pyrimidinones and toxoflavin

A method using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed for the determination of toxoflavin and fervenulin in 6 types of food.The limits of detection (LODs, S/N ≥ 3) of toxoflavin and fervenulin reached 12 µg/kg and 24 µg/kg, respectively.The recoveries ranged from 70.1 % to 108.7 %.Intra-day RSDs (n = 5) and inter-day RSDs (n = 3) ranged from 0.9 % to 9.5 %.The method was successfully applied to analyse 36 samples, and one Tremella fuciformis Berk. sample was found with 7.5 mg/kg toxoflavin and 3.2 mg/kg fervenulin. Toxoflavin and fervenulin were acidic compounds and easily degraded in 0.1 % ammonia solution (v/v),degradation products were identified by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS).

Topics: Bacterial Toxins; Chromatography, High Pressure Liquid; Pyrimidinones; Tandem Mass Spectrometry; Triazines

Toxoflavin contamination was investigated in broken rice produced as a by-product of domestic rice processing complexes (RPCs) in 2011 in South Korea. Of the 68 RPCs investigated, toxoflavin contamination was confirmed in 12 from three provinces: Gangwon, Gyeonggi, and Gyeongsang. Isolation of toxoflavin-producing bacteria independent of toxoflavin contamination was also performed in this study. We obtained 25 toxoflavin-producing bacterial isolates from rice samples; these samples were collected from the same 12 RPCs mentioned above. All 25 toxoflavin-producing bacteria were identified as Burkholderia glumae by 16S rRNA gene sequencing. Toxoflavin-producing ability differed slightly among the 25 isolates, but they all inhibited rice seed germination and induced seed rot. This is the first report of toxoflavin contamination and the toxin-producing bacterium B. glumae in broken rice produced during the rice milling process. Because toxoflavin has stable physical properties even above a boiling temperature of 100°C, it can pose a problem even if rice is cooked or processed. These results will serve as baseline data aiding comprehensive management of toxoflavin contamination during the post-harvest storage and processing of rice.

Topics: Oryza; Pyrimidinones; Quorum Sensing; RNA, Ribosomal, 16S

Bacterial azapteridine-containing phytotoxin toxoflavin is a causal agent of rice grain rot. Here, we heterologously reconstitute

Topics: Escherichia coli; Oxidoreductases; Pyrimidinones; Triazines

Legionella pneumophila is a natural inhabitant of water systems. From there, it can be transmitted to humans by aerosolization resulting in severe pneumonia. Most large outbreaks are caused by cooling towers colonized with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. In our preceding study, the genus Pseudomonas correlated negatively with the presence of L. pneumophila in cooling towers, but it was not clear which species was responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. We found that cooling towers that are free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a gene cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extracts from P. alcaliphila culture found to contain toxoflavin by liquid chromatography coupled with mass spectrometry. In addition, toxoflavin inhibits the growth of Vermameoba vermiformis, a host cell of L. pneumophila. Our study indicates that P. alcaliphila may be important to restrict growth of L. pneumophila in water systems through the production of toxoflavin. A sufficiently high concentration of toxoflavin is likely not achieved in the bulk water but might have a local inhibitory effect such as near or in biofilms.

Topics: Humans; Legionella; Legionella pneumophila; Phylogeny; Pseudomonas; Pyrimidinones; RNA, Ribosomal, 16S; Triazines; Water; Water Microbiology

Bacterial opportunistic pathogens make diverse secondary metabolites both in the natural environment and when causing infections, yet how these molecules mediate microbial interactions and their consequences for antibiotic treatment are still poorly understood. Here, we explore the role of three redox-active secondary metabolites, pyocyanin, phenazine-1-carboxylic acid, and toxoflavin, as interspecies modulators of antibiotic resilience. We find that these molecules dramatically change susceptibility levels of diverse bacteria to clinical antibiotics. Pyocyanin and phenazine-1-carboxylic acid are made by Pseudomonas aeruginosa, while toxoflavin is made by Burkholderia gladioli, organisms that infect cystic fibrosis and other immunocompromised patients. All molecules alter the susceptibility profile of pathogenic species within the "Burkholderia cepacia complex" to different antibiotics, either antagonizing or potentiating their effects, depending on the drug's class. Defense responses regulated by the redox-sensitive transcription factor SoxR potentiate the antagonistic effects these metabolites have against fluoroquinolones, and the presence of genes encoding SoxR and the efflux systems it regulates can be used to predict how these metabolites will affect antibiotic susceptibility of different bacteria. Finally, we demonstrate that inclusion of secondary metabolites in standard protocols used to assess antibiotic resistance can dramatically alter the results, motivating the development of new tests for more accurate clinical assessment.

Topics: Anti-Bacterial Agents; Burkholderia cepacia complex; Humans; Phenazines; Pseudomonas aeruginosa; Pyocyanine; Pyrimidinones; Triazines

Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterized carotenoid biosynthetic genes of the plant-pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcript and crtZ is independently transcribed in the opposite direction. Using splicing by overlap extension with polymerase chain reaction (SOE by PCR) based on asymmetric amplification, we reassembled crtE-B, crtE-B-I, and crtE-B-I-Y. High-performance liquid chromatography confirmed that Escherichia coli expressing the reassembled crtE-B, crtE-B-I, and crtE-B-I-Y operons produced phytoene, lycopene, and β-carotene, respectively. We found that the carotenoids conferred tolerance to UV radiation and toxoflavin. Pantoea ananatis shares rice environments with the toxoflavin producer Burkholderia glumae and is considered to be the first reported example of producing and using carotenoids to withstand toxoflavin. We confirmed that carotenoid production by P. ananatis depends on RpoS, which is positively regulated by Hfq/ArcZ and negatively regulated by ClpP, similar to an important regulatory network of E. coli (Hfq

Topics: Bacterial Proteins; Carotenoids; Endopeptidase Clp; Escherichia coli; Escherichia coli Proteins; Host Factor 1 Protein; Multigene Family; Pantoea; Plasmids; Pyrimidinones; Quorum Sensing; Sigma Factor; Triazines; Ultraviolet Rays

The toxoflavin (Txn), broad host range phytotoxin produced by a variety of bacteria, including Burkholderia glumae, is a key pathogenicity factor of B. glumae in rice and field crops. Two bacteria exhibiting Txn-degrading activity were isolated from healthy rice seeds and identified as Sphingomonas adhaesiva and Agrobacterium sp. respectively. The genes stdR and stdA, encoding proteins responsible for Txn degradation of both bacterial isolates, were identical, indicating that horizontal gene transfer occurred between microbial communities in the same ecosystem. We identified a novel Txn-quenching regulation of bacteria, demonstrating that the LysR-type transcriptional regulator (LTTR) StdR induces the expression of the stdA, which encodes a Txn-degrading enzyme, in the presence of Txn as a coinducer. Here we show that the bacterial StdR

