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.

Histidine-Triad Hydrolases Provide Resistance to Peptide-Nucleotide Antibiotics.

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.

Identification of an endo-chitinase from Corallococcus sp. EGB and evaluation of its antifungal properties.

As the main component of the fungal cell wall, chitin has been regarded as an optimal molecular target for the biocontrol of plant-pathogenic fungi. In this study, the chitin hydrolase CcCti1, which belongs to the glycoside hydrolase family 18 (GH 18) and exhibits potential antifungal activity, was identified from Corallococcus sp. EGB. CcCti1 lacks a fibronectin type-III (FN3) domain that is present in similar enzymes from most genera of myxobacteria, indicating that CcCti1 may have acquired chitinase activity due to the FN3 domain deletion during myxobacterial evolution. CcCti1 was expressed in Escherichia coli BL21 (DE3) with a specific activity of up to 10.5 U/µmol with colloidal chitin as the substrate. Product analysis showed that CcCti1 could hydrolyze chitin into N-acetylated chitohexaose (GlcNAc)6 as the major product, in addition to chitooligosaccharides. The analysis of biochemical properties indicated that the CBD and FN3 domains in CcCti1 determine the substrate affinity and pH stability. Otherwise, CcCti1 exhibited efficient biocontrol activity against the plant pathogen Magnaporthe oryzae in a dose-dependent manner, inhibiting the conidia germination and appressoria formation at a concentration of 0.08 mg/mL. Overall, the chitohexaose-producing chitinase CcCti1 with hydrolytic features may find potential application in chitin conversion and biocontrol of fungal plant diseases.

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

Orientia tsutsugamushi Is Highly Susceptible to the RNA Polymerase Switch Region Inhibitor Corallopyronin A In Vitro and In Vivo.

Scrub typhus is a potentially lethal infection caused by the obligate intracellular bacterium Orientia tsutsugamushi Reports on the emergence of doxycycline-resistant strains highlight the urgent need to develop novel antiinfectives against scrub typhus. Corallopyronin A (CorA) is a novel α-pyrone compound synthesized by the myxobacterium Corallococcus coralloides that was characterized as a noncompetitive inhibitor of the switch region of the bacterial RNA polymerase (RNAP). We investigated the antimicrobial action of CorA against the human-pathogenic Karp strain of O. tsutsugamushiin vitro and in vivo The MIC of CorA against O. tsutsugamushi was remarkably low (0.0078 µg/ml), 16-fold lower than that against Rickettsia typhi In the lethal intraperitoneal O. tsutsugamushi mouse infection model, a minimum daily dose of 100 µg CorA protected 100% of infected mice. Two days of treatment were sufficient to confer protection. In contrast to BALB/c mice, SCID mice succumbed to the infection despite treatment with CorA or tetracycline, suggesting that antimicrobial treatment required synergistic action of the adaptive immune response. Similar to tetracycline, CorA did not prevent latent infection of O. tsutsugamushiin vivo However, latency was not caused by acquisition of antimicrobial resistance, since O. tsutsugamushi reisolated from latently infected BALB/c mice remained fully susceptible to CorA. No mutations were found in the CorA-binding regions of the ß and ß' RNAP subunit genes rpoB and rpoC Inhibition of the RNAP switch region of O. tsutsugamushi by CorA is therefore a novel and highly potent target for antimicrobial therapy for scrub typhus.

The biofilm inhibitor Carolacton inhibits planktonic growth of virulent pneumococci via a conserved target.

New antibacterial compounds, preferentially exploiting novel cellular targets, are urgently needed to fight the increasing resistance of pathogens against conventional antibiotics. Here we demonstrate that Carolacton, a myxobacterial secondary metabolite previously shown to damage Streptococcus mutans biofilms, inhibits planktonic growth of Streptococcus pneumoniae TIGR4 and multidrug-resistant clinical isolates of serotype 19A at nanomolar concentrations. A Carolacton diastereomer is inactive in both streptococci, indicating a highly specific interaction with a conserved cellular target. S. mutans requires the eukaryotic-like serine/threonine protein kinase PknB and the cysteine metabolism regulator CysR for susceptibility to Carolacton, whereas their homologues are not needed in S. pneumoniae, suggesting a specific function for S. mutans biofilms only. A bactericidal effect of Carolacton was observed for S. pneumoniae TIGR4, with a reduction of cell numbers by 3 log units. The clinical pneumonia isolate Sp49 showed immediate growth arrest and cell lysis, suggesting a bacteriolytic effect of Carolacton. Carolacton treatment caused a reduction in membrane potential, but not membrane integrity, and transcriptome analysis revealed compensatory reactions of the cell. Our data show that Carolacton might have potential for treating pneumococcal infections.

