Toxin Synthesis Research Articles

A new strategy: Inhibitory effect and mechanism of Cymbopogon martinii essential oil on Aspergillus flavus

Cymbopogon martinii (C. martini) essential oil can inhibit the growth and toxin synthesis of Aspergillus flavus (A. flavus). In this study, C. martinii essential oil effectively inhibited radial growth, spore production, mycelium formation and aflatoxin B1 (AFB1) synthesis of A. flavus. It also significantly changed the morphology and ultrastructure of mycelium. C. martini essential oil significantly changed the activity of antioxidant enzymes and lipid peroxidation level in mycelium, and ROS level increased significantly. According to proteomic analysis, 1604 membrane proteins of A. flavus have been identified. C. martinii essential oil treatment seriously damaged the integrity of the A. flavus cell membrane, and C. martinii essential oil treatment has destroyed 58 cell membrane proteins and released 157 membrane proteins. This study provided a new explanation for the inhibition mechanism of C. martinii essential oil on A. flavus, and also provided technical support for the application of C. martinii essential oil in mildew prevention.

Antifungal mechanisms of γ-aminobutyric acid against the postharvest pathogen Alternaria alternata

γ-Aminobutyric acid (GABA) is a plant resistance elicitor essential in preventing and controlling postharvest disease in fruits and vegetables. However, limited information is available involving the inhibitory effect of exogenous GABA on postharvest pathogens. This study found that GABA inhibited the postharvest pathogenic fungus, Alternaria alternata (A. alternata). Transcriptome analysis revealed that the genes related to sporulation, toxin synthesis, and cell wall degradation enzymes are downregulated in A. alternata after GABA treatment. Moreover, the effect of GABA on A. alternata was verified using electron microscopy and by determining the activity of the cell wall degradation enzyme. These findings provided information about the underlying antifungal mechanism about GABA against A. alternata and suggested that GABA could be a potential alternative for controlling A. alternata in fruit and vegetable preservation.

Genetic association of toxin production in the dinoflagellate Alexandrium minutum.

Dinoflagellates of the genus Alexandrium are responsible for harmful algal blooms and produce paralytic shellfish toxins (PSTs). Their very large and complex genomes make it challenging to identify the genes responsible for toxin synthesis. A family-based genomic association study was developed to determine the inheritance of toxin production in Alexandrium minutum and identify genomic regions linked to this production. We show that the ability to produce toxins is inheritable in a Mendelian way, while the heritability of the toxin profile is more complex. We developed the first dinoflagellate genetic linkage map. Using this map, several major results were obtained: 1. A genomic region related to the ability to produce toxins was identified. 2. This region does not contain any polymorphic sxt genes, known to be involved in toxin production in cyanobacteria. 3. The sxt genes, known to be present in a single cluster in cyanobacteria, are scattered on different linkage groups in A. minutum. 4. The expression of two sxt genes not assigned to any linkage group, sxtI and sxtG, may be regulated by the genomic region related to the ability to produce toxins. Our results provide new insights into the organization of toxicity-related genes in A. minutum, suggesting a dissociated genetic mechanism for the production of the different analogues and the ability to produce toxins. However, most of the newly identified genes remain unannotated. This study therefore proposes new candidate genes to be further explored to understand how dinoflagellates synthesize their toxins.

Cyanobacteria and Their Metabolites in Mono- and Polidominant Shallow Eutrophic Temperate Lakes.

Monodominant (one species dominates) or polidominant (multiple species dominate) cyanobacterial blooms are pronounced in productive freshwater ecosystems and pose a potential threat to the biota due to the synthesis of toxins. Seasonal changes in cyanobacteria species and cyanometabolites composition were studied in two shallow temperate eutrophic lakes. Data on cyanobacteria biomass and diversity of dominant species in the lakes were combined with chemical and molecular analyses of fifteen potentially toxin-producing cyanobacteria species (248 isolates from the lakes). Anatoxin-a, saxitoxin, microcystins and other non-ribosomal peptides formed the diverse profiles in monodominant (Planktothrix agardhii) and polidominant (Aphanizomenon gracile, Limnothrix spp. and Planktolyngbya limnetica) lakes. However, the harmfulness of the blooms depended on the ability of the dominant species to synthesize cyanometabolites. It was confirmed that P. agardhii produced a greater amount and diverse range of MCs and other NRPs. In the polidominant lake, isolates of the co-dominant A. gracile, L. planctonica and P. limnetica synthesized no or only small amounts of cyanometabolites. In general, the profile of cyanometabolites was greater in cyanobacteria isolates than in environmental samples, indicating a high potential for toxic cyanobacteria bloom.

