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

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

Heterologous production of a new lanthipeptide boletupeptin using a cryptic biosynthetic gene cluster of the myxobacterium Melittangium boletus.

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.

Heterologous biosynthesis of myxobacterial lanthipeptides melittapeptins.

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

Mining the soil myxobacteria and finding sources of anti-diabetic metabolites.

Secondary metabolites produced by myxobacterial genera are often characterized as diverse molecules with unique structural properties which drove us to search for myxobacterial source of anti-diabetic drug discovery. In the present study, from 80 soil samples, out of sixty-five observed isolates, 30 and 16 were purified as Myxococcus and non-Myxococcus, respectively. Isolated strains taxonomically belonged to the genera Myxococcus, Corallococcus and Cystobacter, Archangium, Nanocystis, and Sorangium, and some could not be attributed. Secondary metabolites of selected non-Myxococcus isolates extracted by the liquid-liquid method showed that the myxobacterium UTMC 4530 demonstrated the highest inhibition on the formation of carbonyl group and fructosamine, respectively. In addition, it showed 23% and 15.8% inhibitory activity on α-glucosides and α-amylase compared to acarbose (23%, 18%), respectively. The extract of strain UTMC 4530 showed 35% induction effect on glucose adsorption while showing no radical scavenging activity and no toxic effect on HRBC lysis and HepG2 in cytotoxicity assays. The strain UTMC 4530 (ON808962), with the multiple antidiabetic activity, showed 87.3% similarity to Corallococcus llansteffanensis which indicates its affiliation to a new genus. The results of this study revealed that secondary metabolites produced by strain UTMC 4530 can be considered a promising source to find new therapeutic and pharmaceutical applications perhaps a multi-mechanism anti-diabetic compound.

Myxobacteria: biology and bioactive secondary metabolites.

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.

Genome-Guided Discovery of the Myxobacterial Thiolactone-Containing Sorangibactins.

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.

Heterologous Biosynthesis of Myxobacterial Antibiotic Miuraenamide A.

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

Unmasking of the von Willebrand A-domain surface adhesin CglB at bacterial focal adhesions mediates myxobacterial gliding motility.

The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal-adhesion (bFA) sites to power gliding motility. Using total internal reflection fluorescence and force microscopies, we identify the von Willebrand A domain-containing outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling adhesin of the gliding transducer (Glt) machinery at bFAs. Biochemical and genetic analyses reveal that CglB localizes to the cell surface independently of the Glt apparatus; once there, it is recruited by the OM module of the gliding machinery, a heteroligomeric complex containing the integral OM ß barrels GltA, GltB, and GltH, as well as the OM protein GltC and OM lipoprotein GltK. This Glt OM platform mediates the cell-surface accessibility and retention of CglB by the Glt apparatus. Together, these data suggest that the gliding complex promotes regulated surface exposure of CglB at bFAs, thus explaining the manner by which contractile forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum.

Synthesis of isochromanone containing natural products from myxobacteria.

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

A Disturbed Siderophore Transport Inhibits Myxobacterial Predation.

BACKGROUND: Understanding the intrinsic mechanisms of bacterial competition is a fundamental question. Iron is an essential trace nutrient that bacteria compete for. The most prevalent manner for iron scavenging is through the secretion of siderophores. Although tremendous efforts have focused on elucidating the molecular mechanisms of siderophores biosynthesis, export, uptake, and regulation of siderophores, the ecological aspects of siderophore-mediated competition are not well understood. METHODS: We performed predation and bacterial competition assays to investigate the function of siderophore transport on myxobacterial predation. RESULTS: Deletion of msuB, which encodes an iron chelate uptake ABC transporter family permease subunit, led to a reduction in myxobacterial predation and intracellular iron, but iron deficiency was not the predominant reason for the decrease in the predation ability of the ∆msuB mutant. We further confirmed that obstruction of siderophore transport decreased myxobacterial predation by investigating the function of a non-ribosomal peptide synthetase for siderophore biosynthesis, a TonB-dependent receptor, and a siderophore binding protein in M. xanthus. Our results showed that the obstruction of siderophores transport decreased myxobacterial predation ability through the downregulation of lytic enzyme genes, especially outer membrane vesicle (OMV)-specific proteins. CONCLUSIONS: This work provides insight into the mechanism of siderophore-mediated competition in myxobacteria.

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

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

Thiamyxins: Structure and Biosynthesis of Myxobacterial RNA-Virus Inhibitors.