Isolation and identification of myxobacteria from saline-alkaline soils in Xinjiang, China.

Fifty-eight terrestrial and salt-tolerant myxobacteria were isolated from the saline-alkaline soils collected from Xinjiang, China. Based on the morphologies and the 16S rRNA gene sequences, these isolates were assigned into 6 genera, Myxococcus, Cystobacter, Corallococcus, Sorangium, Nannocystis and Polyangium. All the strains grew better with 1% NaCl than without NaCl. Some Myxococcus strains were able to grow at 2% NaCl concentration, suggesting that these strains may be particular type of terrestrial myxobacteria.

[Isolation and identification of myxobacteria in the saline-alkaline soils of Akesu in Xinjiang].

OBJECTIVE: To isolate myxobacteria and investigate their diversity in saline-alkaline soils from Akesu in Xinjiang. METHODS: Conventional culture-dependent methods, e. g. baiting technique, water agar, soil extract agar and mineral agar, were used to isolate myxobacteria from 25 soil samples collected from Akesu areas of Xinjiang. Combining with physicochemical properties (acidity/alkalinity, salt concentration, vegetation and geographical locations) of the soil samples, myxobacterial diversity was studied. RESULTS: In total 58 strains were isolated, and identified as belonging to 6 different genera, i.e. Myxococcus, Cystobacter, Corallococcus, Sorangium, Nannocystis and Polyangium of Myxococcales. The most frequent genus isolated was Myxococcus which may better adapt in harsh environments. Different myxobacterial diversity was detected in different habitat. CONCLUSION: Myxobacteria diversity was low in saline-alkaline soils of Akesu in Xinjiang.

Phaselicystis flava gen. nov., sp. nov., an arachidonic acid-containing soil myxobacterium, and the description of Phaselicystidaceae fam. nov.

A bacterial strain designated SBKo001(T) was isolated from a forest soil sample from Mt Makiling in Laguna, Philippines. It shows the general characteristics associated with myxobacteria, such as swarming of Gram-negative, rod-shaped vegetative cells, fruiting body formation and bacteriolytic activity. The strain is mesophilic, strictly aerobic and chemoheterotrophic and also exhibits resistance to various antibiotics. Major fatty acids are iso-C(15 : 0), C(17 : 1) 2-OH and C(20 : 4) (arachidonic acid). The G+C content of the genomic DNA is 69.2 mol%. A reference strain, NOSO-1 (=DSM 53757), isolated from the Etosha Basin in Namibia, shares nearly the same characteristics with SBKo001(T). The identical 16S rRNA gene sequences of the two strains show 94 % identity to strains of the cellulose-degrading Byssovorax and Sorangium species. Phylogenetic analysis reveals a novel branch diverging from the Polyangiaceae, Sorangiineae, Myxococcales. Their uniqueness in morphological growth stages, unusual fatty acid profile, broad-spectrum antibiotic resistance and branch divergence from the Polyangiaceae imply that strains SBKo001(T) and NOSO-1 not only represent a novel genus and species, proposed here as Phaselicystis flava gen. nov., sp. nov., but also belong to a new family, Phaselicystidaceae fam. nov. The type strain of Phaselicystis flava is SBKo001(T) (=DSM 21295(T) =NCCB 100230(T)).

Improving conjugation efficacy of Sorangium cellulosum by the addition of dual selection antibiotics.

The conjugation protocols in myxobacterium Sorangium cellulosum are often inapplicable due to the strain-specific sensitivity to the presence of Escherichia coli cells or the resistances to many antibiotics. Here we report that the conjugative transfer of the mobilizable plasmid pCVD442 from E. coli DH5alpha (lambda pir) to Sorangium strains could be greatly increased by the presence of low doses of dual selection antibiotics in the mating medium. The improvement was efficient in either E. coli-tolerant or sensitive Sorangium strains. For those phleomycin and hygromycin tolerant Sorangium strains, chloramphenicol-resistance gene was developed as a new selectable marker by driving the resistance gene with the aphII promoter. Using the improved protocol, the epothilone biosynthetic pathway was inactivated by an insertion mutation in the biosynthetic genes of the producing Sorangium strains.

