Polyphenolic Constituents of the Pericarp of Mangosteen (Garcinia mangostana L.).

Three new polyphenols, together with 14 known compounds, were isolated from a hot water extract of mangosteen (Garcinia mangostana L.) pericarp, a plant that has been used medicinally in Southeast Asia. The three new polyphenols were characterized as a 4-aryl-2-flavanylbenzopyran derivative (tentatively named GM-1), 1, 3,4,3',5'-tetrahydroxy-5-methoxybenzophenone (GM-2), 2, and 2,3-dihydrochromone derivative (GM-3), 3 on the basis of NMR and MS data. The relative stereostructure of GM-1 was assigned to have 2,3-cis-3,4-trans- and 2″,3″-cis configurations on the basis of the coupling constants of heterocyclic ring protons in the (1)H NMR spectrum along with nuclear Overhauser effect correlations. The HPLC analysis indicated that major polyphenolic components in the hot water extract of mangosteen pericarp were epicatechin and procyanidin B2 (epicatechin dimer).

Isolation of a new insertion sequence, IS13655, and its application to Corynebacterium glutamicum genome mutagenesis.

A new functional Corynebacterium glutamicum insertion sequence (IS) element, IS13655, was isolated using a suicide vector. The IS element was 1,293 bp in size and contained 26-bp imperfect inverted repeats (IRs) and 3-bp target site duplication as direct repeats (DRs). IS13655 harbored two ORFs with high similarity to the transposase of IS1206, an IS3 family element. IS13655 revealed relatively high transposition efficiency, with low target site selectivity along the Corynebacterium glutamicum R genome, making it a potentially useful genetic engineering tool.

Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes.

We constructed a mesophilic anaerobic chemostat that was continuously fed with synthetic wastewater containing propionate as the sole source of carbon and energy. Steady-state conditions were achieved below the critical dilution rate of 0.3 d (-1) with almost complete substrate degradation. The propionate-degrading methanogenic communities in the chemostat at dilution rates of 0.01, 0.08, and 0.3 d (-1) were analyzed using molecular biological techniques. Fluorescence in situ hybridization with archaeal and bacterial domain-specific probes showed that archaeal cells predominated throughout the three dilution rates. Archaeal-16S rRNA gene clone library analysis and quantitative real-time polymerase chain reaction studies showed that hydrogenotrophic methanogen rRNA genes closely related to Methanoculleus was detected at a dilution rate of 0.01 d (-1) , whereas rRNA genes closely related to the Methanoculleus and Methanospirillum genera were detected at dilution rates of 0.08 and 0.3 d (-1) . The aceticlastic methanogen, Methanosaeta , was detected throughout the three dilution rates. Bacterial-rRNA gene clone library analysis and denaturing gradient gel electrophoresis demonstrated that rRNA genes affiliated with the genus Syntrophobacter predominated at the low dilution rate, whereas rRNA genes affiliated with the phylum Firmicutes predominated at the higher dilution rates. A significant number of rRNA genes affiliated with the genus Pelotomaculum were detected at dilution rate of 0.3 d (-1) . The diversity of genes encoding acetate kinase agreed closely with the results of the rRNA gene analysis. The dilution rates significantly altered the archaeal and bacterial communities in the propionate-fed chemostat.

A new insertion sequence, IS14999, from Corynebacterium glutamicum.

A new insertion sequence from Corynebacterium glutamicum ATCC 14999 was isolated and characterized. This IS element, designated IS14999, comprised a 1149 bp nucleotide sequence with 22 bp imperfect terminal inverted repeats. IS14999 carries a single open reading frame of 345 amino acids encoding a putative transposase that appears to have partial homology to IS642, an IS630/Tc1 superfamily element, at the C-terminal region in the amino acid sequence. This indicated that IS14999 belonged to the IS630/Tc1 superfamily, which was first identified in C. glutamicum. IS14999 has a unique distance of 38 amino acid residues between the second and third amino acids in the DDE motif, which is well known as the catalytic centre of transposase. This suggested that IS14999 constituted a new subfamily of the IS630/Tc1 superfamily. A phylogenetic tree constructed on the basis of amino acid sequences of transposases revealed that this new transposable element was more similar to eukaryotic Tc1/mariner family elements than to prokaryotic IS630 family elements. Added to the fact that IS14999 was present in only a few C. glutamicum strains, this implies that IS14999 was probably acquired by a recent lateral transfer event from eukaryotic cells. Analysis of the insertion site in C. glutamicum R revealed that IS14999 appeared to transpose at random and always caused a target duplication of a 5'-TA-3' dinucleotide upon insertion, like the other IS630/Tc1 family elements. These findings indicated that IS14999 could be a powerful tool for genetic manipulation of corynebacteria and related species.

Effect of dilution rate on structure of a mesophilic acetate-degrading methanogenic community during continuous cultivation.

The community structures of two mesophilic acetate-degrading methanogenic consortia enriched at dilution rates of 0.025 and 0.6 d(-1) were analyzed by fluorescence in situ hybridization (FISH) and phylogenetic analyses based on 16S rDNA clonal sequences and quantitative real-time polymerase chain reaction (PCR). FISH experiments with archaeal and bacterial domain-specific probes showed that archaeal cells were predominant and only a small number of bacterial cells were detected at both dilution rates. In the domain Archaea, the number of cells closely related to Methanosarcina barkeri was shown to be greater at the high dilution rate using FISH with species-specific probes. Taxonomic analyses based on rDNA clonal sequences obtained at the low and high dilution rates showed that 43% of 100 clones and 72% of 92 clones, respectively, were affiliated with the domain Archaea and the remainders at each dilution rate were affiliated with the domain Bacteria. Within the domain Archaea, all rDNA clones at both dilution rates were affiliated with the genera Methanosaeta or Methanosarcina of the aceticlastic methanogens. Within the domain Bacteria, the rDNA clones obtained at the low dilution rate were affiliated with four phyla, Firmicutes (36%), Bacteroidetes (9%), Chloroflexi (6%) and candidate division OP12 (5%). The rDNA clones obtained at the high dilution rate were affiliated with four phyla, Firmicutes (16%), Bacteroidetes (8%), Proteobacteria (1%) and candidate division OP12 (3%). Real-time quantitative PCR experiments showed that the number of rDNA sequences affiliated with the genus Methanosarcina was greater at the high dilution rate. In addition, a significant number of rDNA sequences affiliated with the genus Methanoculleus were detected only at the low dilution rate. Detection of a hydrogenotrophic methanogen at the low dilution rate suggests that the syntrophic acetate oxidation by hydrogenotrophic methanogens and acetate-oxidizing bacteria could occur at the low dilution rate.