Topics: Burkholderia; Ecosystem; Gene Expression Regulation, Bacterial; Oryza; Pyrimidinones; Quorum Sensing; Sphingomonas; Triazines

Rice is an important source of food for more than half of the world's population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the

Topics: Anti-Bacterial Agents; Bacterial Proteins; Burkholderia; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Onions; Oryza; Plant Diseases; Proton-Motive Force; Pyrimidinones; Sodium Bicarbonate; Triazines; Virulence; Virulence Factors

The human histone demethylases of the KDM4 family have been related to diseases such as prostate and breast cancer. Majority of currently known inhibitors suffer from the low permeability and low selectivity between the enzyme isoforms. In this study, toxoflavin motif was used to design and synthesize new KDM4C inhibitors with improved biological activity and in vitro ADME properties. Inhibitors displayed good passive cellular permeability and metabolic stability. However, diminishing of redox liability and consequently non-specific influence on cell viability still remains a challenge.

Topics: A549 Cells; Animals; Dogs; Dose-Response Relationship, Drug; Drug Design; Enzyme Inhibitors; Humans; Jumonji Domain-Containing Histone Demethylases; Madin Darby Canine Kidney Cells; Mice; Microsomes, Liver; Molecular Structure; Pyrimidinones; Quantum Theory; Structure-Activity Relationship; Triazines

Burkholderia glumae, the primary causative agent of bacterial panicle blight in rice, has been reported as an opportunistic pathogen in patients with chronic infections. This study aimed to re-sequence the clinical isolate B. glumae strain AU6208 and comparatively analyze its genome using B. glumae strain BGR1 from rice plant as the reference. Re-sequencing results revealed that the genome of strain AU6208 comprised 96 contigs corresponding to a 6.1 Mbp genome of the strain AU6208, with 5322 coding sequences and 68.2 % GC content; this is much larger compared to the genome previously sequenced by us and described by Seo et al (2015), which was reported to be 4.1 Mbp comprising >1200 contigs, 4361 coding sequences, and 67.31 % GC content. Moreover, this updated genome shares >80 % identity to the 7.2 Mbp genome of BGR1, which encodes 6491 coding sequences and has 68.3 % GC content. Further computational analysis revealed that the strain AU6208 encodes several bacteriocin biosynthesis genes, antibiotic, as well as virulent genes such as toxoflavin genes, which included 425 specialty genes and 12 toxoflavin genes. Upon further characterization, 12 toxoflavins (ToxA, B, C, D, E, F, G, H, I, J, TofI, and TofR) were found in AU6208 with 70-100 % sequence, family, and domain similarity with that of BGR1. Upon comparison with BGR1, the structural characterizations of selected toxoflavin genes (ToxB, ToxC, ToxG, H, and TofI) revealed variations in 2D and 3D structures such as differences in α-helix, β-sheets, loops, physiological properties of proteins, RMSD values, etc. These variations may play significant role in different mode of action in different hosts thereby indicating that in addition to their respective hosts, toxoflavins could also contribute to exploit other hosts across the kingdom. In addition to understanding the epidemiology of strain AU6208, this updated genomics data will also unfold the pathogenicity of bacteria in diversity of various hosts and anti-virulence.

Topics: Bacterial Proteins; Bacterial Toxins; Burkholderia; Genome, Bacterial; Pyrimidinones; Triazines

The phytopathogenic Burkholderia species B. glumae and B. plantarii are the causal agents of bacterial wilt, grain rot, and seedling blight, which threaten the rice industry globally. Toxoflavin and tropolone are produced by these phytopathogens and are considered the most hostile biohazards with a broad spectrum of target organisms. However, despite their nonspecific toxicity, the effects of toxoflavin and tropolone on bacteria remain unknown. RNA-seq based transcriptome analysis was employed to determine the genome-wide expression patterns under phytotoxin treatment. Expression of 2327 and 830 genes was differentially changed by toxoflavin and tropolone, respectively. Enriched biological pathways reflected the down-regulation of oxidative phosphorylation and ribosome function, beginning with the inhibition of membrane biosynthesis and nitrogen metabolism under oxidative stress or iron starvation. Conversely, several systems such as bacterial chemotaxis, flagellar assembly, biofilm formation, and sulfur/taurine transporters were highly expressed as countermeasures against the phytotoxins. In addition, our findings revealed that three hub genes commonly induced by both phytotoxins function as the siderophore enterobactin, an iron-chelator. Our study provides new insights into the effects of phytotoxins on bacteria for better understanding of the interactions between phytopathogens and other microorganisms. These data will also be applied as a valuable source in subsequent applications against phytotoxins, the major virulence factor.

Topics: Anti-Bacterial Agents; Burkholderia; Escherichia coli; Escherichia coli Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Oryza; Plant Diseases; Pyrimidinones; Transcriptome; Triazines; Tropolone

Toxoflavin (1), fervenulin (2), and reumycin (3), known to be produced by plant pathogen Burkholderia glumae BGR1, are structurally related 7-azapteridine antibiotics. Previous biosynthetic studies revealed that N-methyltransferase ToxA from B. glumae BGR1 catalyzed the sequential methylation at N6 and N1 in pyrimido[5,4-e]-as-triazine-5,7(6H,8H)-dione (4) to generate 1. However, the N8 methylation of 4 in the biosynthesis of fervenulin remains unclear. To explore the N-methyltransferases required for the biosynthesis of 1 and 2, we identified and characterized the fervenulin and toxoflavin biosynthetic gene clusters in S. hiroshimensis ATCC53615. On the basis of the structures of intermediates accumulated from the four N-methyltransferase gene inactivation mutants and systematic enzymatic methylation reactions, the tailoring steps for the methylation order in the biosynthesis of 1 and 2 were proposed. The N-methylation order and routes for the biosynthesis of fervenulin and toxoflavin in S. hiroshimensis are more complex and represent an obvious departure from those in B. glumae BGR1.

