Plasma quercetin metabolites are affected by intestinal microbiota of human microbiota-associated mice fed with a quercetin-containing diet.

Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice has been reported. Recent research has revealed that several intestinal bacteria metabolize quercetin. We hypothesize that the difference in composition of intestinal microbiota affects quercetin absorption from the intestine. Germ-free BALB/cA female mice (18 weeks old) were randomly divided into four groups and orally administered with fecal suspension from four human individuals (HF1, HF2, HF3, HF4) to produce the human microbiota-associated mice. All mice were fed the 0.05% quercetin-containing pelleted diet for four weeks. Significant differences were observed in plasma total cholesterol and cecal microbiota among the four groups. Plasma quercetin concentration was significantly higher in the HF3 group than in the HF1 group. The plasma isorhamnetin/quercetin ratio showed significant negative correlation with visceral fat levels (r = -0.544, p = 0.013). Positive correlation was observed between the Log10 Enterobacteriaceae count and the plasma quercetin metabolites. Principal component analysis showed that all groups were distributed in different regions by using the correlation diagram with the second and third principal component. This study indicates that intestinal microbiota of human microbiota-associated mice inoculated with different fecal suspensions react to dietary quercetin in different ways.

Metal Ions in Activated Carbon Improve the Detection Efficiency of Aflatoxin-Producing Fungi.

Aflatoxins (AF), produced by several Aspergillus species, are visible under ultraviolet light if present in high amounts. AF detection can be improved by adding activated carbon, which enhances the observation efficiency of weakly AF-producing fungi. However, commercial activated carbon products differ in their characteristics, making it necessary to investigate which characteristics affect method reproducibility. Herein, the addition of 10 activated carbon products resulted in different AF production rates in each case. The differences in the production of aflatoxin G1 (AFG1) were roughly correlated to the observation efficiency in the plate culture. Trace element analysis showed that the concentrations of several metal ions differed by factors of >100, and the carbons that most effectively increased AFG1 production contained higher amounts of metal ions. Adding 5 mg L-1 Fe or Mg ions increased AFG1 production even without activated carbon. Furthermore, co-addition of both ions increased AFG1 production stably with the addition of carbon. When varying the concentration of additives, only AFG1 production increased in a concentration-dependent manner, while the production of all the other AFs decreased or remained unchanged. These findings suggest that a key factor influencing AF production is the concentration of several metal ions in activated carbon and that increasing AFG1 production improves AF detectability.

Light-Irradiation Wavelength and Intensity Changes Influence Aflatoxin Synthesis in Fungi.

Fungi respond to light irradiation by forming conidia and occasionally synthesizing mycotoxins. Several light wavelengths, such as blue and red, affect the latter. However, the relationship between light irradiation and mycotoxin synthesis varies depending on the fungal species or strain. This study focused on aflatoxin (AF), which is a mycotoxin, and the types of light irradiation that increase AF synthesis. Light-irradiation tests using the visible region indicated that blue wavelengths in the lower 500 nm region promoted AF synthesis. In contrast, red wavelengths of 660 nm resulted in limited significant changes compared with dark conditions. Irradiation tests with different intensity levels indicated that a low light intensity increased AF synthesis. For one fungal strain, light irradiation decreased the AF synthesis under all wavelength conditions. However, the decrease was mitigated by 525 nm low intensity irradiation. Thus, blue-green low intensity irradiation may increase AF synthesis in fungi.

Addition of Carbon to the Culture Medium Improves the Detection Efficiency of Aflatoxin Synthetic Fungi.