Study on the interaction mechanism between Crocus sativus and Fusarium oxysporum based on dual RNA-seq.

The saffron phenylpropane synthesis pathway and Fusarium oxysporum cell wall-degrading enzymes play key roles in their early interactions. Saffron (Crocus sativus) is a highly important crop with diverse medicinal properties. F. oxysporum is a widely-distributed soil-borne fungus, causing the serious saffron rot disease. Currently, there is no effective management strategy to control this disease because of no resistant cultivars and limited information about the resistance and pathogenic mechanisms. In this study, we first characterized the infection process and physiological responses of saffron infected by F. oxysporum. The molecular mechanism of these infection interactions was revealed by dual RNA-seq analysis. On the 3rd day of infection, the hyphae completely entered, colonized and spread in the corm cells; while on the 6th day of infection, hyphae had appeared in the xylem cells, blocking these vessels. Transcriptome results indicate that within the host, phenylpropanoid metabolism, plant hormone signal transduction and plant pathogen interaction pathways were activated during infection. These pathways were conducive to the enhancement of cell wall, the occurrence of hypersensitivity, and the accumulation of various antibacterial proteins and phytoantitoxins. Meanwhile, in the fungus, many up-regulated genes were related to F. oxysporum cell wall degrading enzymes, toxin synthesis and pathogenicity gene, showing its strong pathogenicity. This study provides new ideas for the control of saffron corm rot, and also provides a theoretical basis for mining the key functional genes.

Complete Genome Sequence of Nocardiopsis exhalans Strain JCM 11759T, Isolated from Indoor Air of a Water-Damaged Private House in Finland.

The genus Nocardiopsis contains pharmaceutically and biotechnologically important species that produce a wide variety of secondary metabolites with a wide range of biological activities. Here, we report the complete genome sequence of Nocardiopsis exhalans JCM 11759T for a better understanding of its metabolic characteristics and toxin synthesis pathway.

Cysteine Inhibits the Growth of Fusarium oxysporum and Promotes T-2 Toxin Synthesis through the Gtr/Tap42 Pathway.

Fusarium oxysporum is ubiquitous and can easily contaminate food during processing and storage, potentially producing T-2 toxin, which can pose a severe health risk to public health. Previous research on the presence of T-2 has focused on starch-rich foods, while protein- and amino acid-rich foods have received relatively little attention. In this study, the effects of amino acids on the growth of F. oxysporum and its T-2 production were investigated by gene deletion and complementation experiments. The results showed that amino acids, including aspartic acid, methionine, isoleucine, serine, phenylalanine, and cysteine, significantly inhibited the growth of F. oxysporum, while promoting T-2 synthesis, with cysteine having the most pronounced effect. The target of rapamycin complex 1 (TORC1) is a key pathway in response to a variety of amino acids, including cysteine. gtr2 and tap42 were found to be negative regulators of T-2 synthesis. The study highlights the elevated risk of T-2 production by F. oxysporum in cysteine-rich foods and the need to take appropriate measures to prevent and control the potential harm that such foods may present to public health. IMPORTANCE F. oxysporum and its T-2 contamination of food not only leads to food wastage but also poses a major food safety challenge to humans. The growth and T-2 production characteristics of F. oxysporum in high-protein substrates are considerably different from those in grains. Here, we show that the abundant free amino acids in a protein-rich food matrix are a key regulatory factor for the growth of, and toxin production by, F. oxysporum. Cysteine has the most pronounced effect on inhibiting mycelial growth and promoting T-2 synthesis through the TORC1 pathway. This implies that consumers tend to overlook T-2 contamination due to the poor growth of F. oxysporum in food rich in protein and amino acids, especially cysteine. Therefore, particular attention should be paid to the protection of those products.