Topics: Biocatalysis; Dose-Response Relationship, Drug; Methyltransferases; Molecular Structure; Pyrimidinones; Streptomyces; Structure-Activity Relationship; Triazines

Toxoflavin, a 7-azapteridine phytotoxin produced by the bacterial pathogens such as Burkholderia glumae and Burkholderia gladioli, has been known as one of the key virulence factors in crop diseases. Because the toxoflavin had an antibacterial activity, a metagenomic E. coli clone capable of growing well in the presence of toxoflavin (30 μg/ml) was isolated and the first metagenome-derived toxoflavin-degrading enzyme, TxeA of 140 amino acid residues, was identified from the positive E. coli clone. The conserved amino acids for metal-binding and extradiol dioxygenase activity, Glu-12, His-8 and Glu-130, were revealed by the sequence analysis of TxeA. The optimum conditions for toxoflavin degradation were evaluated with the TxeA purified in E. coli. Toxoflavin was totally degraded at an initial toxoflavin concentration of 100 μg/ml and at pH 5.0 in the presence of Mn2+, dithiothreitol and oxygen. The final degradation products of toxoflavin and methyltoxoflavin were fully identified by MS and NMR as triazines. Therefore, we suggested that the new metagenomic enzyme, TxeA, provided the clue to applying the new metagenomic enzyme to resistance development of crop plants to toxoflavin-mediated disease as well as to biocatalysis for Baeyer-Villiger type oxidation.

Topics: Amino Acid Sequence; Bacterial Toxins; Burkholderia; Enzymes; Metagenomics; Pyrimidinones; Sequence Homology, Amino Acid; Triazines

Bacterial-fungal interactions are widely found in distinct environments and contribute to ecosystem processes. Previous studies of these interactions have mostly been performed in soil, and only limited studies of aerial plant tissues have been conducted. Here we show that a seed-borne plant pathogenic bacterium, Burkholderia glumae (Bg), and an air-borne plant pathogenic fungus, Fusarium graminearum (Fg), interact to promote bacterial survival, bacterial and fungal dispersal, and disease progression on rice plants, despite the production of antifungal toxoflavin by Bg. We perform assays of toxoflavin sensitivity, RNA-seq analyses, lipid staining and measures of triacylglyceride content to show that triacylglycerides containing linolenic acid mediate resistance to reactive oxygen species that are generated in response to toxoflavin in Fg. As a result, Bg is able to physically attach to Fg to achieve rapid and expansive dispersal to enhance disease severity.

Topics: Air Microbiology; Burkholderia; Drug Resistance, Fungal; Fusarium; Gene Expression Profiling; Gene Expression Regulation, Fungal; Host-Pathogen Interactions; Microbial Interactions; Mutation; Oryza; Phylogeny; Plant Diseases; Pyrimidinones; Seeds; Triazines

Pathogenic and mutualistic bacteria associated with eukaryotic hosts often lack distinctive genomic features, suggesting regular transitions between these lifestyles. Here we present evidence supporting a dynamic transition from plant pathogenicity to insect-defensive mutualism in symbiotic Burkholderia gladioli bacteria. In a group of herbivorous beetles, these symbionts protect the vulnerable egg stage against detrimental microbes. The production of a blend of antibiotics by B. gladioli, including toxoflavin, caryoynencin and two new antimicrobial compounds, the macrolide lagriene and the isothiocyanate sinapigladioside, likely mediate this defensive role. In addition to vertical transmission, these insect symbionts can be exchanged via the host plant and retain the ability to initiate systemic plant infection at the expense of the plant's fitness. Our findings provide a paradigm for the transition between pathogenic and mutualistic lifestyles and shed light on the evolution and chemical ecology of this defensive mutualism.

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Burkholderia gladioli; Coleoptera; Fungi; Glycine max; In Situ Hybridization, Fluorescence; Pyrimidinones; Symbiosis; Triazines

Burkholderia glumae converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to BgToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B. thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase that shows a low level of sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a class I methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine ring system is orthogonal to its orientation in BgToxA. In BthII1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in BgToxA, it is first directed toward an sp

Topics: Bacterial Proteins; Binding Sites; Burkholderia; Catalytic Domain; Crystallography, X-Ray; Histidine; Hydrogen Bonding; Methylation; Methyltransferases; Models, Molecular; Multigene Family; Oxidation-Reduction; Phylogeny; Protein Conformation; Pyrimidinones; Recombinant Proteins; S-Adenosylhomocysteine; S-Adenosylmethionine; Species Specificity; Stereoisomerism; Triazines

Defensive mutualisms are often facultative in nature, and their evolutionary dynamics can be shaped by changes in local antagonist communities or arms races with coevolving antagonists. Under these conditions, selection may favour hosts that flexibly acquire symbionts producing compounds with bioactivity against current antagonists. Here, we study the prevalence, dynamics and strain diversity of Burkholderia gladioli bacteria in Lagria beetles, a recently described protective symbiosis involving vertical transmission and antifungal defense for the host eggs. In Lagria hirta, we investigate the fate of the bacteria during the host life cycle. Despite a transmission route relying solely on the females, the bacteria are present in both sexes during the larval stage, suggesting a potentially multifaceted defensive role. In L. hirta and L. villosa adults, culture-dependent and -independent techniques revealed that individual beetles harbour diverse Burkholderia strains from at least two different phylogenetic clades, yet all closely related to free-living B. gladioli. Interestingly, rearing the beetles in the laboratory strongly impacted symbiont strain profiles in both beetle species. Our findings highlight the dynamic nature of the B. gladioli-Lagria symbiosis and present this as a valuable system for studying multiple strain coinfections, as well as the evolutionary and ecological factors regulating defensive symbiosis.

Topics: Animals; Antibiosis; Biological Evolution; Burkholderia; Coleoptera; Female; Fungi; Larva; Male; Phylogeny; Pyrimidinones; Symbiosis; Triazines

Burkholderia glumae is an emerging plant-pathogenic bacterium that causes disease in rice in several of the major rice-producing areas throughout the world. In the southern United States, B. glumae is the major causal agent of bacterial panicle blight of rice and has caused severe yield losses in recent decades. Despite its importance, few management options are available for diseases caused by B. glumae, and knowledge of how this pathogen causes disease is limited. In an effort to identify novel factors that contribute to the pathogenicity of B. glumae, random mutagenesis using the miniTn5gus transposon was performed on two strains of B. glumae. Resultant mutants were screened in the laboratory for altered phenotypes in various known or putative virulence factors, including toxoflavin, lipase and extracellular polysaccharides. Mutants that exhibited altered phenotypes compared to their parent strain were selected and subsequently characterized using a PCR-based method to identify the approximate location of the transposon insertion. Altogether, approximately 20 000 random mutants were screened and 51 different genes were identified as having potential involvement in the production of toxoflavin, lipase and/or extracellular polysaccharide. Especially, two regulatory genes, ntpR and tepR, encoding a LysR-type transcriptional regulator and a σ54-dependent response regulator, respectively, were discovered in this study as new negative regulatory factors for the production of toxoflavin, the major phytotoxin synthesized by B. glumae and involved in bacterial pathogenesis.