Aflatoxin (AF) is a harmful secondary metabolite that is synthesized by the Aspergillus species. Although AF detection techniques have been developed, techniques for detection of AF synthetic fungi are still required. Techniques such as plate culture methods are continually being modified for this purpose. However, plate culture methods require refinement because they suffer from several issues. In this study, activated charcoal powder (carbon) was added to a culture medium containing cyclodextrin (CD) to enhance the contrast of fluorescence and improve the detection efficiency for AF synthetic fungi. Two culture media, potato dextrose agar and yeast extract sucrose agar, were investigated using both plate and liquid cultures. The final concentrations of CD and carbon in the media were 3 mg/mL and 0.3 mg/mL, respectively. Addition of carbon improved the visibility of fluorescence by attenuating approximately 30% of light scattering. Several fungi that could not be detected with only CD in the medium were detected with carbon addition. The carbon also facilitated fungal growth in the potato dextrose liquid medium. The results suggest that addition of carbon to media can enhance the observation of AF-derived fluorescence.

Acetylated Deoxynivalenol Generates Differences of Gene Expression that Discriminate Trichothecene Toxicity.

Deoxynivalenol (DON), which is a toxic secondary metabolite generated by Fusarium species, is synthesized through two separate acetylation pathways. Both acetylation derivatives, 3-acetyl-DON (3ADON) and 15-acetyl-DON (15ADON), also contaminate grain and corn widely. These derivatives are deacetylated via a variety of processes after ingestion, so it has been suggested that they have the same toxicity as DON. However, in the intestinal entry region such as the duodenum, the derivatives might come into contact with intestinal epithelium cells because metabolism by microflora or import into the body has not progressed. Therefore, the differences of toxicity between DON and these derivatives need to be investigated. Here, we observed gene expression changes in the yeast pdr5Δ mutant strain under concentration-dependent mycotoxin exposure conditions. 15ADON exposure induced significant gene expression changes and DON exposure generally had a similar but smaller effect. However, the glucose transporter genes HXT2 and HXT4 showed converse trends. 3ADON also induced a different expression trend in these genes than DON and 15ADON. These differences in gene expression suggest that DON and its derivatives have different effects on cells.

Low toxicity of deoxynivalenol-3-glucoside in microbial cells.

Host plants excrete a glucosylation enzyme onto the plant surface that changes mycotoxins derived from fungal secondary metabolites to glucosylated products. Deoxynivalenol-3-glucoside (DON3G) is synthesized by grain uridine diphosphate-glucosyltransferase, and is found worldwide, although information on its toxicity is lacking. Here, we conducted growth tests and DNA microarray analysis to elucidate the characteristics of DON3G. The Saccharomyces cerevisiae PDR5 mutant strain exposed to DON3G demonstrated similar growth to the dimethyl sulfoxide control, and DNA microarray analysis revealed limited differences. Only 10 genes were extracted, and the expression profile of stress response genes was similar to that of DON, in contrast to metabolism genes like SER3, which encodes 3-phosphoglycerate dehydrogenase. Growth tests with Chlamydomonas reinhardtii also showed a similar growth rate to the control sample. These results suggest that DON3G has extremely low toxicity to these cells, and the glucosylation of mycotoxins is a useful protective mechanism not only for host plants, but also for other species.

Phytotoxicity evaluation of type B trichothecenes using a Chlamydomonas reinhardtii model system.

Type B trichothecenes, which consist of deoxynivalenol (DON) and nivalenol (NIV) as the major end products, are produced by phytotoxic fungi, such as the Fusarium species, and pollute arable fields across the world. The DON toxicity has been investigated using various types of cell systems or animal bioassays. The evaluation of NIV toxicity, however, has been relatively restricted because of its lower level compared with DON. In this study, the Chlamydomonas reinhardtii testing system, which has been reported to have adequate NIV sensitivity, was reinvestigated under different mycotoxin concentrations and light conditions. The best concentration of DON and NIV, and their derivatives, for test conditions was found to be 25 ppm (2.5 × 10(-2) mg/mL). In all light test conditions, DON, NIV, and fusarenon-X (FusX) indicated significant growth inhibition regardless of whether a light source existed, or under differential wavelength conditions. FusX growth was also influenced by changes in photon flux density. These results suggest that C. reinhardtii is an appropriate evaluation system for type B trichothecenes.

Molecular characterization of a novel hepatitis E virus (HEV) strain obtained from a wild boar in Japan that is highly divergent from the previously recognized HEV strains.