A characteristic of the species composition of pathogenic fungi of the genus Fusarium in corn biocenoses of the Voronezh region.

Corn is one of the main crops of modern world agriculture. It ranks f irst in terms of gross grain harvests and second in terms of acreage, ceding only to the main grain crop of the globe, wheat. The problem of increasing the production of grain and green mass of corn remains one of the urgent tasks of agricultural production. High potential yields very often remain untapped due to diseases, direct losses from which are estimated at 20–50 %. The purpose of this work was to study the species composition of micromycetes on corn collected in different phases of vegetation in May-July 2020 in the Voronezh region, to identify phytopathogenic genus Fusarium fungi, to study pathogenic and phytotoxic strains of the fungi to replenish the collection of the All-Russian Scientific Research Institute of a Phytopathology. Preservation of infectious material of fungi from the genus Fusarium is of no small importance for phytopathological, immunological, breeding, genetic and toxicological studies. As a result of the mycological studies carried out, a lot of fungi isolates from the genera Fusarium, Aspergillus, Cladosporium, Curvularia, Penicillium, Rhizopus, Periconia, Pythium, Trichothecium, etc., isolated from the affected roots, stems and ears of corn in the Voronezh region in 2020 were identif ied. Fungi isolates from seven taxonomic groups: Fusarium fujikuroi Nirenberg (F. moniliforme, F. verticillioides), Fusarium oxysporum Schltdl., Fusarium culmorum (Wm.G. Sm.) Sacc., Fusarium graminearum Schwabe, Fusarium heterosporum Nees & T. Nees (F. lolii ), Fusarium roseum Link (F. sambucinum), Fusarium sporotrichioides Sherb. were tested for pathogenicity and phytotoxicity on seedlings of plant-testers. It has been shown that pathogenic and phytotoxic activity in fungi varies signif icantly between Fusarium species and within the same species. The greatest danger to corn is represented by the species F. sporotrichioides, F. graminearum, F. culmorum, F. fujikuroi, F. oxysporum, F. heterosporum, which have a high intensity of phytotoxic activity associated with the fact that they contribute to the synthesis and accumulation of dangerous toxins in plant tissues. As a result of the conducted studies, 55 strains of fungi from the genus Fusarium belonging to seven species were selected. The isolates, stable in morphological and cultural characteristics and studied for pathogenicity and toxicity, were placed for long-term storage in the Russian State Collection of Plant Pathogenic Microorganisms and Cultivars for Identif ication of Phytopathogenic Microbial Strains at the All-Russian Scientif ic Research Institute of a Phytopathology.

Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus.

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis.

Immunosuppressive activity of Campylobacter jejuni isolates in relation to the cellular link of the body’s immunoprotection

Global environmental changes have caused transformations in the biology of microorganisms, especially among campylobacter, which are currently associated with food toxic infections. The means of influence of these bacteria on susceptible organisms, namely toxins, have not been finally clarified. The purpose of this study was to investigate the genetic conditionality of toxin formation in isolates of Campylobacter jejuni and determination of the degree of inhibition of the body’s protective reactions by toxic fractions of Campylobacter protein compounds. The methodology of this study was based on the polymerase chain reaction using primers to indicate the nucleotide sequences of the Campylobacter jejuni genome that encode the synthesis of toxins. Samples from 4 Campylobacter isolates were examined for the content of protein fractions according to the Lowry assay. The analysis of the electropherogram of the results of DNA amplification in a comparative aspect with the data of standard samples allowed establishing the presence of genome elements that indicate the potential ability to produce toxins in Campylobacter jejuni isolates sampled from the material under study. Toxic fractions separated from the supernatant of Campylobacter jejuni broth culture are represented by protein-carbohydrate substances. The obtained peak toxigenic fractions of the dialysate of the bacterial culture sediment contained protein within 9.5-17 μg/ml. In the dialysate of the broth culture supernatant, where 5 groups of toxigenic fractions were distinguished, their protein content ranged within 10-85 μg/ml. By reproducing the opsono-phagocytic reaction involving toxigenic fractions of Campylobacter jejuni, a sufficiently pronounced immunosuppressive effect of these complexes on the body of warm-blooded animals was established with an opsonic index of 2.6 ± 0.03. The obtained results allow clarifying the connection between toxin formation in Campylobacter jejuni and their immunosuppressive effect on the body of warm-blooded animals and humans, which in the future will positively affect the improvement of measures for the prevention and treatment of animals with this pathology