Topics: Base Sequence; Burkholderia; DNA Transposable Elements; DNA, Bacterial; Gene Expression Regulation, Bacterial; Genes, Regulator; Lipase; Mutagenesis; Mutation; Oryza; Phenotype; Plant Diseases; Polysaccharides, Bacterial; Pyrimidinones; Quorum Sensing; Sequence Analysis, DNA; Triazines; Virulence Factors

Burkholderia gladioli is a causal agent of bacterial panicle blight and sheath/grain browning in rice in many countries. Many strains produce the yellow pigment toxoflavin, which is highly toxic to plants, fungi, animals and microorganisms. Although there have been several studies on the toxoflavin biosynthesis system of B. glumae, it is still unclear how B. gladioli activates toxoflavin biosynthesis. In this study, we explored the genomic organization of the toxoflavin system of B. gladioli and its biological functions using comparative genomic analysis between toxoflavin-producing strains (B. glumae BGR1 and B. gladioli BSR3) and a strain not producing toxoflavin (B. gladioli KACC11889). The latter exhibits normal physiological characteristics similar to other B. gladioli strains. Burkholderia gladioli KACC11889 possesses all the genes involved in toxoflavin biosynthesis, but lacks the quorum-sensing (QS) system that functions as an on/off switch for toxoflavin biosynthesis. These data suggest that B. gladioli has evolved to use the QS signalling cascade of toxoflavin production (TofI/TofR of QS → ToxJ or ToxR → tox operons) similar to that in B. glumae. However, some strains may have evolved to eliminate toxoflavin production through deletion of the QS genes. In addition, we demonstrate that the toxoflavin biosynthetic system enhances the virulence of B. gladioli. These findings provide another line of evidence supporting the differential regulation of the toxoflavin system in Burkholderia strains.

Topics: Biosynthetic Pathways; Burkholderia gladioli; Genes, Bacterial; Genetic Complementation Test; Movement; Onions; Operon; Pyrimidinones; Reproducibility of Results; Triazines; Virulence

Toxoflavin is a major virulence factor of the rice pathogen Burkholderia glumae. The tox operon of B. glumae contains five putative toxoflavin biosynthetic genes toxABCDE. ToxA is a predicted S-adenosylmethionine-dependent methyltransferase, and toxA knockouts of B. glumae are less virulent in plant infection models. In this study, we show that ToxA performs two consecutive methylations to convert the putative azapteridine intermediate, 1,6-didemethyltoxoflavin, to toxoflavin. In addition, we report a series of crystal structures of ToxA complexes that reveals the molecular basis of the dual methyltransferase activity. The results suggest sequential methylations with initial methylation at N6 of 1,6-didemethyltoxoflavin followed by methylation at N1. The two azapteridine orientations that position N6 or N1 for methylation are coplanar with a 140° rotation between them. The structure of ToxA contains a class I methyltransferase fold having an N-terminal extension that either closes over the active site or is largely disordered. The ordered conformation places Tyr7 at a position of a structurally conserved tyrosine site of unknown function in various methyltransferases. Crystal structures of ToxA-Y7F consistently show a closed active site, whereas structures of ToxA-Y7A consistently show an open active site, suggesting that the hydroxyl group of Tyr7 plays a role in opening and closing the active site during the multistep reaction.

Topics: Bacterial Proteins; Burkholderia; Catalysis; Catalytic Domain; Crystallography, X-Ray; Methylation; Methyltransferases; Protein Structure, Secondary; Pyrimidinones; Triazines

Burkholderia glumae is the chief causal agent for bacterial panicle blight of rice. The acyl-homoserine lactone (AHL)-mediated quorum-sensing (QS) system dependent on a pair of luxI and luxR homologs, tofI and tofR, is the primary cell-to-cell signaling mechanism determining the virulence of this bacterium. Production of toxoflavin, a major virulence factor of B. glumae, is known to be dependent on the tofI/tofR QS system. In our previous study, however, it was observed that B. glumae mutants defective in tofI or tofR produced toxoflavin if they grew on the surface of a solid medium, suggesting that alternative signaling pathways independent of tofI or tofR are activated in that growth condition for the production of toxoflavin. In this study, potential genetic components involved in the tofI- and tofR-independent signaling pathways for toxoflavin production were sought through screening random mini-Tn5 mutants of B. glumae to better understand the intercellular signaling pathways of this pathogen. Fifteen and three genes were initially identified as the potential genetic elements of the tofI- and tofR-independent pathways, respectively. Especially, the ORF (bglu_2g06320) divergently transcribed from toxJ, which encodes an orphan LuxR protein and controls toxoflavin biosynthesis, was newly identified in this study as a gene required for the tofR-independent toxoflavin production and named as toxK. Among those genes, flhD, dgcB, and wzyB were further studied to validate their functions in the tofI-independent toxoflavin production, and similar studies were also conducted with qsmR and toxK for their functions in the tofR-independent toxoflavin production. This work provides a foundation for future comprehensive studies of the intercellular signaling systems of B. glumae and other related pathogenic bacteria.

Topics: Bacterial Proteins; Burkholderia; Gene Expression Regulation, Bacterial; Genomics; Onions; Plant Diseases; Pyrimidinones; Quorum Sensing; Signal Transduction; Triazines; Virulence

Pseudomonas spp. are prolific producers of natural products from many structural classes. Here we show that the soil bacterium Pseudomonas protegens Pf-5 is capable of producing trace levels of the triazine natural product toxoflavin (1) under microaerobic conditions. We evaluated toxoflavin production by derivatives of Pf-5 with deletions in specific biosynthesis genes, which led us to propose a revised biosynthetic pathway for toxoflavin that shares the first two steps with riboflavin biosynthesis. We also report that toxM, which is not present in the well-characterized cluster of Burkholderia glumae, encodes a monooxygenase that degrades toxoflavin. The toxoflavin degradation product of ToxM is identical to that of TflA, the toxoflavin lyase from Paenibacillus polymyxa. Toxoflavin production by P. protegens causes inhibition of several plant-pathogenic bacteria, and introduction of toxM into the toxoflavin-sensitive strain Pseudomonas syringae DC3000 results in resistance to toxoflavin.

Topics: Biosynthetic Pathways; Chromatography, Liquid; Cloning, Molecular; Molecular Structure; Multigene Family; Pseudomonas; Pyrimidinones; Triazines

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow-green and purple) pigment compounds in casein-peptone-glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment-deficient mutant derivatives of the virulent and pigment-proficient strain 411gr-6, having mini-Tn5gus insertions in aroA encoding 3-phosphoshikimate 1-carboxyvinyltransferase and aroB encoding 3-dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.

