae, conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora, and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection., (© 2023. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

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.

Tubulysins are bioactive secondary metabolites produced by myxobacteria that promote microtubule disassembly. Microtubules are required for protozoa such as Tetrahymena to form cilia and flagella. To study the role of tubulysins in myxobacteria, we co-cultured myxobacteria and Tetrahymena. When 4000 Tetrahymena thermophila and 5.0 × 10 8 myxobacteria were added to 1 ml of CYSE medium and co-cultured for 48 h, the population of T. thermophila increased to more than 75,000. However, co-culturing tubulysin-producing myxobacteria, including Archangium gephyra KYC5002, with T. thermophila caused the population of T. thermophila to decrease from 4000 to less than 83 within 48 h. Almost no dead bodies of T. thermophila were observed in the culture medium. Co-culturing of T. thermophila and the A. gephyra KYC5002 strain with inactivation of the tubulysin biosynthesis gene led to the population of T. thermophila increasing to 46,667. These results show that in nature, most myxobacteria are preyed upon by T. thermophila, but some myxobacteria prey on and kill T. thermophila using tubulysins. Adding purified tubulysin A to T. thermophila changed the cell shape from ovoid to spherical and caused cell surface cilia to disappear., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

By targeting mRNA transcripts, non-coding small RNAs (sRNAs) regulate the expression of genes governing a wide range of bacterial functions. In the social myxobacterium Myxococcus xanthus , the sRNA Pxr serves as a gatekeeper of the regulatory pathway controlling the life-cycle transition from vegetative growth to multicellular fruiting body development. When nutrients are abundant, Pxr prevents the initiation of the developmental program, but Pxr-mediated inhibition is alleviated when cells starve. To identify genes essential for Pxr function, a developmentally defective strain in which Pxr-mediated blockage of development is constitutively active (strain "OC") was transposon-mutagenized to identify suppressor mutations that inactivate or bypass Pxr inhibition and thereby restore development. One of the four loci in which a transposon insertion restored development is rnd , encoding the Ribonuclease D protein (RNase D). RNase D is an exonuclease important for tRNA maturation. Here, we show that disruption of rnd abolishes the accumulation of Pxr-S, the product of Pxr processing from a longer precursor form (Pxr-L) and the active inhibitor of development. Additionally, the decrease in Pxr-S caused by rnd disruption was associated with increased accumulation primarily of a longer novel Pxr-specific transcript (Pxr-XL) rather than of Pxr-L. The introduction of a plasmid expressing rnd reverted cells back to OC-like phenotypes in development and Pxr accumulation, indicating that a lack of RNase D alone suppresses the developmental defect of OC. Moreover, an in vitro Pxr-processing assay demonstrated that RNase D processes Pxr-XL into Pxr-L; this implies that overall, Pxr sRNA maturation requires a sequential two-step processing. Collectively, our results indicate that a housekeeping ribonuclease plays a central role in a model form of microbial aggregative development. To our knowledge, this is the first evidence implicating RNase D in sRNA processing.

The operation of modern wastewater treatment plants (WWTPs) is driven by activated sludge microbiota, a complex assemblage of trophically interacting microorganisms. Microbial predation is crucial to fundamental understanding of how biological interactions drive microbiome structuring and functioning of WWTPs. However, predatory bacteria have received little attention regarding their diversity, activity, and ecological function in activated sludge, limiting the exploitation of food web interactions for wastewater microbiome engineering. Here, by using rRNA-stable isotope probing of activated sludge microbiota with 13 C-labeled prey bacteria, we uncovered diverse as-yet-uncultivated putative predatory bacteria that actively incorporated 13 C-biomass. Myxobacteria, especially Haliangium and the mle1-27 clade, were found as the dominant active predators, refreshing conventional views based on a few predatory isolates of Bdellovibrionota from WWTPs. The identified predatory bacteria showed more selective predation on prey compared with the protists dominated by ciliates, providing in situ evidence for inter-domain predation behavior divergence in activated sludge. Putative predatory bacteria were tracked over a two-year microbiome monitoring effort at a local WWTP, revealing the predominance of Myxococcota (6.5 ± 1.3%) over Bdellovibrionota (1.0 ± 0.2%) lineages. Phylogenetic analysis unveiled highly diverse myxobacteria inhabiting activated sludge and suggested a habitat filtering effect in global WWTPs. Further mining of a global activated sludge microbiome dataset revealed the prevalence of Myxococcota (5.4 ± 0.1%) species and potential impacts of myxobacterial predation on process performance. Collectively, our findings provided unique insights into the predating activity, diversity, and prevalence of Myxococcota species in activated sludge, highlighting their links with wastewater treatment processes via trophic regulation of enteric and functional bacteria., (© 2023. The Author(s).)

