Biogas production using anaerobic groundwater containing a subterranean microbial community associated with the accretionary prism

In a deep aquifer associated with an accretionary prism, significant methane (CH₄) is produced by a subterranean microbial community. Here, we developed bioreactors for producing CH₄ and hydrogen (H₂) using anaerobic groundwater collected from the deep aquifer. To generate CH₄, the anaerobic groundwater amended with organic substrates was incubated in the bioreactor. At first, H₂ was detected and accumulated in the gas phase of the bioreactor. After the H₂ decreased, rapid CH₄ production was observed. Phylogenetic analysis targeting 16S rRNA genes revealed that the H₂-producing fermentative bacterium and hydrogenotrophic methanogen were predominant in the reactor. The results suggested that syntrophic biodegradation of organic substrates by the H₂-producing fermentative bacterium and the hydrogenotrophic methanogen contributed to the CH₄ production. For H₂ production, the anaerobic groundwater, amended with organic substrates and an inhibitor of methanogens (2-bromoethanesulfonate), was incubated in a bioreactor. After incubation for 24 h, H₂ was detected from the gas phase of the bioreactor and accumulated. Bacterial 16S rRNA gene analysis suggested the dominance of the H₂-producing fermentative bacterium in the reactor. Our study demonstrated a simple and rapid CH₄ and H₂ production utilizing anaerobic groundwater containing an active subterranean microbial community.     

Floral Reversion in Arabidopsis suecica Is Correlated with the Onset of Flowering and Meristem Transitioning

Angiosperm flowers are usually determinate structures that may produce seeds. In some species, flowers can revert from committed flower development back to an earlier developmental phase in a process called floral reversion. The allopolyploid Arabidopsis suecica displays photoperiod-dependent floral reversion in a subset of its flowers, yet little is known about the environmental conditions enhancing this phenotype, or the morphological processes leading to reversion. We have used light and electron microscopy to further describe this phenomenon. Additionally, we have further studied the phenology of flowering and floral reversion in A. suecica. In this study we confirm and expand upon our previous findings that floral reversion in the allopolyploid A. suecica is photoperiod-dependent, and show that its frequency is correlated with the timing for the onset of flowering. Our results also suggest that floral reversion in A. suecica displays natural variation in its penetrance between geographic populations of A. suecica.     

Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria

The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost.     

Morroniside cinnamic acid conjugate as an anti-inflammatory agent

A morroniside cinnamic acid conjugate was prepared and evaluated on E-selectin mediated cell–cell adhesion as an important role in inflammatory processes. 7-O-Cinnamoylmorroniside exhibited excellent anti-inflammatory activity (IC₅₀ = 49.3 μM) by inhibiting the expression of E-selectin; further, it was more active than another cinnamic-acid-conjugated iridoid glycoside (harpagoside; IC₅₀ = 88.2 μM), 7-O-methylmorroniside, and morroniside itself. As a result, 7-O-cinnamoylmorroniside was observed to be a potent inhibitor of TNF-α-induced E-selectin expression.     

A Clostridium perfringens hem gene cluster contains a cysG(B) homologue that is involved in cobalamin biosynthesis

The hem gene cluster, which consists of hemA, cysG(B), hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl-tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N-terminal region of C. perfringens HemD is homologous to those reported for the C-terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C. perfringens CysG(B) is a predicted 220-residue protein which shows homology to the N-terminal region of S. typhimurium CysG. Disruption of the cysG(B) gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12-deficient medium, the mutant strain showed a four-fold increase in its doubling time compared with that of the wild-type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysG(B) is involved in the chelation of cobalt to precorrin II as suggested for the CysG(B) domain of S. typhimurium CysG, enabling the synthesis of cobalamin.     

High-performance liquid chromatographic determination of phospholipid peroxidation products of rat liver after carbon tetrachloride administration

A method to detect and determine phospholipid peroxidation products in a biological system was developed using reversed-phase high performance liquid chromatography and normal-phase HPLC. Reversed-phase HPLC could separate phosphatidylcholine (PC) hydroperoxides and phosphatidylethanolamine (PE) hydroperoxides of rat liver from the respective phospholipids. A linear relationship was observed between these hydroperoxides and their peak areas on the chromatogram. In the experiment with rats administered CCl4, reversed-phase HPLC gave prominent, large peaks attributable to the peroxidation of phospholipids, and the peroxide level of the liver phospholipids was tentatively determined. Normal-phase HPLC analysis confirmed that both PC and PE in the liver phospholipids were peroxidized after CCl4 treatment. Neither the thiobarbituric acid value of the liver homogenate nor the fatty acid composition of the liver phospholipid fraction showed any significant difference between CCl4-treated and control rats. It is concluded that normal-phase HPLC and reversed-phase HPLC can complement each other to serve as a direct and sensitive method for the determination of lipid peroxide levels in a biological source. However, it was difficult to distinguish phospholipid hydroperoxides from their hydroxy derivatives.     

Membrane-bound, 2-keto-D-gluconate-yielding D-gluconate dehydrogenase from "Gluconobacter dioxyacetonicus" IFO 3271: molecular properties and gene disruption

Most Gluconobacter species produce and accumulate 2-keto-d-gluconate (2KGA) and 5KGA simultaneously from d-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from "Gluconobacter dioxyacetonicus" IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-delta-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, d-arabitol, and d-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that "G. dioxyacetonicus" IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.     

Inhibitiory effects of gold(III) ions on ribonuclease and deoxyribonuclease

Inhibitory effects of gold(III) ions (Au(III)) on ribonuclease A (RNase A) and deoxyribonuclease I (DNase I) were investigated at neutral pH. RNase A was completely inhibited by 3 molar equivalents of Au(III) ions. DNase I was inhibited by 10 molar equivalents of Au(III) ions. Stoichiometric analyses suggest that Au(III) ions were coordinated to RNase A molecules. The Au(III)-inhibited RNase A and DNase I were renatured to exhibit 80% and 60% of their intrinsic activity, when the bound Au(III) ions were eliminated from the nucleases by addition of thiourea, which forms a strong complex with gold ions. This suggests that RNase A and DNase I were not oxidized to lose their activity, but reversibly complexed with Au(III) ions to lose their activity. Au(III) ions were probably considered to be bound to histidine and methionine residues in the nucleases, resulting in the inhibition of their activity. CD spectra revealed that the Au(III)-induced inhibition caused a conformational change in RNase A molecules and that the addition of thiourea induced refolding of the Au(III)-inhibited RNase A.