Topics: Adaptation, Physiological; Burkholderia; Genes, Bacterial; Glycine; Glyphosate; Metabolic Networks and Pathways; Microbial Viability; Mutation; Onions; Oryza; Pigments, Biological; Plant Diseases; Pyrimidinones; Shikimic Acid; Triazines; Ultraviolet Rays; Virulence

The recently discovered enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) has been implicated in the topoisomerase-mediated repair of DNA damage. In the clinical setting, it has been hypothesized that TDP2 may mediate drug resistance to topoisomerase II (topo II) inhibition by etoposide. Therefore, selective pharmacological inhibition of TDP2 is proposed as a novel approach to overcome intrinsic or acquired resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoflavins and deazaflavins were identified as the first reported sub-micromolar and selective inhibitors of this enzyme. Toxoflavin derivatives appeared to exhibit a clear structure-activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further exploration. The deazaflavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display redox activity; however low cell permeability proved to be a challenge.

Topics: Phosphoric Diester Hydrolases; Pyrimidinones; Structure-Activity Relationship; Topoisomerase II Inhibitors; Triazines

In this study, we developed a simple and sensitive biosensor for the determination of toxoflavin (which is toxic to various plants, fungi, animals, and bacteria) in natural samples based on β-galactosidase activity. The proposed toxoflavin detection method for toxin-producing bacteria or toxin-contaminated foods is simple and cost effective. Burkholderia glumae, a species known to cause rice grain rot and wilt in various field crops, produces toxoflavin under the control of a LysR-type transcriptional regulator ToxR and its ligand toxoflavin. As the expression of toxoflavin biosynthetic genes requires toxoflavin as a co-activator of ToxR, a novel biosensor stain was constructed based on lacZ reporter gene integration into the first gene of the toxoflavin biosynthesis operon, toxABCDE of B. glumae. The biosensor was composed of a sensor strain (COK71), substrates (X-gal or ONPG), and culture medium, without any complex preparation process. We demonstrated that the biosensor strain is highly specific to toxoflavin, and can quantify relative amounts of toxoflavin compared with known concentrations of toxoflavin. The proposed method was reliable and simple; samples containing 50-500 nM of toxoflavin could be analyzed. More importantly, the proposed biosensor strain could identify toxoflavin-producing bacteria in real samples. The excellent performance of this biosensor is useful for diagnostic purposes, such as detecting toxoflavin-contaminated foods and environmental samples.

Topics: beta-Galactosidase; Biosensing Techniques; Burkholderia; Lac Operon; Oryza; Pyrimidinones; Quorum Sensing; Triazines

SIRT1 and SIRT2 are deacetylase enzymes that belong to the sirtuin family and are involved in tumorigenesis. In our screen for small molecules inhibiting SIRT1/2 toxoflavin was identified. Toxoflavin potently inhibited SIRT1 activity in in vitro deacetylase assay using purified SIRT1 protein. SIRT2 activity was also inhibited by toxoflavin less potently than SIRT1 in deacetylase assay in vitro. Toxoflavin exhibited growth inhibition of various cancer cell lines including A549 lung cancer cells with a GI(50) of 48 nM. Toxoflavin treatment in A549 cells increased the acetylated form of p53, which is a substrate of SIRT1. The acetylation levels of α-tubulin, a SIRT2 substrate, were also increased by toxoflavin treatment dose-dependently. Several toxoflavin derivatives were synthesized to determine the preliminary structure-activity relationship of toxoflavin. Some of the toxoflavin derivatives showed highly selective inhibition against SIRT1. In conclusion, this study presented toxoflavin as a potent SIRT1/2 inhibitor with anticancer activity.

Topics: Acetylation; Antineoplastic Agents; Benzamides; Cell Line, Tumor; Humans; Neoplasms; Pyrimidinones; Sirtuin 1; Sirtuin 2; Small Molecule Libraries; Structure-Activity Relationship; Triazines; Tubulin

Toxoflavin (an azapteridine) is degraded to a single product by toxoflavin lyase (TflA) in a reaction dependent on reductant, Mn(II), and oxygen. The isolated product was fully characterized by NMR and MS and was identified as a triazine in which the pyrimidine ring was oxidatively degraded. A mechanism for toxoflavin degradation based on the identification of the enzymatic product and the recently determined crystal structure of toxoflavin lyase is proposed.

Topics: Lyases; Manganese; Models, Molecular; Molecular Structure; Oxidation-Reduction; Oxygen; Pyrimidinones; Triazines

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.

Topics: Antifungal Agents; Burkholderia; DNA Fingerprinting; Onions; Pigmentation; Pyrimidinones; Rhizoctonia; Triazines; Virulence

This study established a new system for potato transformation using toxoflavin as selection agent and toxoflavin lyase (tflA) as selectable marker gene. Potato plants expressing tflA was successfully transformed on toxoflavin medium with 27% efficiency, similar to that for the hygromycin/hpt selection system. The transgenic potato expressing tflA also showed resistance to Burkholderia glumea infection.

Topics: Genetic Engineering; Genetic Markers; Lyases; Pyrimidinones; Solanum tuberosum; Transformation, Genetic; Triazines

Burkholderia glumae causes bacterial panicle blight of rice and produces major virulence factors, including toxoflavin, under the control of the quorum-sensing (QS) system mediated by the luxI homolog, tofI, and the luxR homolog, tofR. In this study, a series of markerless deletion mutants of B. glumae for tofI and tofR were generated using the suicide vector system, pKKSacB, for comprehensive characterization of the QS system of this pathogen. Consistent with the previous studies by other research groups, ΔtofI and ΔtofR strains of B. glumae did not produce toxoflavin in Luria-Bertani (LB) broth. However, these mutants produced high levels of toxoflavin when grown in a highly dense bacterial inoculum (∼ 10(11) CFU/ml) on solid media, including LB agar and King's B (KB) agar media. The ΔtofI/ΔtofR strain of B. glumae, LSUPB201, also produced toxoflavin on LB agar medium. These results indicate the presence of previously unknown regulatory pathways for the production of toxoflavin that are independent of tofI and/or tofR. Notably, the conserved open reading frame (locus tag: bglu_2g14480) located in the intergenic region between tofI and tofR was found to be essential for the production of toxoflavin by tofI and tofR mutants on solid media. This novel regulatory factor of B. glumae was named tofM after its homolog, rsaM, which was recently identified as a novel negative regulatory gene for the QS system of another rice pathogenic bacterium, Pseudomonas fuscovaginae. The ΔtofM strain of B. glumae, LSUPB286, produced a less amount of toxoflavin and showed attenuated virulence when compared with its wild type parental strain, 336gr-1, suggesting that tofM plays a positive role in toxoflavin production and virulence. In addition, the observed growth defect of the ΔtofI strain, LSUPB145, was restored by 1 µM N-octanoyl homoserine lactone (C8-HSL).