A better understanding of the genetic regulation of the biosynthesis of microbial compounds could accelerate the discovery of new biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the net production rates of known natural compounds, indicating that their biosynthesis was critically regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for boosting compound yields from myxobacterial producer strains., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

In this study, an unprecedented myxobacterial siderophore termed sorangibactin was discovered by heterologous expression of a coelibactin-like nonribosomal peptide synthetase (NRPS) gene cluster from the Sorangiineae strain MSr11367 in the host Myxococcus xanthus DK1622. De novo structure elucidation uncovered a linear polycyclic structure consisting of an N-terminal phenol group, an oxazole, tandem N -methyl-thiazolidines, and an unusual C-terminal γ-thiolactone moiety. Except for the unprecedented oxazoline dehydrogenation to form an oxazole, which we show to be catalyzed by a cytochrome P450-dependent enzyme, other tailoring steps were found necessary for efficient downstream processing. The unusual thioesterase (TE) domain is proposed to select homocysteine or methionine for offloading involving an intramolecular γ-thiolactone formation. Its active site comprises a rare cysteine, which was found essential for product formation by point mutation to alanine or serine, which both abolished its activity. This unusual release mechanism and the resulting rare thiolactone structure can serve as a starting point for detailed biochemical investigations.

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.

Social diversification in microbes is an evolutionary process where lineages bifurcate into distinct populations that cooperate with themselves but not with other groups. In bacteria, this is frequently driven by horizontal transfer of mobile genetic elements (MGEs). Here, the resulting acquisition of new genes changes the recipient's social traits and consequently how they interact with kin. These changes include discriminating behaviors mediated by newly acquired effectors. Since the producing cell is protected by cognate immunity factors, these selfish elements benefit from selective discrimination against recent ancestors, thus facilitating their proliferation and benefiting the host. Whether social diversification benefits the population at large is less obvious. The widespread use of next-generation sequencing has recently provided new insights into population dynamics in natural habitats and the roles MGEs play. MGEs belong to accessory genomes, which often constitute the majority of the pangenome of a taxon, and contain most of the kin-discriminating loci that fuel rapid social diversification. We further discuss mechanisms of diversification and its consequences to populations and conclude with a case study involving myxobacteria.

Antimicrobial resistance (AMR) is a global concern, and as soon as new antibiotics are introduced, resistance to those agents emerges. Therefore, there is an increased appetite for alternative antimicrobial agents to traditional antibiotics. Here, we used in silico methods to investigate potential antimicrobial peptides (AMPs) from predatory myxobacteria. Six hundred seventy-two potential AMP sequences were extracted from eight complete myxobacterial genomes. Most putative AMPs were predicted to be active against Klebsiella pneumoniae with least activity being predicted against Staphylococcus aureus. One hundred seventeen AMPs (defined here as 'potent putative AMPs') were predicted to have very good activity against more than two bacterial pathogens, and these were characterized further in silico. All potent putative AMPs were predicted to have anti-inflammatory and antifungal properties, but none was predicted to be active against viruses. Twenty six (22%) of them were predicted to be hemolytic to human erythrocytes, five were predicted to have anticancer properties, and 56 (47%) were predicted to be biofilm active. In vitro assays using four synthesized AMPs showed high MIC values (e.g. So&#95;ce&#95;56&#95;913 250 µg/ml and Coral&#95;AMP411 125 µg/ml against E. coli). However, antibiofilm assays showed a substantial reduction in numbers (e.g. Coral&#95;AMP411 and Myxo&#95;mac104 showed a 69% and 73% reduction, respectively, at the lowest concentration against E. coli) compared to traditional antibiotics. Fourteen putative AMPs had high sequence similarity to proteins which were functionally associated with proteins of known function. The myxobacterial genomes also possessed a variety of biosynthetic gene clusters (BGCs) that can encode antimicrobial secondary metabolites, but their numbers did not correlate with those of the AMPs. We suggest that AMPs from myxobacteria are a promising source of novel antimicrobial agents with a plethora of biological properties., (© 2022. The Author(s).)