GTPase Rac Regulates Conidiation, AFB1 Production and Stress Response in Pathogenic Fungus Aspergillus flavus.

As a member of the Rho family, Rac plays important roles in many species, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism, angiogenesis, and immunosuppression. In this study, by constructing Rac-deleted mutants in Aspergillus flavus, it was found that the deletion of Rac gene led to the decline of growth and development, conidia production, AFB1 toxin synthesis, and seed infection ability of A. flavus. The deletion of Rac gene also caused the disappearance of A. flavus sclerotium, indicating that Rac is required for sclerotium formation in A. flavus. The sensitivity of Rac-deficient strains responding to cell wall stress and osmotic pressure stress increased when compared to A. flavus WT. The Western blot result showed that mitogen-activated serine/threonine-protein kinase Slt2 and mitogen-activated protein kinase Hog1 proteins were no longer phosphorylated in Rac-deficient strains of A. flavus, showing that Rac may be used as a molecular switch to control the Slt2-MAPK cascade pathway and regulate the osmotic Hog-MAPK cascade pathway in A. flavus in response to external stress. Altogether, these results indicated that Rac was involved in regulating the growth and development, conidia formation and AFB1 synthesis, and response to cell wall stress and osmotic pressure stress in A. flavus.

Consistency between the ichthyotoxicity and allelopathy among strains and ribotypes of Margalefidinium polykrikoides suggests that its toxins are allelochemicals

Harmful algal blooms (HABs) of the ichthytoxic dinoflagellate Margalefidinium polykrikoides have caused mass mortality of marine life around the world. While its toxic effects can impact fish, bivalves, coral, zooplankton, and even other phytoplankton, the toxin(s) and allelochemical(s) eliciting these impacts have yet to be definitely identified, leaving open the question as to whether its toxicity and allelopathic effects are caused by the same chemical agents. In this study, we investigated the ability of 10 strains of M. polykrikoides with different geographic origins and ribotypes to cause mortality in two strains of the dinoflagellate, Akashiwo sanguinea (allelopathy), and the sheepshead minnow, Cyprinodon variegatus (toxicity). Results showed that the potency of allelopathy against both strains of A. sanguinea and toxicity to the fish were significantly correlated across strains of M. polykrikoides (p < 0.001 for all). These results strongly support the notion that the major allelochemicals and toxins of M. polykrikoides are identical chemicals, an ecological strategy that may be more energetically efficient than the separate synthesis of toxins and allelochemicals as has been reported in other HABs. Our results also highlight the vital significance of the definitive identification of allelochemicals and toxins of M. polykrikoides and of the quantitative characterization of these compounds in the field where HABs of M. polykrikoides occur during blooms.

Role of serine/threonine protein phosphatase PrpN in the life cycle of Bacillus anthracis.