Topics: Burkholderia; Gene Expression Regulation, Bacterial; Pyrimidinones; Quorum Sensing; Triazines

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.

Topics: Arabidopsis; Biomarkers; Cinnamates; Genes, Bacterial; Genetic Techniques; Hygromycin B; Light; Oryza; Paenibacillus; Phosphotransferases (Alcohol Group Acceptor); Plant Leaves; Plants; Plants, Genetically Modified; Pyrimidinones; Recombinant Fusion Proteins; Transformation, Genetic; Triazines

High-resolution crystal structures are reported for apo, holo, and substrate-bound forms of a toxoflavin-degrading metalloenzyme (TflA). In addition, the degradation reaction is shown to be dependent on oxygen, Mn(II), and dithiothreitol in vitro. Despite its low sequence identity with proteins of known structure, TflA is structurally homologous to proteins of the vicinal oxygen chelate superfamily. Like other metalloenzymes in this superfamily, the TflA fold contains four modules that associate to form a metal binding site; however, the fold displays a rare rearrangement of the structural modules indicative of domain permutation. Moreover, unlike the 2-His-1-carboxylate facial triad commonly utilized by vicinal oxygen chelate dioxygenases and other dioxygen-activating non-heme Fe(II) enzymes, the metal center in TflA consists of a 1-His-2-carboxylate facial triad. The substrate-bound complex shows square-pyramidal geometry in which one position is occupied by O5 of toxoflavin. The open coordination site is predicted to be the dioxygen binding site. TflA appears to stabilize the reduced form of toxoflavin through second-sphere interactions. This anionic species is predicted to be the electron source responsible for reductive activation of oxygen to produce a peroxytoxoflavin intermediate.

Topics: Bacterial Proteins; Binding Sites; Carboxylic Acids; Catalysis; Crystallography, X-Ray; Histidine; Hydrogen Bonding; Ligands; Lyases; Models, Molecular; Oxygen; Pyrimidinones; Triazines

Pathogenic bacteria synthesize and secrete toxic low molecular weight compounds as virulence factors. These microbial toxins play essential roles in the pathogenicity of bacteria in various hosts, and are emerging as targets for antivirulence strategies. Toxoflavin, a phytotoxin produced by Burkholderia glumae BGR1, has been known to be the key factor in rice grain rot and wilt in many field crops. Recently, toxoflavin-degrading enzyme (TxDE) was identified from Paenibacillus polymyxa JH2, thereby providing a possible antivirulence strategy for toxoflavin-mediated plant diseases. Here, we report the crystal structure of TxDE in the substrate-free form and in complex with toxoflavin, along with the results of a functional analysis. The overall structure of TxDE is similar to those of the vicinal oxygen chelate superfamily of metalloenzymes, despite the lack of apparent sequence identity. The active site is located at the end of the hydrophobic channel, 9 Å in length, and contains a Mn(II) ion interacting with one histidine residue, two glutamate residues, and three water molecules in an octahedral coordination. In the complex, toxoflavin binds in the hydrophobic active site, specifically the Mn(II)-coordination shell by replacing a ligating water molecule. A functional analysis indicated that TxDE catalyzes the degradation of toxoflavin in a manner dependent on oxygen, Mn(II), and the reducing agent dithiothreitol. These results provide the structural features of TxDE and the early events in catalysis.

Topics: Amino Acid Sequence; Bacterial Proteins; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Molecular Sequence Data; Paenibacillus; Pyrimidinones; Toxins, Biological; Triazines

The primary objective of this study was to determine the effects of quorum quenching in the pathogenicity and toxoflavin production of Burkholderia glumae causing bacterial rice grain rot.. An acyl-homoserine lactonase (aiiA) gene from Bacillus sp. was expressed in B. glumae under the control of a constitutive promoter. Acyl-homoserine lactone production in the aiiA-transformants was reduced significantly, and the aiiA-expressing B. glumae strain reduced the severity of soft rot when the strain was co-inoculated with a soft-rot pathogen, Pectobacterium carotovorum ssp. carotovorum SCCI. However, the aiiA-transformant still caused rice seedling rot and rice grain rot. The aiiA-expressing strains had wild-type levels of transcription from the genes in the toxoflavin biosynthetic operon, and as well as wild-type levels of toxin production.. Our results show that aiiA-mediated quorum quenching does not affect virulence or toxoflavin production in B. glumae.. Indirect quorum quenching may prove an ineffective approach to the control of rice grain rot, because it reduces, but does not eliminate entirely homoserine lactones in B. glumae. Virulence of rice grain rot was retained despite reduction in homoserine lactones by the expression of aiiA in B. glumae.

Topics: Bacterial Proteins; Burkholderia; Carboxylic Ester Hydrolases; Cloning, Molecular; Gene Expression Regulation, Bacterial; Metalloendopeptidases; Operon; Oryza; Pectobacterium carotovorum; Plant Diseases; Promoter Regions, Genetic; Pyrimidinones; Quorum Sensing; Seedlings; Transformation, Genetic; Triazines; Virulence; Virulence Factors

Quorum sensing (QS) plays important roles in the pathogenicity of Burkholderia glumae, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in B. glumae. The QS-defective mutant of B. glumae exhibited less catalase activity than wild-type B. glumae. A beta-glucuronidase assay of a katG::Tn3-gusA78 reporter fusion protein revealed that katG expression is under the control of QS. Furthermore, katG expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates katG expression. The katG mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 micromol(-1) m(-2), the survival rate of the katG mutant was 10(5)-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the katG mutant.

Topics: Bacterial Proteins; Burkholderia; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Bacterial; Light; Microbial Viability; Mutation; Oryza; Pyrimidinones; Quorum Sensing; Triazines

Burkholderia glumae produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the toxABCDE (toxoflavin biosynthesis genes) and toxFGHI (toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for tox gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both tox operons. ToxR and ToxJ both bound to the toxA and toxF regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both tox operons possessed T-N(11)-A motifs. Following random mutagenesis of toxR, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (toxR'A'::Omega toxF::Tn3-gusA34) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred toxF expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that B. glumae cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR.

Topics: Bacterial Proteins; Binding Sites; Burkholderia; Chromatography, Gel; DNA Footprinting; DNA Mutational Analysis; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Bacterial; Molecular Structure; Mutagenesis; Operon; Protein Binding; Protein Multimerization; Pyrimidinones; Quorum Sensing; Transcription Factors; Triazines

Eight novel toxoflavin glycosides, which are potential prodrugs in antibody directed enzyme prodrug therapy (ADEPT), were synthesized. The structures of all toxoflavin glycosides were characterized by (13)C NMR spectroscopy, elemental analysis, and MS. Their enzymatic hydrolysis activities were tested against beta-glucosidase (EC.3.2.1.21).