Microbial diversity of soil bacteria in agricultural field contaminated with heavy metals.

In this study we evaluated the bacterial diversity in a soil sample from a site next to a chemical industrial factory previously contaminated with heavy metals. Analysis of 16S rDNA sequences amplified from DNA directly extracted from the soil revealed 17 different bacterial types (genera and/or species). They included Polyangium spp., Sphingomonas spp., Variovorax spp., Hafina spp., Clostridia, Acidobacteria, the enterics and some uncultured strains. Microbes able to tolerate high concentrations of cadmium (500 micromol/L and above) were also isolated from the soil. These isolates included strains of Acinetobacter (strain CD06), Enterobacter sp. (strains CD01, CD03, CD04 and CD08) (similar strains also identified in culture-independent approach) and a strain of Stenotrophomonas sp. The results indicated that the species identified from direct analysis of 16S rDNA of the soil can be quite different from those strains obtained from enrichment cultures and the microbial activities for heavy metal resistance might be more appropriately addressed by the actual isolates.

Cloning and characterization of an rRNA methyltransferase from Sorangium cellulosum.

A locus (kmr) responsible for aminoglycosides-resistance of Sorangium cellulosum was cloned and characterized in Myxococcus xanthus. The gene kmr encodes a putative rRNA methyltransferase. Expression of the complete ORF endowed the Myxococcus transformants with the resistance to aminoglycosidic antibiotics of kanamycin, apramycin, gentamycin, neomycin, and tobramycin at an extraordinary high-level (MIC, higher than 500 microg/ml). However, the gene did not function in Escherichia coli cells. In Sorangium genome, the gene kmr was followed by a putative integrase gene, and was highly homologous in different Sorangium strains. The Sorangium rRNA methyltransferase sequence was in low similarity to the reported 16S rRNA methyltransferases, and their resistance spectrums were also different. The results indicate that the rRNA methyltransferase (Kmr) in Sorangium strains is a new member of the rRNA methyltransferases family.

The biosynthetic genes for disorazoles, potent cytotoxic compounds that disrupt microtubule formation.

Disorazoles are polyketides produced by the myxobacterium Sorangium cellulosum So ce12. Their mode of action is to inhibit tubulin polymerization and destabilize microtubules. Using transposon mutagenesis, two mutant strains were identified that produced no disorazoles. Sequencing the DNA flanking the insertions revealed a polyketide synthase gene cluster that would encode three polypeptides, DszA, DszB, and DszC, with DszC containing both nonribosomal peptide synthetase and polyketide synthase modules. The disorazole polyketide synthase modules lack an acyltransferase domain. Instead, a separate gene, dszD, encodes an AT protein, thus revealing that the disorazole gene cluster falls into the trans-AT Type I family of PKS enzymes.

Characteristics and living patterns of marine myxobacterial isolates.

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.

Enhygromyxa salina gen. nov., sp. nov., a slightly halophilic myxobacterium isolated from the coastal areas of Japan.

Six isolates of novel marine myxobacteria, designated strains SHK-1T, SMK-1-1, SMK-1-3, SMK-10, SKK-2, and SMP-6, were obtained from various coastal samples (mud, sands and algae) collected around Japan. All of the isolates had Gram-negative rod-shaped cells, motile by gliding and grew aerobically. They showed bacteriolytic action, fruiting body formation, and NaCl requirement for growth with an optimum concentration of 1.0-2.0% (w/v). In addition, divalent cationic components of seawater, such as Mg2+ or Ca2+, were also needed for growth. The major respiratory quinone was MK-7. The G+C content of genomic DNA ranged from 65.6 to 67.4 mol% (by HPLC). The isolates shared almost identical 16S rDNA sequences, and clustered with a recently described marine myxobacterium, Plesiocystis pacifica, as their closest relative on a phylogenetic tree (95.9-96.0% similarity). Physiological and chemotaxonomic differences between the new strains and strains of the genus Plesiocystis justify the proposal of a new genus. Therefore, we propose to classify the six isolates into a new taxon of marine myxobacteria with the name, Enhygromyxa salina gen. nov., sp. nov. The type strain is SHK-1(T) (JCM 11769(T) = DSM 15217(T) = AJ 110011(T)).