Reversible protein phosphorylation at serine/threonine residues is one of the most common protein modifications, widely observed in all kingdoms of life. The catalysts controlling this modification are specific serine/threonine kinases and phosphatases that modulate various cellular pathways ranging from growth to cellular death. Genome sequencing and various omics studies have led to the identification of numerous serine/threonine kinases and cognate phosphatases, yet the physiological relevance of many of these proteins remain enigmatic. In Bacillus anthracis, only one ser/thr phosphatase, PrpC, has been functionally characterized; it was reported to be non-essential for bacterial growth and survival. In the present study, we characterized another ser/thr phosphatase (PrpN) of B. anthracis by various structural and functional approaches. To examine its physiological relevance in B. anthracis, a null mutant strain of prpN was generated and shown to have defects in sporulation and reduced synthesis of toxins (PA and LF) and the toxin activator protein AtxA. We also identified CodY, a global transcriptional regulator, as a target of PrpN and ser/thr kinase PrkC. CodY phosphorylation strongly controlled its binding to the promoter region of atxA, as shown using phosphomimetic and phosphoablative mutants. In nutshell, the present study reports phosphorylation-mediated regulation of CodY activity in the context of anthrax toxin synthesis in B. anthracis by a previously uncharacterized ser/thr protein phosphatase-PrpN.

Antifungal and antimycotoxic activities of 3 essential oils against 3 mycotoxinogenic fungi.

Fungal toxins can have various adverse health effects, including carcinogenic, teratogenic or hepatotoxic impacts. In addition, fungal alteration has also a negative impact on agricultural plant production. The use of chemical fungicides to control mycotoxin contamination is increasingly controversial and regulated. More environmentally friendly methods are therefore being explored. Essential oils, as compounds extracted from plants, are liquids whose specific aromatic compounds give each essential oil its own unique characteristics. Due to their rich chemical composition, essential oils (EOs) have many interesting properties, including antifungal activities. The objective of the present study was to analyze volatile chemical composition of EOs (Cymbopogon schoenanthus, Cymbopogon nardus and Eucalyptus camaldulensis) by GC/MS and to investigate their effects on the growth, sporulation and mycotoxin production of Aspergillus flavus, Aspergillus carbonarius and Fusarium verticillioides (aflatoxin B1, ochratoxin A and fumonisin B1, respectively). In addition, EOs influence on aflatoxin B1 (AFB1) and fumonisin B1 (FB1) biosynthesis pathways was explored using real-time qRT-PCR. The results obtained in vitro, by direct contact with the EOs and by diffusion of their volatile compounds, showed that the essential oils had inhibitory effects on the growth and the production of mycotoxins of the 3 fungal strains and modified the expression of some toxin synthesis genes. We conclude that the recorded effects were dependent on the combined effects of the EOs type, the fungal strains and the doses studied.

Polymorphisms of dtxR Gene of Corynebacterium diphtheriae Isolated from Diphtheria Outbreak in Indonesia

Background: In Corynebacterium diphtheriae, the dtxR gene plays a role in regulating diphtheria toxin synthesis. The dtxR gene is often used as a marker for identifying C. diphtheriae by the polymerase chain reaction (PCR) method because it is present in all strains of this bacterium. Mutations in the dtxR gene can cause the over-synthesis of diphtheria toxin and reduce PCR assays' sensitivity. Objectives: This study aimed to describe the polymorphisms in the dtxR gene of C. diphtheriae isolated from a diphtheria outbreak in Indonesia. Methods: Forty-eight isolates of C. diphtheriae were obtained from clinical samples (throat/nasopharyngeal swabs) of diphtheria cases and close contacts. The isolates were revived on a blood agar plate (BAP), bacterial colonies were harvested, and deoxyribonucleic acid (DNA) was extracted. The DNA sequencing was carried out using a Whole-genome Sequencing (WGS) approach. The data were converted and analyzed with U-gene software. The dtxR gene analysis was performed with C. diphtheriae PW8 as references. Results: There were 59-point mutation locations in 48 isolates examined. None of these single nucleotide polymorphisms (SNPs) coded for amino acid changes. Based on the mutation pattern, seven clades/groups of the dtxR gene of 48 C. diphtheriae isolates were examined. Conclusions: At least seven types of DNA sequences and more than 50 SNPs of the dtxR gene were identified in 48 C. diphtheriae isolates from a diphtheria outbreak in Indonesia. Although all of them are silent mutations, they must be considered in the design of PCR examination in diphtheria laboratories.

Regulatory Networks Controlling Neurotoxin Synthesis in Clostridium botulinum and Clostridium tetani.

