Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability

The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBM_Xyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBM_Xyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBM_EG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBM_Xyn10B-GFP were twofold higher than those of ThCBM_EG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBM_Xyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBM_Y52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBM_EG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.     

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na₂SiO₃, 600 mM Na₂MoO₄, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.     

Further Study on the Relation of Polyoxin Structure to Chitin Synthetase Inhibition

As a part of studies on the mechanism of action of antibiotics polyoxins, effects of various N-aminoacyl derivatives of polyoxin C and other polyoxins on chitin-UDP acetylglucosaminyl-transferase (EC 2.4.1.16, chitin synthetase) prepared from phytopathogenic fungus Piricularia oryzae were investigated. It was found that they inhibited the enzyme in competition with the substrate UDP-N-acetylglucosamine, Inhibitor constants, Ki, for these polyoxins were determined and the values of binding affinity, ?ΔG_bind of the inhibitors to the enzyme were calculated from the Ki values. In addition, by using these ?ΔG_bind values the values of partial binding affinity, ?Δg for the several atoms and atomic groups or the several moieties contained in the polyoxin J molecule were estimated. From the results obtained, it was concluded that the carbamoylpolyoxamic acid moiety of polyoxins helps to stabilize the polyoxin-enzyme complex through the contributions of its oxygen atom at C?1″, amino group at C?2″, hydroxyl groups at C?3″ and C?4″, aliphatic carbon chain and terminal carbamoyloxy group.

The results obtained by the kinetic investigation using various nucleotides and nucleotide sugars suggested that there was a specific binding site on the enzyme corresponding to the uridine moiety of UDP-N-acetylglucosamine, and that the pyrimidine nucleoside moiety of polyoxins was also bound to this site.     

Studies on the Low Temperature Fermentation

An attempt has been made to isolate the bacteria capable of accumulating amino acids during the growth at low temperature from various natural sources. A psychrophilic strain P 145 forming glutamic acid at 5°C was obtained and identified as a Brevibacterium sp. The bacterium grew in the range of 0° to 37°C and exhibited the optimum growth at 15°C. The bacterium was defined as a facultative psychrophile.

The strain strictly required methionine only at above 28°C; below this temperature it grew normally without the amino acid. When methionine was added thiamine and biotin stimulated the growth of this strain at 28°C.

With the Brevibacterium sp. P 145 isolated from soil, the effect of incubation temperature on the extracellular amino acid accumulation has been examined from cultural and enzymological points of view. The strain was found to accumulate l-glutamic acid up to 5.88 mg/ml and l-alanine 0.38 mg/ml at 5°C, whereas it formed 0.21 mg/ml of l-glutamic acid and 2.54 mg/ml of l-alanine at 28°C.

The accumulation of l-alanine in the medium at 28°C seemed to be related to the thiamine requirement of the strain. In the case of thiamine deficiency, l-alanine was the main product in the culture at 28°C. When the incubation temperature was abruptly shifted from 28° to 5°C or from 5° to 28°C, the amino acid accumulation was also changed to that of the final temperature. l-Alanine dehydrogenase existed even in the cells grown at 5°C but was not active at this low temperature. These results were in accord with the informations obtained from cultural experiments.     

Fatty Acid Hydroxamate Formation by Intact Cells of Micrococcus sp.

When azelaic acid was used as a sole carbon source on the growth of Micrococcus sp. which was isolated from soil, intact cells of the organism catalyzed the enzymic condensation of fatty acids with hydroxylamine. Some of the characteristics of fatty acid hydroxamate formation were investigated.

The enzyme activity was tested with azelaic acid compared to other fatty acids. Azelylhydroxamate formation was activated with the addition of reduced glutathione or 2-mercaptoethanol. The reaction was inhibited by p-chloromercuribenzoate (PCMB), ethylene diamine tetraacetate (EDTA), NaF and benzoate.     

High‐resolution prediction of leaf onset date in Japan in the 21st century under the IPCC A1B scenario

Reports indicate that leaf onset (leaf flush) of deciduous trees in cool‐temperate ecosystems is occurring earlier in the spring in response to global warming. In this study, we created two types of phenology models, one driven only by warmth (spring warming [SW] model) and another driven by both warmth and winter chilling (parallel chill [PC] model), to predict such phenomena in the Japanese Islands at high spatial resolution (500 m). We calibrated these models using leaf onset dates derived from satellite data (Terra/MODIS) and in situ temperature data derived from a dense network of ground stations Automated Meteorological Data Acquisition System. We ran the model using future climate predictions created by the Japanese Meteorological Agency's MRI‐AGCM3.1S model. In comparison to the first decade of the 2000s, our results predict that the date of leaf onset in the 2030s will advance by an average of 12 days under the SW model and 7 days under the PC model throughout the study area. The date of onset in the 2090s will advance by 26 days under the SW model and by 15 days under the PC model. The greatest impact will occur on Hokkaido (the northernmost island) and in the central mountains.     

Combining mapping of physiological quantitative trait loci and transcriptome for cold tolerance for counteracting male sterility induced by low temperatures during reproductive stage in rice

Male sterility induced by low temperatures (LTs) during the reproductive stage is a major constraint for temperate zone rice. To detect physiological quantitative trait loci (QTLs), we modeled genotypic variation in the physiological processes involved in low temperature spikelet sterility on the basis of anther length (AL), a proxy for microspore and pollen grain number per anther. The model accounted for 83% of the genotypic variation in potential AL at normal temperature and the ability to maintain AL at LT. We tested the model on 208 recombinant inbred lines of cold‐tolerant ‘Tohoku‐PL3’ (PL3) × cold‐sensitive ‘Akihikari’ (AH) for 2 years. QTLs for spikelet fertility (FRT) at LT were detected on chromosomes 5 (QTL for Cold Tolerance at Reproductive stage, qCTR5) and 12 (qCTR12). qCTR12 was annotated with the ability to maintain AL under LTs. qCTR5 was in a region shared with QTLs for culm length and heading date. Genome‐wide expression analysis showed 798 genes differentially expressed in the spikelets between the parents at LTs. Of these, 12 were near qCTR5 and 23 were near qCTR12. Gene expression analysis confirmed two candidate genes for qCTR5 (O‐methyltransferase ZRP4, Os05g0515600; beta‐1,3‐glucanase‐like protein, Os05g0535100) and one for qCTR12 (conserved hypothetical protein, Os12g0550600). Nucleotide polymorphisms (21 deletions, 2 insertions and 10 single nucleotide polymorphisms) in PL3 were found near the candidate conserved hypothetical protein (Os12g0550600) and upstream in PL3, but not in AH. Haplotype analysis revealed that this gene came from ‘Kuchum’. The combination of mapping physiological QTLs with gene expression analysis can be extended to identify other genes for abiotic stress response in cereals.