Although a consensus classification system for hepatitis E virus (HEV) genotypes is currently unavailable, HEV variants (JBOAR135-Shiz09 and wbJOY_06) from wild boars (Sus scrofa leucomystax) have provisionally been classified into two novel genotypes (5 and 6). While performing a survey of HEV infections among 566 wild boars that were captured in Japan between January 2010 and August 2013, we found 24 boars (4.2%) with ongoing HEV infections: 13 had genotype 3 HEV, 10 had genotype 4 HEV and the remaining boar possessed a novel HEV variant (designated wbJNN_13). The entire wbJNN_13 genome comprised 7247 nucleotides excluding the poly(A) tail, and was highly divergent from known genotype 1 to 4 HEV isolates derived from humans, swine, wild boars, deer, mongoose and rabbits by 22.4-28.2%, JBOAR135-Shiz09 and wbJOY_06 by 19.6-21.9% and rat, ferret, bat and avian HEV isolates by 40.9-46.1% over the entire genome. Phylogenetic trees confirmed that wbJNN_13 is distantly related to all known HEV isolates. A Simplot analysis revealed no significant recombination among the existing HEV strains. These results indicate the presence of at least three genetic lineages of presumably boar-indigenous HEV strains. Further studies to fully understand the extent of the genomic heterogeneity of HEV variants infecting wild boars are warranted.

Regulation of metabolic products and gene expression in Fusarium asiaticum by agmatine addition.

The metabolic products resulting from the cultivation of F. asiaticum in agmatine were identified using capillary electrophoresis-time of flight mass spectrometry. Glyoxylic acid was detected from fungal cultures grown in agmatine, while it was absent in control cells. The abundance of other metabolic products of the glycolytic pathway also increased because of agmatine; however, there was no increase in the amounts of pyruvic acid or metabolites from the tricarboxylic acid cycle. Moreover, gene expression levels within Fusarium asiaticum exposed to agmatine were analyzed by DNA microarray. Changes in gene expression levels directed the changes in metabolic products. Our results suggest that acetyl coenzyme A, which is a starting substrate for the biosynthesis of deoxynivalenol (DON), was simultaneously produced by activated ß-oxidation. Furthermore, the content of 4-aminobutyrate (GABA) was increased in the agmatine addition culture medium. GABA can be synthesized from agmatine through putrescine and might influence the regulation of DON-related genes.

RNA preparation of Saccharomyces cerevisiae using the digestion method may give misleading results.

Zymolyase (lyticase) is used for cell wall digestion in yeast experiments and is needed for incubation processes under moderate experimental conditions. This has been thought to cause unfavorable effects, and many researchers are aware that the enzyme method is unsuitable for RNA preparation following several reports of stress responses to the enzyme process. However, RNA preparation with enzyme digestion continues to be used. This may be because there have been insufficient data directly comparing RNA preparation conditions with previous studies. We investigated the influence of enzyme processes in RNA preparation using a DNA microarray, and compared superoxide dismutase (SOD) activities with a non-treated control and the results of previous research. Gene expressions were commonly changed by enzyme processes, and SOD activities increased only during short-term incubation. Meanwhile, both SOD gene expressions and SOD activity during RNA preparation indicated different results than gained under conditions of long-term incubation. These results suggest that zymolyase treatment surely influences gene expressions and enzyme activity, although the effect assumed by previous studies is not necessarily in agreement with that of RNA preparation.

Comprehensive gene expression analysis of type B trichothecenes.