Dipeptidyl peptidase III (DPP III) is a zinc-dependent enzyme that specifically hydrolyzes dipeptides from the N-terminal of different-length peptides, and it is involved in a number of physiological processes. Here, DPP III with an atypical pentapeptide zinc binding motif (HELMH) was identified from Corallococcus sp. EGB. It was shown that the activity of recombined Co DPP III was optimal at 50 °C and pH 7.0 with high thermostability up to 60 °C. Unique to Co DPP III, the crystal structure of the ligand-free enzyme was determined as a dimeric and closed form. The relatively small inter-domain cleft creates a narrower entrance to the substrate binding site and the unfavorable binding of the bulky naphthalene ring. The ectopic expression of Co DPP III in M. xanthus DK1622 resulted in a 12 h head start in fruiting body development compared with the wild type. Additionally, the A-signal prepared from the starving DK1622- Co DPP III rescued the developmental defect of the asgA mutant, and the fruiting bodies were more numerous and closely packed. Our data suggested that Co DPP III played a role in the fruiting body development of myxobacteria through the accumulation of peptides and amino acids to act as the A-signal.

Mangroves are unique intertidal ecosystems that provide ecological niches to different microbes, which play various roles in nutrient recycling and diverse environmental activities. The association between myxobacteria and mangroves are hitherto poorly understood. The aim of our study was to evaluate the myxobacterial community composition as well as isolate myxobacteria and to characterize the antimicrobial activity of myxobacteria isolates from Indonesian mangroves. Twenty-five cultivable myxobacteria were affiliated in six genera: Myxococcus, Corallococcus, Archangium, Chondromyces, Racemicystis and Nannocystis of the order Myxococcales based on partial 16S rRNA gene sequences. Thirteen crude extracts showed moderate activities against at least one of human pathogenic microorganisms. The crude extract of Racemicystis sp. strain 503MSO indicated a novel compound, which has not been reported in the database yet and the identification of this compound needs further study. The myxobacterial communities of three different sampling sites were analyzed using primers adapted for the myxobacteria group identification. The results showed that myxobacterial communities are more diverse than assumed. Therefore, our study has highlighted the importance of the mangrove habitat as promising harbor of myxobacteria as well as novel antimicrobial compounds with activity against pathogenic microorganisms., (© 2022. The Author(s).)

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- β -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.

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 K m 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., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

Light-induced carotenogenesis in Myxococcus xanthus is controlled by the B 12 -based CarH repressor and photoreceptor, and by a separate intricate pathway involving singlet oxygen, the B 12 -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 B 12 -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 B 12 -based photoregulation with CarA-like DNA binding and inhibition by an antirepressor. This study provides new molecular insights into B 12 -dependent photoreceptors. It further establishes the B 12 -dependent pathway for photoregulated carotenogenesis as broadly prevalent across myxobacteria and its evolution, exclusively in one suborder, into a parallel complex B 12 -independent circuit., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

A metabolome-guided screening approach in the novel myxobacterium Corallococcus sp. MCy9072 resulted in the isolation of the unprecedented natural product myxofacycline A, which features a rare isoxazole substructure. Identification and genomic investigation of additional producers alongside targeted gene inactivation experiments and heterologous expression of the corresponding biosynthetic gene cluster in the host Myxococcus xanthus DK1622 confirmed a noncanonical megaenzyme complex as the biosynthetic origin of myxofacycline A. Induced expression of the respective genes led to significantly increased production titers enabling the identification of six further members of the myxofacycline natural product family. Whereas myxofacyclines A-D display an isoxazole substructure, intriguingly myxofacyclines E and F were found to contain 4-pyrimidinole, a heterocycle unprecedented in natural products. Lastly, myxofacycline G features another rare 1,2-dihydropyrol-3-one moiety. In addition to a full structure elucidation, we report the underlying biosynthetic machinery and present a rationale for the formation of all myxofacyclines. Unexpectedly, an extraordinary polyketide synthase-nonribosomal peptide synthetase hybrid was found to produce all three types of heterocycle in these natural products., (© 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Genome mining has revealed that myxobacteria contain a myriad of cryptic biosynthetic gene clusters (BGCs). Here, we report the characterization of a panel of myxobacterial promoters with variable strength that are applicable in the engineering of BGCs in myxobacteria. The screened strongest constitutive promoter was used to efficiently enhance the expression of two complex BGCs governing the biosynthesis of myxochromide and DKxanthene in the model myxobacterium Myxococcus xanthus DK1622. We also showcased the combination of promoter engineering and MS 2 -based spectral networking as an effective strategy to shed light on the previously overlooked chemistry in the family of myxochromide-type lipopeptides. The enriched promoter library substantially expanded the synthetic biology toolkit available for myxobacteria.

