Microbial Diversity in Sediments Collected from the Deepest Cold-Seep Area, the Japan Trench

The Japan Trench land slope at a depth of 6,400 m is the deepest cold-seep environment with Calyptogena communities. Sediment samples from inside and beside the Calyptogena communities were collected, and the microbial diversity in the sediment samples was studied by molecular phylogenetic techniques. From DNA extracted directly from the sediment samples, 16S rDNAs were amplified by the polymerase chain reaction method. The sequences of the amplified 16S rDNAs selected by restriction fragment length polymorphism analysis were determined and compared with sequences in DNA databases. The results showed that 33 different bacterial 16S rDNA sequences from the two samples analyzed fell into similar phylogenetic categories, the α-, γ-, δ-, and ɛ-subdivisions of Proteobacteria, Cytophaga, and gram-positive bacteria; some of the 16S rDNA sequences were common to both samples. δ- and ɛ-Proteobacteria-related sequences were abundant in both sediments. These sequences are mostly related to sulfate-reducing or sulfur-reducing bacteria and epibionts, respectively. Eight different archaeal 16S rDNA sequences were cloned from the sediments. The majority of the archaeal 16S rDNA sequences clustered in Crenarchaeota and showed high similarities to marine group I archaeal rDNA. A Methanococcoides burtonii–related sequence obtained from the sediment clustered in the Euryarchaeota indicating that M. burtonii–related strains in the area of Calyptogena communities may contribute to production of methane in this environment. From these results, we propose a possible model of sulfur circulation within the microbial community and that of Calyptogena clams in the cold-seep environment. Received June 15, 1998; accepted November 10, 1998.     

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80°C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53ml/mol at 25°C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.     

Homo-d-lactic acid production from mixed sugars using xylose-assimilating operon-integrated Lactobacillus plantarum

In order to achieve efficient D-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ΔldhL1-xpk1::tkt-Δxpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of ΔldhL1-xpk1::tkt-Δxpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-D-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of D-lactic acid of 99.5%. Finally, we successfully demonstrated homo-D-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-D-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.     

Chemical and genetic study of Ligularia duciformis and related species in Sichuan and Yunnan Provinces of China

The chemical constituents of the root extracts and the evolutionarily neutral DNA base sequences were studied for 28 samples of Ligularia duciformis, L. kongkalingensis, and L. nelumbifolia collected in Sichuan and Yunnan Provinces of China. The samples could be classified into four chemotypes (1-4). Sesquiterpenoids having eremophilane and oplopane skeletons were isolated from two (Chemotype?1) and three (Chemotype?2) samples, respectively. Two new oplopane derivatives were isolated and their structures were determined. In 18 samples, phenylpropenoids were the major components (Chemotype?3). In five samples, neither phenylpropenoids nor sesquiterpenoids were found (Chemotype?4). Despite this large chemical variety, no correlation was found between the chemotype and the morphological criteria of species identification. The analysis of the evolutionarily neutral DNA regions also indicated that the samples were not separated into distinct clades and that introgression was extensive.     

A Novel Method for Detection of Endo-Xyloglucan Transferase

A new approach has been developed for quantification of theactivity of endo-xyloglucan transferase, a novel enzyme thatmediates the transfer of a segment of one xyloglucan moleculeto another xyloglucan molecule. Purified xyloglucans with definedmolecular-weight distributions and their fluorescent derivatives(pyridylamino xyloglucans) were used as substrates for the enzymaticreaction. The transferase activity was quantified by monitoringchanges in molecular-weight distributions of substrates by analkali compatible gel permeation chromatographic system, equippedwith a pulsed amperometric detector and a fluorescence detector.This new method was applied to the rapid detection and characterizationof a novel transferase derived from plant tissues. (Received February 28, 1992; Accepted September 28, 1992)     

Immunoelectron Microscopic Localization of Sperm-specific Nuclear Basic Proteins during Spermatogenesis in Anuran Amphibians

Antisera were raised in rabbits against sperm-specific nuclear basic proteins (SPs) of Bufo japonicus and Xenopus laevis . The localizations of these proteins in spermatogenic cells were then studied by electron microscopy with colloidal gold labeled antibodies as probes. The numbers of gold particles counted on ultra-thin sections of cells at various spermatogenic stages were corrected for the density per unit area, on the basis of areas determined with a digitizer. No grains were deposited during early nuclear elongation stages. Grains appeared on nuclei at the beginning of chromatin granulation, and their density increased first gradually and then sharply at the last step of spermiogenesis. Recalculation of grain counts according to the estimated nuclear volumes of Bufo spermatogenic cells also indicated a sharp increase in the amount of SPs per nucleus in the last step of spermiogenesis. No significant localization of grains in the cytoplasm was observed at any stage of spermatogenesis.     

Lateral gene transfer in the deep sea of Mariana Trench: identification of nar gene cluster encoding membrane-bound nitrate reductase from Pseudomonas sp. strain MT-1.

The genes encoding membrane-bound nitrate reductase and its locus from Pseudomonas sp. strain MT-1, which is isolated from the sediment of Mariana Trench, were identified. To some extent, the gene organization in the cluster was different from those of other Pseudomonads. Quite interestingly, two genes encoding putative nitrate transporter (narK and narM) showed higher homologies to counterparts of organisms belonging to other genera than those of Pseudomonads. Especially, narM showed no significant homology to the genes for nitrate transporter of Pseudomonads, and was homologous to those of some marine bacteria. Further, arrangements of NarL- and Fnr-binding motifs in the cluster were different from those of P. stutzeri, closely related strain with MT-1. These observations clearly indicated that lateral transfer of genes in nar gene cluster had occurred in deep sea, and it may contribute to bacterial adaptation to environment of there.