During our search for novel myxobacterial natural products, we discovered the thiamyxins: thiazole- and thiazoline-rich non-ribosomal peptide-polyketide hybrids with potent antiviral activity. We isolated four congeners of this unprecedented natural product family with the non-cyclized thiamyxin D fused to a glycerol unit at the C-terminus. Alongside their structure elucidation, we present a concise biosynthesis model based on biosynthetic gene cluster analysis and isotopically labelled precursor feeding. We report incorporation of a 2-(hydroxymethyl)-4-methylpent-3-enoic acid moiety by a GCN5-related N-acetyltransferase-like decarboxylase domain featuring polyketide synthase. The thiamyxins show potent inhibition of RNA viruses in cell culture models of corona, zika and dengue virus infection. Their potency up to a half maximal inhibitory concentration of 560 nM combined with milder cytotoxic effects on human cell lines indicate the potential for further development of the thiamyxins.

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

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

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

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

Structural and Functional Characterization of a New Bacterial Dipeptidyl Peptidase III Involved in Fruiting Body Formation in Myxobacteria.

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 CoDPP III was optimal at 50 °C and pH 7.0 with high thermostability up to 60 °C. Unique to CoDPP 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 CoDPP 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-CoDPP III rescued the developmental defect of the asgA mutant, and the fruiting bodies were more numerous and closely packed. Our data suggested that CoDPP 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.

Characterization of Constitutive Promoters for the Elicitation of Secondary Metabolites in Myxobacteria.

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 MS2-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 and their products: current trends and future perspectives in industrial applications.

Myxobacteria belong to a group of bacteria that are known for their well-developed communication system and synchronized or coordinated movement. This typical behavior of myxobacteria is mediated through secondary metabolites. They are capable of producing secondary metabolites belonging to several chemical classes with unique and wide spectrum of bioactivities. It is predominantly significant that myxobacteria specialize in mechanisms of action that are very rare with other producers. Most of the metabolites have been explored for their medical and pharmaceutical values while a lot of them are still unexplored. This review is an attempt to understand the role of potential metabolites produced by myxobacteria in different applications. Different myxobacterial metabolites have demonstrated antibacterial, antifungal, and antiviral properties along with cytotoxic activity against various cell lines. Beside their metabolites, these myxobacteria have also been discussed for better exploitation and implementation in different industrial sectors.

Structure and biosynthesis of sorangipyranone - a new γ-dihydropyrone from the myxobacterial strain MSr12020.

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 Sandarazols are Cryptic and Structurally Unique Plasmid-Encoded Toxins from a Rare Myxobacterium*.

Herein, we describe a new plasmid found in Sandaracinus sp. MSr10575 named pSa001 spanning 209.7 kbp that harbors a cryptic secondary metabolite biosynthesis gene cluster (BGC). Activation of this BGC by homologous-recombination-mediated exchange of the native promoter sequence against a vanillate inducible system led to the production and subsequent isolation and structure elucidation of novel secondary metabolites, the sandarazols A-G. The sandarazols contain intriguing structural features and very reactive functional groups such as an α-chlorinated ketone, an epoxyketone, and a (2R)-2-amino-3-(N,N-dimethylamino)-propionic acid building block. In-depth investigation of the underlying biosynthetic machinery led to a concise biosynthetic model for the new compound family, including several uncommon biosynthetic steps. The chlorinated congener sandarazol C shows an IC50  value of 0.5 µm against HCT 116 cells and a MIC of 14 µm against Mycobacterium smegmatis, which points at the sandarazols' potential function as defensive secondary metabolites or toxins.

Supercritical Fluid Extraction Enhances Discovery of Secondary Metabolites from Myxobacteria.

Supercritical fluid extraction (SFE) is widely used for the isolation of natural products from plants, but its application in efforts to identify structurally and physicochemically often dissimilar microbial natural products is limited to date. In this study, we evaluated the impact of SFE on the extractability of myxobacterial secondary metabolites, aiming to improve the prospects of discovering novel natural products. We investigated the influence of different co-solvents on the extraction efficiency of secondary metabolites from three myxobacterial strains and the antimicrobial activity profiles of the corresponding extracts. For each known secondary metabolite, we found extraction conditions using SFE leading to superior yields in the extracts compared to conventional solvent extraction. Compounds with a logP higher than 3 showed the best extraction efficiency using 20% EtOAc as a co-solvent, whereas compounds with logP values lower than 3 were better extractable using more polar co-solvents such as MeOH. Extracts generated with SFE showed increased antimicrobial activities including the presence of activities not explained by known myxobacterial secondary metabolites, highlighting the advantage of SFE for bioactivity-guided isolation. Moreover, non-targeted metabolomics analysis revealed a group of chlorinated metabolites produced by the well-studied model myxobacterium Myxococcus xanthus DK1622, which were not accessible previously due to their low concentration in conventional extracts. The enriched SF extracts were used for isolation and subsequent structure elucidation of chloroxanthic acid A as the founding member of a novel secondary metabolite family. Our findings encourage the increased utilization of SFE as a part of future screening workflows of microbial natural products.