Myxobacteria are a prolific source of structurally diverse natural products, and one of the best-studied myxobacterial products is the siderophore myxochelin. Herein, we report two new compounds, myxochelins N ( 1 ) and O ( 2 ), that are nicotinic paralogs of myxochelin A, from the terrestrial myxobacterium Archangium sp. SDU34; 2 is functionalized with a rare 2-oxazolidinone. A precursor-feeding experiment implied that the biosynthesis of 1 or 2 was due to altered substrate specificity of the loading module of MxcE, which likely accepts nicotinic acid and benzoic acid instead of more conventional 2,3-dihydroxybenzoic acid. We also employed a phylogenomic approach to map the evolutionary relationships of the myxochelin biosynthetic gene clusters (BGCs) in all the available myxobacterial genomes, to pave the way for the future discovery of potentially hidden myxochelin derivatives. Although the biological function of 1 and 2 is unclear yet, this work underpins that even extensively studied BGCs in myxobacteria can still produce new chemistry.

Corallococcus sp. strain EGB is a Gram-negative myxobacteria isolated from saline soil, and has considerable potential for the biocontrol of phytopathogenic fungi. However, the detailed mechanisms related to development and predatory behavior are unclear. To obtain a comprehensive overview of genetic features, the genome of strain EGB was sequenced, annotated, and compared with 10 other Corallococcus species. The strain EGB genome was assembled as a single circular chromosome of 9.4 Mb with 7916 coding genes. Phylogenomics analysis showed that strain EGB was most closely related to Corallococcus interemptor AB047A, and it was inferred to be a novel species within the Corallococcus genus. Comparative genomic analysis revealed that the pan-genome of Corallococcus genus was large and open. Only a small proportion of genes were specific to strain EGB, and most of them were annotated as hypothetical proteins. Subsequent analyses showed that strain EGB produced abundant extracellular enzymes such as chitinases and β-(1,3)-glucanases, and proteases to degrade the cell-wall components of phytopathogenic fungi. In addition, 35 biosynthetic gene clusters potentially coding for antimicrobial compounds were identified in the strain EGB, and the majority of them were present in the dispensable pan-genome with unexplored metabolites. Other genes related to secretion and regulation were also explored for strain EGB. This study opens new perspectives in the greater understanding of the predatory behavior of strain EGB, and facilitates a potential application in the biocontrol of fungal plant diseases in the future.

Background: Myxobacteria harbor numerous biosynthetic gene clusters that can produce a diverse range of secondary metabolites. Minicystis rosea DSM 24000 T is a soil-dwelling myxobacterium belonging to the suborderSorangiineae and family Polyangiaceae and is known to produce various secondary metabolites as well as polyunsaturated fatty acids (PUFAs). Here, we use whole-genome sequencing to explore the diversity of biosynthetic gene clusters in M. rosea., Results: Using PacBio sequencing technology, we assembled the 16.04 Mbp complete genome of M. rosea DSM 24000 T , the largest bacterial genome sequenced to date. About 44% of its coding potential represents paralogous genes predominantly associated with signal transduction, transcriptional regulation, and protein folding. These genes are involved in various essential functions such as cellular organization, diverse niche adaptation, and bacterial cooperation, and enable social behavior like gliding motility, sporulation, and predation, typical of myxobacteria. A profusion of eukaryotic-like kinases (353) and an elevated ratio of phosphatases (8.2/1) in M. rosea as compared to other myxobacteria suggest gene duplication as one of the primary modes of genome expansion. About 7.7% of the genes are involved in the biosynthesis of a diverse array of secondary metabolites such as polyketides, terpenes, and bacteriocins. Phylogeny of the genes involved in PUFA biosynthesis (pfa) together with the conserved synteny of the complete pfa gene cluster suggests acquisition via horizontal gene transfer from Actinobacteria., Conclusion: Overall, this study describes the complete genome sequence of M. rosea, comparative genomic analysis to explore the putative reasons for its large genome size, and explores the secondary metabolite potential, including the biosynthesis of polyunsaturated fatty acids., (© 2021. The Author(s).)