Topics: Antibodies; beta-Galactosidase; Glycosides; Immunotherapy; Magnetic Resonance Spectroscopy; Neoplasms; Prodrugs; Pyrimidinones; Triazines

Burkholderia glumae is an emerging rice pathogen in several areas around the world. Closely related Burkholderia species are important opportunistic human pathogens for specific groups of patients, such as patients with cystic fibrosis and patients with chronic granulomatous disease. Here we report that the first clinical isolate of B. glumae, strain AU6208, has retained its capability to be very pathogenic to rice. As previously reported for rice isolate B. glumae BGR1 (and also for the clinical isolate AU6208), TofI or TofR acyl homoserine lactone (AHL) quorum sensing played a pivotal role in rice virulence. We report that AHL quorum sensing in B. glumae AU6208 regulates secreted LipA lipase and toxoflavin, the phytotoxin produced by B. glumae. B. glumae AU6208 lipA mutants were no longer pathogenic to rice, indicating that the lipase is an important virulence factor. It was also established that type strain B. glumae ATCC 33617 did not produce toxoflavin and lipase and was nonpathogenic to rice. It was determined that in strain ATCC 33617 the LuxR family quorum-sensing sensor/regulator TofR was inactive. Introducing the tofR gene of B. glumae AU6208 in strain ATCC 33617 restored its ability to produce toxoflavin and the LipA lipase. This study extends the role of AHL quorum sensing in rice pathogenicity through the regulation of a lipase which was demonstrated to be a virulence factor. It is the first report of a clinical B. glumae isolate retaining strong rice pathogenicity and finally determined that B. glumae can undergo phenotypic conversion through a spontaneous mutation in the tofR regulator.

Topics: Bacterial Proteins; Burkholderia; Burkholderia Infections; Gene Expression Regulation, Bacterial; Humans; Lipase; Molecular Sequence Data; Oryza; Plant Diseases; Pyrimidinones; Quorum Sensing; Sequence Analysis, DNA; Triazines

Burkholderia glumae BGR1 produces a broad-host range phytotoxin, called toxoflavin, which is a key pathogenicity factor in rice grain rot and wilt in many field crops. Our molecular and genetic analyses of toxoflavin-deficient mutants demonstrated that gene clusters for toxoflavin production consist of four transcriptional units. The toxoflavin biosynthesis genes were composed of five genes, toxA to toxE, as Suzuki et al. (2004) reported previously. Genes toxF to toxI, which are responsible for toxoflavin transport, were polycistronic and similar to the genes for resistance-nodulation-division (RND) efflux systems. Using Tn3-gusA reporter fusions, we found that ToxR, a LysR-type regulator, regulates both the toxABCDE and toxFGHI operons in the presence of toxoflavin as a coinducer. In addition, the expression of both operons required a transcriptional activator, ToxJ, whose expression is regulated by quorum sensing. TofI, a LuxI homologue, was responsible for the biosynthesis of both N-hexanoyl homoserine lactone and N-octanoyl homoserine lactone (C8-HSL). C8-HSL and its cognate receptor TofR, a LuxR homologue, activated toxJ expression. This is the first report that quorum sensing is involved in pathogenicity by the regulation of phytotoxin biosynthesis and its transport in plant pathogenic bacteria.

Topics: Bacterial Proteins; Biological Transport; Burkholderia; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Genes, Reporter; Multiprotein Complexes; Operon; Oryza; Pyrimidinones; Recombinant Fusion Proteins; Signal Transduction; Transcription Factors; Triazines

1. It has been suggested that the toxic effect of toxoflavin (TXF) produced by Pseudomonas cocovenenas is mainly due to the impairment of electron transfer of the mitochondrial respiratory chain. However, the cardiovascular effect of TXF is unknown. In the present study, the effect of TXF on the isometric contraction of rat isolated tail artery strips and the underlying mechanisms were investigated. 2. The basal force of the tissues was not affected by the toxin. However, the application of TXF before or during KCl (60 mM) stimulation potentiated KCl-induced vasocontraction, specifically the tonic phase of the contraction. 3. When the vessel strips were precontracted with phenylephrine (Phe), TXF further enhanced the tonic contraction of the tissue. Pretreatment of tissues with TXF also potentiated subsequent vasocontraction induced by Phe. The vasocontractor effects of TXF and Phe, however, were not additive. 4. The vascular effect of TXF was not mediated by oxygen-derived free radicals since catalase and SOD did not affect TXF-enhanced vasocontraction. In contrast, the vasocontractor effect of TXF was dependent on extracellular Ca2+ and abolished by nifedipine (a Ca2+ antagonist). TXF also had no effect on caffeine- or U46619-induced vasocontraction. 5. It is suggested that TXF may potentially contract blood vessels via its effect on Ca2+ channels. This effect of TXF depends on the contractile status of the vascular tissues.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arteries; Caffeine; Calcium; Calcium Channels; Free Radical Scavengers; In Vitro Techniques; Isometric Contraction; Male; Muscle, Smooth, Vascular; Phenylephrine; Potassium Chloride; Prostaglandin Endoperoxides, Synthetic; Pyrimidinones; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Tail; Thromboxane A2; Triazines; Vasoconstriction; Vasoconstrictor Agents

6-Phenyl analogs of toxoflavin (1-methyl-6-phenylpyrimido[5,4-e]-1,2,4-triazine-5,7(1H,6H)-diones ) (7a--f) and their 4-oxides (8a-f) were synthesized by nitrosative or nitrative cyclization of the aldehyde hydrazones (6a-f) of 6-(1-methylhydrazino)-3-phenyluracil (5). Both sets of compounds, 7a-f and 8a-f, gave the corresponding 1-demethyl derivatives (10a-f) upon treatment with nucleophiles such as dimethylformamide (DMF) and acetic acid under heating. The activities of toxoflavins (1a-e), toxoflavin 4-oxides (3a-e) and their 6-phenyl analogs (7a-f and 8a-f) against a variety of bacterial and fungal strains were examined. Most of the compounds showed strong inhibitory activities against gram-positive bacteria. Among the compounds, 1c, 1d, 1e, and 3c exhibited the strongest inhibitions of Micrococcus lutea (0.5 micrograms/ml minimal growth-inhibitory concentration) and Staphylococcus aureus 4R (1 microgram/ml), as well as Bacillus subtilis and Staphylococcus aureus (1-2 micrograms/ml). Most of the compounds had strong antifungal activity (2-100 micrograms/ml minimal growth-inhibitory concentration) against Candida albicans and Saccharomyces cerevisiae.

