Chromosomal organization of biosynthetic gene clusters suggests plasticity of myxobacterial specialized metabolism including descriptions for nine novel species: Archangium lansinium sp. nov., Myxococcus landrumus sp. nov., Nannocystis bainbridgea sp. nov., Nannocystis poenicansa sp. nov., Nannocystis radixulma sp. nov., Polyangium mundeleinium sp. nov., Pyxidicoccus parkwaysis sp. nov., Sorangium aterium sp. nov., Stigmatella ashevillena sp. nov.

Natural products discovered from bacteria provide critically needed therapeutic leads for drug discovery, and myxobacteria are an established source for metabolites with unique chemical scaffolds and biological activities. Myxobacterial genomes accommodate an exceptional number and variety of biosynthetic gene clusters (BGCs) which encode for features involved in specialized metabolism. Continued discovery and sequencing of novel myxobacteria from the environment provides BGCs for the genome mining pipeline. Herein, we describe the collection, sequencing, and genome mining of 20 myxobacteria isolated from rhizospheric soil samples collected in North America. Nine isolates where determined to be novel species of myxobacteria including representatives from the genera Archangium, Myxococcus, Nannocystis, Polyangium, Pyxidicoccus, Sorangium, and Stigmatella. Growth profiles, biochemical assays, and descriptions are provided for all proposed novel species. We assess the BGC content of all isolates and observe differences between Myxococcia and Polyangiia clusters. Utilizing complete or near complete genome sequences we compare the chromosomal organization of BGCs of related myxobacteria from various genera and suggest spatial proximity of hybrid, modular clusters contributes to the metabolic adaptability of myxobacteria.

Concepts and conjectures concerning predatory performance of myxobacteria.

Myxobacteria are excellent model organisms for investigation of predator-prey interactions and predatory shaping of microbial communities. This review covers interdisciplinary topics related to myxobacterial predation and provides current concepts and challenges for determining predatory performance. Discussed topics include the role of specialized metabolites during predation, genetic determinants for predatory performance, challenges associated with methodological differences, discrepancies between sequenced and environmental myxobacteria, and factors that influence predation.

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.

Functional genomics study of Pseudomonas putida to determine traits associated with avoidance of a myxobacterial predator.

Predation contributes to the structure and diversity of microbial communities. Predatory myxobacteria are ubiquitous to a variety of microbial habitats and capably consume a broad diversity of microbial prey. Predator-prey experiments utilizing myxobacteria have provided details into predatory mechanisms and features that facilitate consumption of prey. However, prey resistance to myxobacterial predation remains underexplored, and prey resistances have been observed exclusively from predator-prey experiments that included the model myxobacterium Myxococcus xanthus. Utilizing a predator-prey pairing that instead included the myxobacterium, Cystobacter ferrugineus, with Pseudomonas putida as prey, we observed surviving phenotypes capable of eluding predation. Comparative transcriptomics between P. putida unexposed to C. ferrugineus and the survivor phenotype suggested that increased expression of efflux pumps, genes associated with mucoid conversion, and various membrane features contribute to predator avoidance. Unique features observed from the survivor phenotype when compared to the parent P. putida include small colony variation, efflux-mediated antibiotic resistance, phenazine-1-carboxylic acid production, and increased mucoid conversion. These results demonstrate the utility of myxobacterial predator-prey models and provide insight into prey resistances in response to predatory stress that might contribute to the phenotypic diversity and structure of bacterial communities.

Assessment of Evolutionary Relationships for Prioritization of Myxobacteria for Natural Product Discovery.

Discoveries of novel myxobacteria have started to unveil the potentially vast phylogenetic diversity within the family Myxococcaceae and have brought about an updated approach to myxobacterial classification. While traditional approaches focused on morphology, 16S gene sequences, and biochemistry, modern methods including comparative genomics have provided a more thorough assessment of myxobacterial taxonomy. Herein, we utilize long-read genome sequencing for two myxobacteria previously classified as Archangium primigenium and Chondrococcus macrosporus, as well as four environmental myxobacteria newly isolated for this study. Average nucleotide identity and digital DNA-DNA hybridization scores from comparative genomics suggest previously classified as A. primigenium to instead be a novel member of the genus Melittangium, C. macrosporus to be a potentially novel member of the genus Corallococcus with high similarity to Corallococcus exercitus, and the four isolated myxobacteria to include another novel Corallococcus species, a novel Pyxidicoccus species, a strain of Corallococcus exiguus, and a potentially novel Myxococcus species with high similarity to Myxococcus stipitatus. We assess the biosynthetic potential of each sequenced myxobacterium and suggest that genus-level conservation of biosynthetic pathways support our preliminary taxonomic assignment. Altogether, we suggest that long-read genome sequencing benefits the classification of myxobacteria and improves determination of biosynthetic potential for prioritization of natural product discovery.