Type B trichothecenes, deoxynivalenol (DON) and nivalenol (NIV), are secondary metabolites of Fusarium species and are major pollutants in food and feed products. Recently, the production trend of their derivatives, 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), and 4-acetylnivalenol (4-AcNIV or fusarenon-X), has been changing in various regions worldwide. Although in vivo behavior has been reported, it is necessary to acquire more detailed information about these derivatives. Here, the yeast PDR5 mutant was used for toxicity evaluation, and the growth test revealed that DON, 15-AcDON, and 4-AcNIV had higher toxicity compared to 3-AcDON and NIV. 15-AcDON exerted the most significant gene expression changes, and cellular localization clustering exhibited repression of mitochondrial ribosomal genes. This study suggests that the toxicity trends of both DON products (DON and its derivatives) and NIV products (NIV and its derivatives) are similar to those observed in mammalian cells, with a notable toxic response to 15-AcDON.

Gene expression profiles of yeast Saccharomyces cerevisiae sod1 caused by patulin toxicity and evaluation of recovery potential of ascorbic acid.

Patulin (PAT) is a fungal secondary metabolite and exhibits various toxicities including DNA damage and oxidative stress. These toxicities are eased by ascorbic acid (AsA). Although a number of studies regarding the mitigating effect of AsA against PAT toxicity have been reported, a comprehensive study about gene expressions is currently underway. Here, we carried out a detailed evaluation of PAT toxicity by co-incubation with AsA using the superoxide dismutase (SOD) mutant. DNA microarray results extracted the alterations in iron transporter and Fe/S cluster assembly genes; some of the genes that constitute the cellular iron transporter systems remained dysfunctional even in the presence of AsA. Meanwhile, AsA treatment reduced the alterations of G1/S phase cell cycle regulation genes. These results suggest that oxidative stress-derived DNA damage still exists, although AsA treatment effectively reduces PAT toxicity. This implies that a combined condition is required for complete blockade of PAT toxicity.

Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during Sesbania rostrata stem nodule development.

A parA gene in-frame deletion mutant of Azorhizobium caulinodans ORS571 (ORS571-ΔparA) was constructed to evaluate the roles of the chromosome-partitioning gene on various bacterial traits and on the development of stem-positioned nodules. The ΔparA mutant showed a pleiomorphic cell shape phenotype and was polyploid, with differences in nucleoid sizes due to dramatic defects in chromosome partitioning. Upon inoculation of the ΔparA mutant onto the stem of Sesbania rostrata, three types of immature nodule-like structures with impaired nitrogen-fixing activity were generated. Most showed signs of bacteroid early senescence. Moreover, the ΔparA cells within the nodule-like structures exhibited multiple developmental-stage phenotypes. Since the bacA gene has been considered an indicator for bacteroid formation, we applied the expression pattern of bacA as a nodule maturity index in this study. Our data indicate that the bacA gene expression is parA dependent in symbiosis. The presence of the parA gene transcript was inversely correlated with the maturity of nodule; the transcript was switched off in fully mature bacteroids. In summary, our experimental evidence demonstrates that the parA gene not only plays crucial roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in S. rostrata stem nodules.

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571.

BACKGROUND: Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. RESULTS: The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. CONCLUSION: The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

An outer membrane autotransporter, AoaA, of Azorhizobium caulinodans is required for sustaining high N2-fixing activity of stem nodules.

In this study, we investigated the function of a putative high-molecular-weight outer membrane protein, azorhizobial outer membrane autotransporter A (AoaA), of Azorhizobium caulinodans ORS571. Sequence analysis revealed that AoaA was an autotransporter protein belonging to the type V protein secretion system. Azorhizobium caulinodans forms N(2)-fixing nodules on the stems and roots of Sesbania rostrata. The sizes of stem nodules formed by an aoaA mutant having transposon insertion within this ORF were as large as those in the wild-type strain, but the N(2)-fixing activity of the nodules by the aoaA mutant was lower than that of wild-type nodules. cDNA-amplified fragment length polymorphism and reverse transcriptase-PCR analysis revealed that the expressions of several pathogen-related genes of host plants were induced in the aoaA mutant nodules. Furthermore, exopolysaccharide production was defective in the aoaA mutant under free-living conditions. These results indicate that AoaA may have an important role in sustaining the symbiosis by suppressing plant defense responses. The exopolysaccharide production controlled by AoaA might mediate this suppression mechanism.

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis.

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.