Bacteria are globally distributed in various environments on earth, but a global view of the geographic diversity and distribution of a single taxon is lacking. The Earth Microbiome Project (EMP) has established a global collection of microbial communities, providing the possibility for such a survey. Myxococcales is a bacterial order with a potent ability to produce diverse natural products and have wide application potential in agriculture, biomedicine, and environmental protection. In this study, through a comparative analysis of the EMP data and public information, we determined that myxobacteria account for 2.34% of the total bacterial operational taxonomic units (OTUs), and are one of the most diverse bacterial groups on Earth. Myxococcales OTUs are globally distributed and prefer nonsaline soil and sediments, followed by saline environments, but rarely appear in host-associated environments. Myxobacteria are among the least-investigated bacterial groups. The presently cultured and genome-sequenced myxobacteria are most likely environmentally widespread and abundant taxa, and account for approximately 10% and 7% of the myxobacterial community (>97% similarity), respectively. This global panoramic view of the geographic distribution and diversity of myxobacteria, as well as their cultured and genome-sequenced information, will enable us to explore these important bioresources more reasonably and efficiently. The diversity and distribution of myxobacteria beyond the EMP data are further discussed. IMPORTANCE The diversity and distribution of bacteria are crucial for our understanding of their ecological importance and application potential. Myxobacteria are fascinating prokaryotes with multicellular behaviors and a potent capacity for producing secondary metabolites, and have a wide range of potential applications. The ecological importance of myxobacteria in major ecosystems is becoming established, but the global geographic diversity and distribution remain unclear. From a global survey we revealed that Myxococcales OTUs are globally distributed and prefer nonsaline soil and sediments, followed by saline environments, but rarely appear in host-associated environments. The global panoramic view of the geographic distribution and diversity of myxobacteria, as well as their cultured and genome-sequenced information, will enable us to explore these important bioresources more reasonably and efficiently.

Trophic interactions are crucial for carbon cycling in food webs. Traditionally, eukaryotic micropredators are considered the major micropredators of bacteria in soils, although bacteria like myxobacteria and Bdellovibrio are also known bacterivores. Until recently, it was impossible to assess the abundance of prokaryotes and eukaryotes in soil food webs simultaneously. Using metatranscriptomic three-domain community profiling we identified pro- and eukaryotic micropredators in 11 European mineral and organic soils from different climes. Myxobacteria comprised 1.5-9.7% of all obtained SSU rRNA transcripts and more than 60% of all identified potential bacterivores in most soils. The name-giving and well-characterized predatory bacteria affiliated with the Myxococcaceae were barely present, while Haliangiaceae and Polyangiaceae dominated. In predation assays, representatives of the latter showed prey spectra as broad as the Myxococcaceae. 18S rRNA transcripts from eukaryotic micropredators, like amoeba and nematodes, were generally less abundant than myxobacterial 16S rRNA transcripts, especially in mineral soils. Although SSU rRNA does not directly reflect organismic abundance, our findings indicate that myxobacteria could be keystone taxa in the soil microbial food web, with potential impact on prokaryotic community composition. Further, they suggest an overlooked, yet ecologically relevant food web module, independent of eukaryotic micropredators and subject to separate environmental and evolutionary pressures., (© 2021. The Author(s).)

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1-N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.

Sorangipyranone was isolated as a novel natural product featuring a unique 2,3-dihydro-γ-4H-pyrone scaffold from cultures of the myxobacterial strain MSr12020. We report here the full structure elucidation of sorangipyranone by spectroscopic techniques including 2D NMR and high-resolution mass spectrometry together with the analysis of the biosynthetic pathway. Determination of the absolute configuration was performed by time-dependent density functional theory-electronic circular dichroism calculations and determination of the applicability of the Snatzke's helicity rule, to correlate the high-wavelength n→π* electronic circular dichroism (ECD) transition and the absolute configuration of the 2,3-dihydro-4H-γ-pyrone, was done by the analysis of low-energy conformers and the Kohn-Sham orbitals. Sorangipyranone outlines a new class of a γ-dihydropyrone-containing natural product comprised of malonyl-CoA-derived building blocks and features a unique polyketide scaffold. In silico analysis of the genome sequence of the myxobacterial strain MSr12020 complemented with feeding experiments employing stable isotope-labeled precursors allowed the identification and annotation of a candidate biosynthetic gene cluster that encodes a modular polyketide synthase assembly line. A model for the biosynthetic pathway leading to the formation of the γ-dihydropyrone scaffold is presented in this study., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Considered a key taxon in soil and marine microbial communities, myxobacteria exist as coordinated swarms that utilize a combination of lytic enzymes and specialized metabolites to facilitate predation of microbes. This capacity to produce specialized metabolites and the associated abundance of biosynthetic pathways contained within their genomes have motivated continued drug discovery efforts from myxobacteria. Of all myxobacterial biosynthetic gene clusters deposited in the antiSMASH database, only one putative acylhomoserine lactone (AHL) synthase, agpI, was observed, in genome data from Archangium gephyra. Without an AHL receptor also apparent in the genome of A. gephyra, we sought to determine if AgpI was an uncommon example of an orphaned AHL synthase. Herein we report the bioinformatic assessment of AgpI and discovery of a second AHL synthase from Vitiosangium sp. During axenic cultivation conditions, no detectible AHL metabolites were observed in A. gephyra extracts. However, heterologous expression of each synthase in Escherichia coli provided detectible quantities of 3 AHL signals including 2 known AHLs, C8-AHL and C9-AHL. These results suggest that A. gephyra AHL production is dormant during axenic cultivation. The functional, orphaned AHL synthase, AgpI, is unique to A. gephyra, and its utility to the predatory myxobacterium remains unknown.