Draft Genome Sequence of Aneurinibacillus sp. Strain BA2021, Isolated as a Contaminant of a Laboratory-Cultivated Predatory Myxobacterium.

During laboratory cultivation of the myxobacterium Archangium violaceum strain Cb vi76, a reoccurring contaminant was isolated and sequenced. Comparative taxonomic analysis of the draft genome suggested the contaminant to be a novel species, currently designated Aneurinibacillus sp. strain BA2021, from the genus Aneurinibacillus, members of which are considered promising biocontrol agents.

Identification of a solo acylhomoserine lactone synthase from the myxobacterium Archangium gephyra.

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.

Myxobacterial Response to Methyljasmonate Exposure Indicates Contribution to Plant Recruitment of Micropredators.

Chemical exchanges between plants and microbes within rhizobiomes are critical to the development of community structure. Volatile root exudates such as the phytohormone methyljasmonate (MeJA) contribute to various plant stress responses and have been implicated to play a role in the maintenance of microbial communities. Myxobacteria are competent predators of plant pathogens and are generally considered beneficial to rhizobiomes. While plant recruitment of myxobacteria to stave off pathogens has been suggested, no involved chemical signaling processes are known. Herein we expose predatory myxobacteria to MeJA and employ untargeted mass spectrometry, motility assays, and RNA sequencing to monitor changes in features associated with predation such as specialized metabolism, swarm expansion, and production of lytic enzymes. From a panel of four myxobacteria, we observe the most robust metabolic response from plant-associated Archangium sp. strain Cb G35 with 10 µM MeJA impacting the production of at least 300 metabolites and inducing a ≥ fourfold change in transcription for 56 genes. We also observe that MeJA induces A. sp. motility supporting plant recruitment of a subset of the investigated micropredators. Provided the varying responses to MeJA exposure, our observations indicate that MeJA contributes to the recruitment of select predatory myxobacteria suggesting further efforts are required to explore the microbial impact of plant exudates associated with biotic stress.

Survey of Biosynthetic Gene Clusters from Sequenced Myxobacteria Reveals Unexplored Biosynthetic Potential.

Coinciding with the increase in sequenced bacteria, mining of bacterial genomes for biosynthetic gene clusters (BGCs) has become a critical component of natural product discovery. The order Myxococcales, a reputable source of biologically active secondary metabolites, spans three suborders which all include natural product producing representatives. Utilizing the BiG-SCAPE-CORASON platform to generate a sequence similarity network that contains 994 BGCs from 36 sequenced myxobacteria deposited in the antiSMASH database, a total of 843 BGCs with lower than 75% similarity scores to characterized clusters within the MIBiG database are presented. This survey provides the biosynthetic diversity of these BGCs and an assessment of the predicted chemical space yet to be discovered. Considering the mere snapshot of myxobacteria included in this analysis, these untapped BGCs exemplify the potential for natural product discovery from myxobacteria.

GEX1A, a Polyketide from Streptomyces chromofuscus, Corrects the Cellular Defects Associated with Niemann-Pick Type C1 in Human Fibroblasts.

We report the first evidence of GEX1A, a polyketide known to modulate alternative pre-mRNA splicing, as a potential treatment for Niemann-Pick type C disease. GEX1A was isolated from its producing organism, Streptomyces chromofuscus, and screened in NPC1 mutant cells alongside several semisynthetic analogues. We found that GEX1A and analogues are capable of restoring cholesterol trafficking in NPC1 mutant fibroblasts, as well as altering the expression of NPC1 isoforms detected by Western blot. These results, along with the compound's favorable pharmacokinetic properties, highlight the potential of spliceosome-targeting scaffolds such as GEX1A for the treatment of genetic diseases.

Marine Myxobacteria: A Few Good Halophiles.

