Characterization of a facultatively psychrophilic bacterium, vibrio rumoiensis sp. nov., that exhibits high catalase activity

A novel facultatively psychrophilic bacterium, strain S-1, which exhibits extraordinarily high catalase activity was isolated from the drain pool of a fish product processing plant that uses H2O2 as a bleaching and microbicidal agent. The catalase activity of the isolate was 1 or 2 orders of magnitude higher than those of Corynebacterium glutamicum, Staphylococcus aureus, Pseudomonas fluorescens, and five other species tested in this study. The strain seemed to possess only one kind of catalase, according to the results of polyacrylamide gel electrophoresis of the cell extract. The optimum temperature for catalase activity was about 30 degreesC, which was about 20 degreesC lower than that for bovine catalase activity. Electron microscopic observation revealed that the surface of the microorganism was covered by blebs. Although the isolate was nonflagellated, its taxonomic position on the basis of physiological and biochemical characteristics and analysis of 16S rRNA sequence and DNA-DNA relatedness data indicated that strain S-1 is a new species belonging to the genus Vibrio. Accordingly, we propose the name Vibrio rumoiensis. The type strain is S-1 (FERM P-14531).

Gene cloning and expression of the catalase from the hydrogen peroxide-resistant bacterium Vibrio rumoiensis S-1 and its subcellular localization.

The catalase gene, vktA, of the hydrogen peroxide-resistant bacterium Vibrio rumoiensis S-1 has been isolated and sequenced. Plasmid pBSsa1 was obtained by genome library screening and it complemented a catalase-less mutant, Escherichia coli strain UM2, indicating that pBSsa1 contained the catalase gene (vktA). The vktA gene consisted of an open reading frame of 1530 bp encoding a 508 amino-acid protein with a calculated size of 57657.79 Da. The deduced amino acid sequence showed high homology with that of bacterial group III catalases. Investigation of the subcellular catalase localization using an immuno-electron microscopical technique revealed that a larger amount of VktA catalase is present in the periplasmic space than in the cytoplasm. The periplasmic space of this bacterium can therefore be regarded as a reservoir of VktA catalase in V. rumoiensis S-1.

Lytic activity and biochemical properties of lysozyme in the coelomic fluid of the sea urchin strongylocentrotus intermedius

Lysozyme was identified in the coelomic fluid including coelomocytes of the sea urchin Strongylocentrotus intermedius, and its lytic activity and biochemical properties were examined in this study. The urchin lysozyme was electrophoretically fractionated to a single lytic band of about 14 kDa. No distinct difference in the lytic activity of this enzyme was found between urchins held at two temperatures, 11 degrees and 25 degrees C. The lysozyme of this species was purified through several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with a Biogel column, and an affinity chromatography with a heparin Sepharose column. The combination method of Biogel filtration and affinity chromatography resulted in the most purified lysozyme fraction, but we could not obtain a single protein band in SDS-PAGE. In addition, anti-hen egg white lysozyme (HEWL) antibody was produced and confirmed to react specifically with the urchin lysozyme in this study. Therefore, the HEWL antibody may be available for examining the lytic activity of lysozyme at an individual level to determine the biodefense activity of sea urchins. Copyright 1999 Academic Press.

Image area extraction of biological objects from a thin section image by statistical texture analysis.

The statistical method in texture image analysis was applied to area extraction of biological objects from thin section electron microscope images. Four standard estimators defined from the gray level co-occurrence matrix, called 'inverse difference moment,' 'angular second moment,' 'entropy' and 'contrast,' were especially examined using a test pattern consisting of 6 artificial textures and to practical biological thin section images. In the examination on the test pattern, among the estimators the 'contrast' discriminated all textures, but differences of some texture feature levels were small. To clearly discriminate textures, a modified estimator combining 'angular second moment' and 'contrast' was devised. As a result it discriminated all better than the 'contrast.' Electron microscope images used for the image processing are yeast morphological ones showing spherical autophagic bodies in the vacuole. Although the four standard estimators discriminated many organelles, they could not exactly extract images of the autophagic body. However, the modified estimator was able to extract all autophagic bodies from the vacuole image area except for minor points, some of which were not clearly detected by man observation. It as found out that the texture analysis-based method can be used to discriminate slight differences between image textures due to spatial staining granularity.

Metabolism of glycosylphosphatidylinositol-anchored proteins in Arabidopsis.

Although glycosylphosphatidylinositol (GPI)-anchored proteins have now been found in several plants, very little is known regarding their metabolism there. This report describes studies of the biosynthesis and turnover of arabinogalactan proteins, a class of abundant GPI-anchored proteins secreted by cultured Arabidopsis cells.

An auto-tuning method for focusing and astigmatism correction in HAADF-STEM, based on the image contrast transfer function.

An auto-tuning method for high-angle annular detector dark field scanning transmission electron microscopy (HAADF-STEM) is proposed which corrects the defocus to the optimum Scherzer focus and compensates the astigmatism. Because the method is based on the image contrast transfer function formulated for the HAADF-STEM, the defocus and the astigmatism are accurately measured from input of two different defocus images. The method is designed to work independent of object function in the linear imaging model by analysing the spectral ratio between two Fourier spectra of their images, which is useful for cases where the spectrum of object function is not uniformly spread out over the reciprocal space. The method was preliminarily tested in a Hitachi HD-2000 STEM, and successful results of the auto-tunings from the viewpoint of verification of the algorithm were obtained using general specimens of Au fine particles and a thin section of a semiconductor device.

A mechanism of resistance to hydrogen peroxide in Vibrio rumoiensis S-1.

A possible mechanism of resistance to hydrogen peroxide (H2O2) in Vibrio rumoiensis, isolated from the H2O2-rich drain pool of a fish processing plant, was examined. When V. rumoiensis cells were inoculated into medium containing either 5 mM or no H2O2, they grew in similar manners. A spontaneous mutant strain, S-4, derived from V. rumoiensis and lacking catalase activity did not grow at all in the presence of 5 mM H2O2. These results suggest that catalase is inevitably involved in the resistance and survival of V. rumoiensis in the presence of H2O2. Catalase activity was constitutively present in V. rumoiensis cells grown in the absence of H2O2, and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of Escherichia coli. The presence of the HP II-type catalase in V. rumoiensis cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism. A notable difference between V. rumoiensis and E. coli is that catalase is accumulated at very high levels ( approximately 2% of the total soluble proteins) in V. rumoiensis, in contrast to the case for E. coli. When V. rumoiensis cells which had been exposed to 5 mM H2O2 were centrifuged, most intracellular proteins, including catalase, were recovered in the medium. On the other hand, when V. rumoiensis cells were grown on plates containing various concentrations of H2O2, individual cells had a colony-forming ability inferior to those of E. coli, Bacillus subtilis, and Vibrio parahaemolyticus. Thus, it is suggested that when V. rumoiensis cells are exposed to high concentrations of H2O2, most cells will immediately be broken by H2O2. In addition, the cells which have had little or no damage will start to grow in a medium where almost all H2O2 has been decomposed by the catalase released from broken cells.

Purification and characterization of a catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1(T) exhibiting high catalase activity.

Catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1(T), which was isolated from an environment exposed to H(2)O(2) and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U. mg of protein(-1) under standard reaction conditions at 40 degrees C, 1.5 and 4.3 times faster, respectively, than those of the Micrococcus luteus and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1(T) is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1(T) catalase and M. luteus and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1(T) should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1(T) is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.