Over the past decade, the one strain many compounds (OSMAC) approach has been established for the activation of biosynthetic gene clusters (BGCs), which mainly encode the enzymes of secondary metabolite (SM) biosynthesis pathways. These BGCs were successfully activated by altering various culture conditions, such as aeration rate, temperature, and nutrient composition. Here, we determined the biosynthetic potential of 43 bacteria using the genome mining tool antiSMASH. Based on the number of BGCs, biological safety, availability of deposited cultures, and literature coverage, we selected five promising candidates: Bacillus amyloliquefaciens DSM7, Corallococcus coralloides DSM2259, Pyxidicoccus fallax HKI727, Rhodococcus jostii DSM44719, and Streptomyces griseochromogenes DSM40499. The bacteria were cultivated under a broad range of OSMAC conditions (nutrient-rich media, minimal media, nutrient-limited media, addition of organic solvents, addition of biotic additives, and type of culture vessel) to fully assess the biosynthetic potential. In particular, we investigated so far scarcely applied OSMAC conditions to enhance the diversity of SMs. We detected the four predicted compounds bacillibactin, desferrioxamine B, myxochelin A, and surfactin. In total, 590 novel mass features were detected in a broad range of investigated OSMAC conditions, which outnumber the predicted gene clusters for all investigated bacteria by far. Interestingly, we detected mass features of the bioactive compounds cyclo-(Tyr-Pro) and nocardamin in extracts of DSM7 and DSM2259. Both compounds were so far not reported for these strains, indicating that our broad OSMAC screening approach was successful. Remarkably, the infrequently applied OSMAC conditions in defined medium with and without nutrient limitation were demonstrated to be very effective for BGC activation and for SM discovery.

Members of the predatory Myxococcales (myxobacteria) possess large genomes, undergo multicellular development, and produce diverse secondary metabolites, which are being actively prospected for novel drug discovery. To direct such efforts, it is important to understand the relationships between myxobacterial ecology, evolution, taxonomy, and genomic variation. This study investigated the genomes and pan-genomes of organisms within the Myxococcaceae, including the genera Myxococcus and Corallococcus, the most abundant myxobacteria isolated from soils. Previously, ten species of Corallococcus were known, whereas six species of Myxococcus phylogenetically surrounded a third genus (Pyxidicoccus) composed of a single species. Here, we describe draft genome sequences of five novel species within the Myxococcaceae (Myxococcus eversor, Myxococcus llanfairpwllgwyngyllgogerychwyrndrobwllllantysiliogogogochensis, Myxococcus vastator, Pyxidicoccus caerfyrddinensis, and Pyxidicoccus trucidator) and for the Pyxidicoccus type species strain, Pyxidicoccus fallax DSM 14698T. Genomic and physiological comparisons demonstrated clear differences between the five novel species and every other Myxococcus or Pyxidicoccus spp. type strain. Subsequent analyses of type strain genomes showed that both the Corallococcus pan-genome and the combined Myxococcus and Pyxidicoccus (Myxococcus/Pyxidicoccus) pan-genome are large and open, but with clear differences. Genomes of Corallococcus spp. are generally smaller than those of Myxococcus/Pyxidicoccus spp. but have core genomes three times larger. Myxococcus/Pyxidicoccus spp. genomes are more variable in size, with larger and more unique sets of accessory genes than those of Corallococcus species. In both genera, biosynthetic gene clusters are relatively enriched in the shell pan-genomes, implying they grant a greater evolutionary benefit than other shell genes, presumably by conferring selective advantages during predation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Objective: To produce high concentrations of 13-hydroxy-14,15-epoxy-eicosatrienoic acid (14,15-hepoxilin B 3 , 14,15-HXB 3 ) and 13,14,15-trihydroxy-eicosatrienoic acid (13,14,15-trioxilin B 3 , 13,14,15-TrXB 3 ) from arachidonic acid (ARA) using microbial 15-lipoxygenase (15-LOX) without and with epoxide hydrolase (EH), respectively., Results: The products obtained from the bioconversion of ARA by recombinant Escherichia coli cells containing Archangium violaceum 15-LOX without and with Myxococcus xanthus EH were identified as 14,15-HXB 3 and 13,14,15-TrXB 3 , respectively. Under the optimal conditions of 30 g cells L -1 , 200 mM ARA, 25 °C, and initial pH 7.5, the cells converted 200 mM ARA into 192 mM 14,15-HXB 3 and 100 mM 13,14,15-TrXB 3 for 150 min, with conversion yields of 96 and 51% and productivities of 77 and 40 mM h -1 , respectively., Conclusion: These are the highest concentrations, productivities, and yields of hepoxilin and trioxilin from ARA reported thus far.