Currently considered an excellent candidate source of novel chemical diversity, the existence of marine myxobacteria was in question less than 20 years ago. This review aims to serve as a roll call for marine myxobacteria and to summarize their unique features when compared to better-known terrestrial myxobacteria. Characteristics for discrimination between obligate halophilic, marine myxobacteria and halotolerant, terrestrial myxobacteria are discussed. The review concludes by highlighting the need for continued discovery and exploration of marine myxobacteria as producers of novel natural products.

Draft Genome Sequence of Archangium sp. Strain Cb G35.

In an effort to explore myxobacterial natural product biosynthetic pathways, the draft genome sequence of Archangium sp. strain Cb G35 has been obtained. Analysis of the genome using antiSMASH predicts 49 natural product biosynthetic pathways. This genome will contribute to the investigation of myxobacterial secondary metabolite biosynthetic pathways.

Draft Genome Sequence of Cystobacter ferrugineus Strain Cbfe23.

In an effort to explore myxobacterial natural product biosynthetic pathways, the draft genome sequence of Cystobacter ferrugineus strain Cbfe23 has been obtained. Analysis of the genome using antiSMASH suggests a multitude of unique natural product biosynthetic pathways. This genome will contribute to the investigation of secondary metabolism in other myxobacterial species.

α-Methylation follows condensation in the gephyronic acid modular polyketide synthase.

C-methyltransferases (MTs) from modular polyketide synthase assembly lines are relatively rare and unexplored domains that are responsible for installing α-methyl groups into nascent polyketide backbones. The stage at which these synthase-embedded enzymes operate during polyketide biosynthesis has yet to be conclusively demonstrated. In this work we establish the activity and substrate preference for six MTs from the gephyronic acid polyketide synthase and demonstrate their ability to methylate both N-acetylcysteamine- and acyl carrier protein-linked ß-ketoacylthioester substrates but not malonyl thioester equivalents. These data strongly indicate that MT-catalyzed methylation occurs immediately downstream of ketosynthase-mediated condensation during polyketide assembly. This work represents the first successful report of MT-catalyzed mono- and dimethylation of simple thioester substrates and provides the groundwork for future mechanistic and engineering studies on this important but poorly understood enzymatic domain.

Draft Genome Sequence of Gephyronic Acid Producer Cystobacter violaceus Strain Cb vi76.

A draft genome sequence of Cystobacter violaceus strain Cb vi76, which produces the eukaryotic protein synthesis inhibitor gephyronic acid, has been obtained. The genome contains numerous predicted secondary metabolite clusters, including the gephyronic acid biosynthetic pathway. This genome will contribute to the investigation of secondary metabolism in other Cystobacter strains.

Elucidation of gephyronic acid biosynthetic pathway revealed unexpected SAM-dependent methylations.

Gephyronic acid, a cytostatic polyketide produced by the myxobacterium Cystobacter violaceus Cb vi76, exhibits potent and selective eukaryotic protein synthesis inhibition. Next-generation sequencing of the C. violaceus genome revealed five type I polyketide synthases and post-PKS tailoring enzymes including an O-methyltransferase and a cytochrome P450 monooxygenase. Seven methyltransferase (MT) domains embedded within the PKS subunits were found to install the methyl branches throughout the gephyronic acid skeleton. A rare loading domain from the GNAT superfamily also contains an embedded MT domain that catalyzes the in situ production of an isobutyryl starter unit. Phylogenetic analysis identified new motifs that distinguish MT domains located in PKS pathways with in cis acyltransferase (AT) domains from MT domains located in PKS pathways with trans AT enzymes. The identification of the gene cluster sets the stage for the generation of a heterologous expression system, which will allow further investigation of selective eukaryotic protein synthesis inhibitors through the generation of gephyronic acid analogues.

The role of transcription in heterologous expression of polyketides in bacterial hosts.

Heterologous expression of biosynthetic pathways is an indispensable tool in the discovery, production, engineering, and characterization of bacterial polyketides and the complex enzymology involved in their biosynthesis. Ensuring transcription of polyketide biosynthetic gene clusters in heterologous hosts is a pressing problem. This review evaluates the two strategies used to ensure transcription. The first is a promoter replacement approach where promoters known to function in the heterologous host are inserted into the biosynthetic gene cluster. The second is an approach that relies on the heterologous host recognizing and utilizing promoters native to the gene cluster. Both have been successful methodologies and have different strengths and weaknesses, which are highlighted and discussed.

Heterologous expression of the oxytetracycline biosynthetic pathway in Myxococcus xanthus.

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a "universal" host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential "universal" host for heterologous expression of polyketide biosynthetic gene clusters.