Yoshihiro Takubo

Isolation and Characterization of Outermost Layer Deficient Mutant Spores of Bacillus megaterium

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by ‘salt aggregation test,’ which would facilitate selection of such mutants.

Germination of the decoated spores of Bacillus megaterium

Decoated spores of Bacillus megaterium ATCC 12872 were prepared by extracting the inner coat components with an alkaline solution containing sodium dodecyl sulfate and dithiothreitol (SDS‐DTT) from outer coat‐deficient mutant spores, which were produced from one of the mutants isolated and named MAE‐05 by us. The decoated mutant spores germinated as well as the intact spores of the mutant and the parent, indicating that the outer and inner spore cats cannot be essential structures for the initiation of germination.

Characterization and Deposition of the Proteins in the Outermost Layer of Bacillus megaterium Spore

It was proved that three spore coat proteins of 48, 36, and 22 kDa (P48, P36, and P22) were the components of the outermost layer (OL) of Bacillus megaterium ATCC 12872 spore by analysis of the isolated OL. And it was indicated that these proteins were deposited not by disulfide bond, but by ionic and/or hydrophobic bonds on the spore. Among them, P36 and P22 were expected to be located on the very surface of the spore by immunological analysis. In the OL deficient mutant of B. megaterium ATCC 12872, MAE05, whose spore was lacking in these OL proteins and galactosamine‐6‐phosphate polymer, both P36 and P22 were present in the mother cell cytoplasm and deposited on the forespores, but they disappeared with the lysis of mother cells. An OL protein‐releasing factor having proteolytic activity was detected in the culture supernatant at the late sporulating stage of both the wild‐type and the mutant strains. But the factor could not act on the proteins of the mature spores and the forespores at t10 (tn indicates n hr after the end of exponential growth) of the wild‐type strain. Moreover, P36 and P22 were found in the spores of a revertant of MAE05 which could form galactosamine‐6‐phosphate polymer, suggesting that this sugar polymer played the role in protecting the OL proteins against the protease‐like substance after the deposition.

Appearance of Uridine 5′‐Diphospho‐N‐Acetylglucosamine‐4‐Epimerase during Sporulation of Bacillus megaterium

In a biosynthetic study of the spore coat of Bacillus megaterium ATCC 12872 spore with galactosamine phosphate as a major component of the outer coat, high‐performance liquid chromatography (HPLC) and enzyme immunoassay were applied for the measurement of UDP‐N‐acetylglucosamine‐4‐epimerase [EC 5.1.3.7] activity and the enzyme protein concentration, respectively. The new HPLC system using an ion‐pair (or anion‐exchange) column allowed us to determine successfully the enzyme activity and its application, proving that the specific activity of the enzyme in the cells increased at the later stage of sporulation. This increase in activity was parallel to the induction of enzyme protein synthesis, which was detected by sandwich enzyme immunoassay using antiserum to the purified enzyme. These results suggested that the regulation of this enzyme is at the genetic level and it plays an important role in the outer coat synthesis in the later sporulation stage of B. megaterium.

Studies on automated survey system of environmental chemicals by gas chromatography-mass spectrometry-computer III. Construction of an automated survey system and its application

Abstract An automated survey system on the chemicals in environments by a gas chromatography-mass spectrometry-computer system has been constructed of Three Dimensional Mass Chromatography, Modified Probability Based Matching method, and Self Training Interpretive and Retrieval method. And it was applied to analyze environmental materials, with interesting results.

Identification of germination gene of Bacillusmegaterium

Glucose, KNO3, proline and leucine initiate the spore germination of B. megaterium ATCC 12872, but not of B. megaterium ATCC 19213. In order to isolate the gene concerning germination of B. megaterium ATCC 12872, we constructed its gene library in plasmid vector, and introduced into B. megaterium ATCC 19213. We obtained a transformant whose spores differed from those of the wild type strain with respect to germinability. Spores of this transformant could be germinated by glucose, proline or leucine. The recombinant plasmid prepared from this transformant was found to carry 2 kilobase pairs fragment of B. megaterium ATCC 12872 DNA. This fragment may contain the gene encoding the protein which plays an important role in germination.

Studies on automated survey system of environmental chemicals by gas chromatography-mass spectrometry-computer. II: Three dimensional mass chromatography

Abstract A three dimensional mass chromatography (TDMC), which is the extended method of mass chromatography, has been developed for the analysis of environmental chemicals. A considerable performance was suggested by the combination of TDMC to gas chromatography-mass spectrometry-computer system.

Studies on automated survey system of environmental chemicals by gas chromatography-mass spectrometry-computer I. Retrieval system of mass spectra

Abstract A retrieval system of mass spectra based upon the Probability Based Matching method was studied in order to apply it to survey the chemicals in environments by a gas chromatography-mass spectrometry-computer system. As retrieval indices, peak missing probability and relative confidence value were proposed.

Comparison of biodegradation of aniline in two rivers

Samples of water from two rivers in Osaka, one of which is considered to be unpolluted and the other moderately polluted by man made chemicals were shown to have different capabilities in the biodegradation of aniline. Microorganisms in the unpolluted Mino River water were not able to biodegrade aniline, whereas those in the moderately polluted Ina River water could do so. The microflora differed significantly in the two rivers. The MPN counts of the aniline degrading microorganisms of these two river waters also indicated less chance of aniline biodegradation in the Mino River. This would suggest that aniline degradation in river waters may reveal many facts on degree of adaptation to and alteration of natural populations under pollutant stress.

Isolation of the saprophytic strain of MC-3 and participation of the cell surface structure in predation.

From a predatory bacterium, MC‐3, a mutant strain which lost predation ability was isolated by chance selection. Biological properties of the mutant were the same as the parent except only saprophytic property. Properties of the parent and the mutant strains of MC‐3, such as bacteriolytic activity of the culture supernatant, digestion of peptidoglycan of the host bacteria, and growth by utilizing the host cells or their cytoplasmic substances, suggested that cell surface structure of the host cell plays an important role in predation and host specificity.