Myxobacteria are one of the most promising secondary metabolites producers. However, they are difficult to isolate and cultivate. To obtain more myxobacteria and know the effects of environmental factors on myxobacterial community, we characterized myxobacterial communities in Dinghushan acidic forest soils of pH 3.6-4.5 with culture-dependent and -independent techniques, and analyzed environmental factors shaping myxobacterial communities. A total of 21 myxobacteria were isolated using standard cultivation methods, including eleven isolates of Corallococcus, nine isolates of Myxococcus and one isolate of Archangium, and contained three potential novel species. In addition, a total of 67 unknown myxobacterial operational taxonomic units (OTUs) were obtained using high-throughput sequencing method. The abundance of Myxococcales account for 0.9-2.2% of bacterial communities, and Sorangium is the most abundant genus (60.1%) in Myxococcales. Correlation analysis demonstrated that bacterial diversity and soil pH are the key factors shaping myxobacterial community. These results revealed an abundant myxobacterial community which is shaped by other bacteria and pH in Dinghushan acidic forest soils., Competing Interests: The authors have declared that no competing interests exist.

Drug resistance is a critical issue in future clinical treatment. Methicillin-resistant Staphylococcus aureus (MRSA) is among the pathogens that need indispensable drug-discovery efforts. The myxobacteria are a unique group of bacteria that have recently been regarded for their potency to produce new drugs with high chemical diversity and unusual mode of actions. The present study was conducted to isolate and screen myxobacteria for the first time from Iran habitats and evaluate their antibacterial activity against the multidrug-resistant strain of S. aureus. Out of 62 soil and rotten plant samples, 51 myxobacteria were isolated. The isolates belonged to Myxococcus, Corallococcus, Pyxidicoccus, and Cystobacter genera based on morphology and 16S rRNA gene sequencing. Secondary metabolites of the selected strains were screened for activity on MDR strain with resistance to multiple antibiotic classes. The semi-purified fraction from Cystobacter sp. UTMC 4086 showed potent activity against MDR S. aureus with minimum inhibitory effect at 5 ≥ μg per mL compared with vancomycin (5 μg per mL) as well as no toxicity against Artemia salina. Hence, the strain Cystobacter sp. UTMC 4086 can be a valuable candidate for antibiotic discovery against MRSA and its metabolites can be subjected to further purification and analysis aimed at the identification of the effective chemical entity.

Myxobacteria conduct complex social traits that requires populations to be highly related and devoid of exploiters. To enrich for clonal cells in populations, they employ kin discrimination mechanisms. One key system involves a polymorphic cell surface receptor, TraA, which recognizes self by homotypic interactions with neighboring myxobacterial cells. Recent studies revealed that TraA and its partner TraB are fluid outer membrane proteins that coalesce into foci upon recognition of kin. The formation of foci leads to transient membrane fusion junctions and the bidirectional exchange of outer membrane components that facilitates cooperative behaviors. Additionally, expansive suites of polymorphic lipoprotein toxins are exchanged, which act as self-identity barcodes that exquisitely discriminate against nonself to assemble homogenous populations., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The reduction of nitrous oxide (N 2 O) to N 2 represents the key terminal step in canonical denitrification. Nitrous oxide reductase (NosZ), the enzyme associated with this biological step, however, is not always affiliated with denitrifying microorganisms. Such organisms were shown recently to possess a Clade II (atypical) nosZ gene, in contrast to Clade I (typical) nosZ harbored in more commonly studied denitrifiers. Subsequent phylogenetic analyses have shown that Clade II NosZ are affiliated with a much broader diversity of microorganisms than those with Clade I NosZ, the former including both non-denitrifiers and denitrifiers. Most studies attempting to characterize the nosZ gene diversity using DNA-based PCR approaches have only focused on Clade I nosZ, despite recent metagenomic sequencing studies that have demonstrated the dominance of Clade II nosZ genes in many ecosystems, particularly soil. As a result, these studies have greatly underestimated the genetic potential for N 2 O reduction present in ecosystems. Because the high diversity of Clade II NosZ makes it impossible to design a universal primer set that would effectively amplify all nosZ genes in this clade, we developed a suite of primer sets to specifically target seven of ten designated subclades of Clade II nosZ genes. The new primer sets yield suitable product sizes for paired end amplicon sequencing and qPCR, demonstrated here in their use for both conventional single-reaction and multiplex array platforms. In addition, we show the utility of these primers for detecting nosZ gene transcripts from mRNA extracted from soil., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The Escherichia coli microcin C (McC) and related compounds are potent Trojan horse peptide-nucleotide antibiotics. The peptide part facilitates transport into sensitive cells. Inside the cell, the peptide part is degraded by nonspecific peptidases releasing an aspartamide-adenylate containing a phosphoramide bond. This nonhydrolyzable compound inhibits aspartyl-tRNA synthetase. In addition to the efficient export of McC outside the producing cells, special mechanisms have evolved to avoid self-toxicity caused by the degradation of the peptide part inside the producers. Here, we report that histidine-triad (HIT) hydrolases encoded in biosynthetic clusters of some McC homologs or by standalone genes confer resistance to McC-like compounds by hydrolyzing the phosphoramide bond in toxic aspartamide-adenosine, rendering them inactive. IMPORTANCE Uncovering the mechanisms of resistance is a required step for countering the looming antibiotic resistance crisis. In this communication, we show how universally conserved histidine-triad hydrolases provide resistance to microcin C, a potent inhibitor of bacterial protein synthesis., (Copyright © 2020 Yagmurov et al.)