Topics: Anti-Bacterial Agents; Bacteria; Fungi; Microbial Sensitivity Tests; Pyrimidinones; Triazines

In order to study the toxication of toxoflavin produced by P. cocovenenans to immunocytes and detoxication, the effects of toxoflavin on rabbit blood culture in the presence of PHA and detoxication function of seven drugs towards this system have been observed by 3H-TdR incorporation assay. The more toxoflavin used, the more reduction of cpm value. The results indicated that toxoflavin in 0.5 microgram/ml or more suppressed the growth of cells to even still lower than that of control in which PHA was absent; and when 0.1 microgram/ml of toxoflavin were used, the cpm value began to rise again. The effects of toxoflavin on lymphocyte and other blood cells were studied under the microscope and the naked-eye. The detoxication effects of the seven drugs on toxoflavin were observed and shown that SOD, GSH, Cyt. C and VC were most effective in protecting the lymphocytes from poisoning.

Topics: Animals; Antidotes; Cell Division; Cytochrome c Group; Glutathione; Lymphocytes; Pseudomonas; Pyrimidinones; Rabbits; Superoxide Dismutase; Triazines

A linear combination derivative spectrophotometric method is described. The method overcomes the problem of overlapping in derivative spectrophotometry and allows the maximum use of quantitative information. In addition, the method can be used to increase the selectivity, sensitivity and accuracy of the simultaneous analysis of multicomponent mixtures. The application of the method to the simultaneous determination of bongkrekic acid and toxoflavin, the toxic metabolites produced by Pseudomonas farinofermentans, is described.

Topics: Bongkrekic Acid; Food Contamination; Pseudomonas; Pyrimidinones; Spectrophotometry, Ultraviolet; Triazines

Tempe bongkrek was prepared from partially defatted coconut by fermentation with the mould Rhizopus oligosporus. The addition of 2% sodium chloride decreased but did not inhibit formation of bongkrek acid by Pseudomonas cocovenenans strain ITB in either coconut culture medium (CCM) or tempe bongkrek, while 2% sodium chloride or acidification of CCM to pH 4.5 with acetic acid inhibited formation of toxoflavin. Toxin formation by three strains of Ps. cocovenenans in tempe bongkrek was inhibited by the combination of 2% sodium chloride and reduction of pH to 4.5 with acetic acid. Mycelial growth of R. oligosporus was sometimes affected by toxin production by Ps. cocovenenans, but not by the addition of 2% sodium chloride and/or acidification of CCM to pH 4.5 with acetic acid.

Topics: Acetates; Acetic Acid; Anti-Bacterial Agents; Bacterial Toxins; Bongkrekic Acid; Chromatography, High Pressure Liquid; Cocos; Fermentation; Food Microbiology; Hydrogen-Ion Concentration; Pseudomonas; Pyrimidinones; Rhizopus; Sodium Chloride; Triazines

Fermented corn flour containing toxoflavin and bongkretic acid produced by P. farinofermentans can cause acute food poisoning, but study on mutagenicity of toxoflavin has not been reported. We studied the mutagenicity of toxoflavin by Tradescantia Paludosa and animal micronucleus tests. The results showed that toxoflavin possesses distinct mutagenic action in both plant and animal cells. Mutagenic action of toxoflavin in cells may have important bearing on food hygiene examination.

Topics: Animals; Anti-Bacterial Agents; Bongkrekic Acid; Female; Fermentation; Flour; Male; Mice; Micronucleus Tests; Pseudomonas; Pyrimidinones; Triazines

Reaction of xanthothricin demethylation by amines in acetone as a solvent yielded a new compound with a formula of C10H13N5O3. Mass spectrometry, 1H NMR-spectroscopy and x-ray analysis showed that the compound was 4H-1,6-dimethyl-4a(2-oxopropyl)-pyrimido-[5,4-e]-1 ,2,4-triazine-5,7-dione.

Topics: Acetone; Chromatography, Thin Layer; Drug Interactions; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methylation; Pyrimidinones; Triazines; X-Ray Diffraction

The structures of xanthothricin (I) and fervenulin (II), pyrimido-[5,4-e]-1,2,4-triazine antibiotics were determined by x-ray analysis. The crystals of I and II are monoclinic: a 9.667(6), 18.270(7), b 14.029(9), 5.723(2), c 6.914(3), 18.058(7) A, beta 116.47(3), 118.90(2) degrees, Z 4,8; space group P21/c. Analysis of bond length distribution in I and II was indicative of significant alternation of the double and ordinary bonds.

Topics: Chemical Phenomena; Chemistry; Pyrimidinones; Triazines; X-Ray Diffraction

A comparative study of the NMR 1H and 13C spectra of reumycin, fervenulin and xanthothricin in aqueous acid-base media showed that at pH or pD ranging from 8.0 to 1.0 the antibiotics were chemically stable. By the ratio of the 1H and 13C chemical shifts of reumycin at pH 4.0-10.0 the pKa values of this antibiotic were determined: 6.7 in aqueous (D2O) solution and 8.76 in dimethylsulfoxide media. Alkalization of the solutions of reumycin (pH 12.0), fervenulin (pH 9.0) and xanthothricin (pH 8.0) resulted in irreversible chemical transformation of the antibiotics. The analysis of the chemical shifts in the PMR spectra of the transformation products revealed transformation of the uracil ring in reumycin and uracil and triazine rings in fervenulin and xanthothricin. Alkalization of the xanthothricin solutions resulted also in demethylation with formation of reumycin.

Topics: Anti-Bacterial Agents; Chemical Phenomena; Chemistry; Drug Stability; Hydrogen-Ion Concentration; In Vitro Techniques; Magnetic Resonance Spectroscopy; Pyrimidinones; Spectrum Analysis; Triazines

Topics: Adenosine Triphosphatases; Animals; Anti-Bacterial Agents; Antimycin A; Candida; Carcinoma, Ehrlich Tumor; Cyanides; Electron Transport; Hydrogen-Ion Concentration; Ketoglutaric Acids; Malonates; Mitochondria; Mitochondria, Liver; NAD; Oxidation-Reduction; Oxygen Consumption; Phenylhydrazines; Polarography; Potentiometry; Pyrimidinones; Rats; Rotenone; Species Specificity; Spectrometry, Fluorescence; Succinates; Triazines

Topics: Anti-Bacterial Agents; Dermatologic Agents; Heterocyclic Compounds; Pseudomonas; Pyrimidinones; Triazines

Topics: Anti-Bacterial Agents; Antibiotics, Antitubercular; Dermatologic Agents; Humans; Pyrimidinones; Triazines