Clostridium botulinum and Clostridium tetani are Gram-positive, spore-forming, and anaerobic bacteria that produce the most potent neurotoxins, botulinum toxin (BoNT) and tetanus toxin (TeNT), responsible for flaccid and spastic paralysis, respectively. The main habitat of these toxigenic bacteria is the environment (soil, sediments, cadavers, decayed plants, intestinal content of healthy carrier animals). C. botulinum can grow and produce BoNT in food, leading to food-borne botulism, and in some circumstances, C. botulinum can colonize the intestinal tract and induce infant botulism or adult intestinal toxemia botulism. More rarely, C. botulinum colonizes wounds, whereas tetanus is always a result of wound contamination by C. tetani. The synthesis of neurotoxins is strictly regulated by complex regulatory networks. The highest levels of neurotoxins are produced at the end of the exponential growth and in the early stationary growth phase. Both microorganisms, except C. botulinum E, share an alternative sigma factor, BotR and TetR, respectively, the genes of which are located upstream of the neurotoxin genes. These factors are essential for neurotoxin gene expression. C. botulinum and C. tetani share also a two-component system (TCS) that negatively regulates neurotoxin synthesis, but each microorganism uses additional distinct sets of TCSs. Neurotoxin synthesis is interlocked with the general metabolism, and CodY, a master regulator of metabolism in Gram-positive bacteria, is involved in both clostridial species. The environmental and nutritional factors controlling neurotoxin synthesis are still poorly understood. The transition from amino acid to peptide metabolism seems to be an important factor. Moreover, a small non-coding RNA in C. tetani, and quorum-sensing systems in C. botulinum and possibly in C. tetani, also control toxin synthesis. However, both species use also distinct regulatory pathways; this reflects the adaptation of C. botulinum and C. tetani to different ecological niches.

Diguanylate Cyclase (DGC) Implicated in the Synthesis of Multiple Bacteriocins via the Flagellar-Type III Secretion System Produced by Pectobacterium carotovorum subsp. carotovorum.

The plant pathogen Pectobacterium carotovorum subsp. carotovorum (previously Erwinia carotovora subsp. carotovora) causes soft rot and stem rot diseases in a variety of crops, including Chinese cabbage, potato, and tomato. The flagellar-type III secretion systems were used by Pcc’s virulence mechanism to export proteins or bacteriocins to the outside of the cell. DGC, a virulence factor that cyclizes c-di-GMP, a common secondary signal in physiological processes and toxin control systems of many bacteria, was discovered in Pcc’s genomic DNA. The dgc gene in Pcc was blocked using the method of homologous recombination in our study. In the in vivo setting, the results demonstrated that the dgc knockout strain does not release low molecular weight bacteriocins. The bacteriocin gene (carocin S2, carocin S3, carocin S4) and the flagellar-type III secretion system genes were also unable to be transcribed by the dgc knockout strain in the transcription experiment. We also observed that the amount of bacteriocin expressed changed when the amount of L-glutamine in the environment exceeded a particular level. These data suggested that L-glutamine influenced physiological processes in Pcc strains in some way. We hypothesized a relationship between dgc and the genes involved in Pcc LMWB external export via the flagellar-type secretion system based on these findings. In this study, the current findings led us to propose a mechanism in which DGC’s cyclic di-GMP might bind to receptor proteins and positively regulate bacteriocin transcription as well as the synthesis, mobility, and transport of toxins.

Mycolicibacterium lacusdiani sp. nov., an Attached Bacterium of Microcystis aeruginosa