Background: Myxobacteria are micropredators in the soil ecosystem with the capacity to move and feed cooperatively. Some myxobacterial strains have been used to control soil-borne fungal phytopathogens. However, interactions among myxobacteria, plant pathogens, and the soil microbiome are largely unexplored. In this study, we aimed to investigate the behaviors of the myxobacterium Corallococcus sp. strain EGB in the soil and its effect on the soil microbiome after inoculation for controlling cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC)., Results: A greenhouse and a 2-year field experiment demonstrated that the solid-state fermented strain EGB significantly reduced the cucumber Fusarium wilt by 79.6% (greenhouse), 66.0% (2015, field), and 53.9% (2016, field). Strain EGB adapted to the soil environment well and decreased the abundance of soil-borne FOC efficiently. Spatiotemporal analysis of the soil microbial community showed that strain EGB migrated towards the roots and root exudates of the cucumber plants via chemotaxis. Cooccurrence network analysis of the soil microbiome indicated a decreased modularity and community number but an increased connection number per node after the application of strain EGB. Several predatory bacteria, such as Lysobacter, Microvirga, and Cupriavidus, appearing as hubs or indicators, showed intensive connections with other bacteria., Conclusion: The predatory myxobacterium Corallococcus sp. strain EGB controlled cucumber Fusarium wilt by migrating to the plant root and regulating the soil microbial community. This strain has the potential to be developed as a novel biological control agent of soil-borne Fusarium wilt. Video abstract.

Myxobacteria are fascinating micro-predators due to their extraordinary social lifestyle, which is unique in the bacterial domain. These taxa are metabolically active in the soil microbial food web and control populations of soil microbes. However, the effects of fertilisation treatments on predatory myxobacteria in agricultural systems are often overlooked. Here, the high-throughput absolute abundance quantification (HAAQ) method was employed to investigate the abundance and cell density of myxobacteria in the Red Soil Experimental Station fields following 29 years of fertilisation. Using 16S rRNA gene amplicons, we detected a total of 419 myxobacterial operational taxonomic units (OTUs), accounting for 0.25-2.70% of the total bacterial abundance. Significantly different myxobacterial communities were found between nitrogen-fertilised (N&#95;cluster) and manure-fertilised (M&#95;cluster) samples by principal coordinate analysis (PCoA), analysis of similarities (ANOSIM), and Manhattan analysis (p < 0.05). N fertiliser treatments significantly decreased the myxobacterial abundance and copy number, species accumulation index (S), and Shannon index (p < 0.05). Furthermore, UpSet plots showed that the OTU number in the N fertiliser treatment was only 24.4% of that in the M treatment, as the application of N decreased the number of low-abundance myxobacterial OTUs. In addition, network analysis, redundancy analysis (RDA), and random forest (RF) analysis showed that myxobacterial abundance and copy number were the most important variables predicting the soil bacterial community and functional gene α- and β-diversity (P < 0.05). Our findings imply that soil acidification caused by the application of nitrogen fertilisers is the most important driver of the decrease in the myxobacterial abundance and copy number in the soil. We suggest that the changes in the abundance and number of myxobacteria are strongly correlated with the overall bacterial α- and β-diversity indices. In addition, such changes may be an important factor in the overall changes in microbial communities., (Copyright © 2019 Elsevier B.V. All rights reserved.)