Nutrient regulation of epothilone biosynthesis in heterologous and native production strains.

Fermentation media with different initial concentrations of ammonium and phosphate salts were used to study the inhibitory effects of those ions on growth and production of epothilone in Sorangium cellulosum and Myxococcus xanthus. The native epothilone producer, S. cellulosum was more sensitive to ammonium and phosphate than the heterologous producer, M. xanthus. An ammonium concentration of 12 mM reduced epothilone titers by 90% in S. cellulosum but by only 40% in M. xanthus. When 5 mM phosphate was added to the medium, production in both strains was 60% lower. Higher phosphate concentrations had little additional effect on M. xanthus titers, but epothilone production with 17 mM extra-cellular phosphate in S. cellulosum was 95% lower than in the control condition. The effect of iron supplementation to the fermentation medium was also investigated. Both strains showed best production with 20 microM iron added to the medium.

Characterization of Fe(III) reduction by chlororespiring Anaeromyxobacter dehalogenans.

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [(14)C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.

[Viability of lyophilized cells of Myxococcus xanthus UKM 10041 and Polyangium cellulosum UKM 10043 in presence of different antioxidants]. / Zhiznesposobnost' liofilizirovannykh kletok Myxococcus xanthus UKM 10041 i Polyangium cellulosum UKM 10043 v prisutstvii razlichnykh antioksidantov.

It has been established that addition of antioxidants: cystamine, ionol, alpha-tocopherol to protective media (saccharose-gelatin agar) does not affect considerably viability of lyophillized cells of cultures Myxococcus xanthus UCM 10041 and Polyangium cellulosum UCM 10043. Experimentally obtained and predicted data on the survival of cells of M. xanthus UCM 10041 and P. cellulosum UCM 10043 are the values of the same order which evidences for the possibility of the use of quick test for prediction of myxobacteria cells survivability under long-term storage.

Neem (Azadirachta indica) extract as an antibacterial agent against fish pathogenic bacteria.

Aquaneem, an emulsified product prepared from the neem (A. indica) kernel was tested against four pathogenic bacteria of fish (i.e. Aeromonas hydrophila, Pseudomonas fluorescens, Escherichia coli and Myxobacteria spp.) to test its efficacy as an antibacterial agent. Growth inhibitory property of the product at 10, 15 and 20 ppm has been noticed and recorded. The percentage reduction of bacterial cell population was noted to be maximum on 9th day at 20 ppm concentration (i.e. 70.14%, 74.15% and 61.75% for A. hydrophila, P. fluorescens and E. coli respectively) with the only exception of myxobacteria which showed maximum reduction percentage (63.90%) on 15th day. Among all the bacteria tested A. hydrophila, P. fluorescens and Myxobacteria spp. exhibited maximum sensitivity to Aquaneem in terms of percentage reduction of bacterial cell population in comparison to E. coli.

[Effect of serotonin (5-hydroxytryptamine) on the growth and differentiation of microorganisms]. / Deistvie serotonina (5-oksitriptamina) na rost i differentsiatsiiu mikroorganizmov.

Serotonin (5-hydroxytryptamine), a neurotransmitter and social behavior factor in higher animals, accelerates culture growth and induces cell aggregation in Escherichia coli and Rhodospirillum rubrum at concentrations of 2 x 10(-7)-2 x 10(-5)M. In the myxobacterium Polyangium sp., 10(-6)-10(-5)M serotonin stimulates cell aggregation and myxospore formation. At concentrations over 20 microM, serotonin induces the opposite effect: it inhibits cell aggregation and microbial culture growth. Serotonin at these concentrations also inhibits the light-dependent membrane potential generation in Rsp. rubrum (the data were obtained by the method of penetrating ions). Therefore, the above effects can be due to the elimination of the transmembrane electrical gradient by serotonin. As for micromolar serotonin concentrations, their effects presumably result from the specific action of serotonin as an intercellular communication agent accelerating and possibly synchronizing the development of the cell population.