In eutrophic water, attached bacteria of Microcystis play an important role in the formation, development, and degradation of Microcystis blooms. A novel actinobacterium, designated as JXJ CY 35T, was isolated from the culture mass of Microcystis aeruginosa FACHB-905 (Maf) collected from Lake Dianchi, Yunnan Province, China. Strain JXJ CY 35T was gram-positive, acid-fast staining, aerobic, with short rod-shaped cells, positive for catalase, and negative for oxidase. The isolate was able to grow at 10.0–36.0°C, pH 4.0–10.0, and tolerate up to 5.0% (w/v) NaCl, with optimal growth at 28°C, pH 7.0–8.0, and 0% (w/v) NaCl. Cell-wall peptidoglycan contains aspartic acid, glutamic acid, glycine, and alanine, with mannose, ribose, galactose, and arabinose as whole-cell sugars. Polar lipids consist of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), glycolipid (GL1-3), phosphoglycolipid (PGL), phosphatidylinositol (PI), and unidentified lipid (L1). The predominant menaquinone was MK-9. Major fatty acids (>10%) were C17:1ω7c (37.0%) and C18:1ω9c (18.9%). The complete genome sequence of strain JXJ CY 35T was 6,138,096 bp in size with a DNA G + C content of 68.3%. Based on 16S rRNA gene sequences, it has 98.2% similarity to Mycolicibacterium arabiense JCM 18538T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain JXJ CY 35T and the closest five type strains M. arabiense JCM 18538T, M. goodii ATCC 700504T, M. mageritense DSM 44476T, M. austroafricanum DSM 44191T, and Mycobacterium neglectum CECT 8778T were 52.1, 20.3, 20.3, 20.6, and 19.8%, and 92.7, 75.5, 75.6, 76.0, and 75.2%, respectively. On the basis of the above taxonomic data and differences in physiological characteristics from the closely related type strain, strain JXJ CY 35T was determined to represent a novel species of genus Mycolicibacterium, for which the name Mycolicibacterium lacusdiani sp. nov., is proposed. The type strain is JXJ CY 35T (=KCTC 49379T = CGMCC 1.17501T). Different inoculation dosages of the type strain JXJ CY 35T could exhibit different effects on the growth of Maf and its toxin synthesis and release. Strain JXJ CY 35T could promote the growth of Maf by providing it with available phosphorus, nitrogen, probably vitamins, and plant growth hormones.

Luteolin Inhibits the Biofilm Formation and Cytotoxicity of Methicillin-Resistant Staphylococcus aureus via Decreasing Bacterial Toxin Synthesis.

Owing to the fact that luteolin has antibacterial activity against Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), its specific mechanism in MRSA is worthy of investigation, which is the focus of this study. Initially, the collected S. aureus strains were treated with luteolin. Then, the minimum inhibitory concentration (MIC) of luteolin against the S. aureus strains was measured by the broth microdilution. The growth curves, biofilm formation, and cytotoxicity of treated S. aureus were detected using a microplate reader. The live and dead bacteria were evaluated using confocal laser scanning microscopy, the bacterial morphology was observed using scanning electron microscopy, and the S. aureus colony-forming unit (CFU) numbers were assessed. The levels of alpha hemolysin (α-hemolysin), delta hemolysin (δ-hemolysin), and hlaA were detected via western blot and RT-PCR. The mortality of mouse model with S. aureus systemic infection was analyzed, and the levels of IL-6, IL-8, IL-10, and TNF-α were quantitated using ELISA. Concretely, the MIC of luteolin against MRSA N315 was 64 μg/mL. Luteolin at 16 μg/mL did not affect the growth of MRSA N315, but inhibited the biofilm formation and CFU, and promoted the morphological changes and death of MRSA N315. Luteolin decreased the cytotoxicity and the levels of α-hemolysin, δ-hemolysin, and hlaA in MRSA N315, elevated MRSA-reduced mice survival rate, and differentially modulated the inflammatory cytokine levels in MRSA-infected mice. Collectively, luteolin inhibits biofilm formation and cytotoxicity of MRSA via blocking the bacterial toxin synthesis.

2-Hydroxy-4-methoxybenzyl as a Thiol-Protecting Group for Directed-Disulfide Bond Formation.

The chemical synthesis of disulfide-rich peptides such as toxins can be accomplished by using numerous orthogonal cysteine-protecting groups. Herein we report the use of the Hmboff/on protecting group for directed disulfide bond formation. Its combination with classical Trt, Acm, and Mob groups was studied for the synthesis of NMB-1 and phlotoxin-1 toxins highlighting new orthogonal strategies for directed disulfide bond formation.