Yoshimi Ishihara

Differences in the surface membranes and water content between the vegetative cells and spores of Bacillus subtilis

Bacillus subtilis forms both vegetative cells and spores. The fluidity of the membranes in these forms was measured by using fluorescent anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH). The spores were more rigid than the vegetative cells, suggesting that the structure of the spores and vegetative cells was different. This difference was thought to be due to the structure of the cell membranes. The anisotrophy of DPH in the cell membranes of spores gave higher values at all temperatures. The anisotrophy of DPH in the cell membranes of vegetative cells was lower than that of the spores and the value depended upon the temperature. Time Domain Reflectometry (TDR) was used to measure the quantities of bound and free water in the vegetative cells and spores. The spores were dehydrated, and the amount of bound and free water in the spores was about two‐thirds of the levels in the vegetative cells. The spores have fewer sugars molecules on their cell surface membranes, but contained as much sugars within the cell. Almost 100 per cent of the vegetative cells wee absorbed toward chitin, but the spores were not absorbed toward it at all. It was felt that the surface membrane of the vegetative cell had a high mobility because it was sugar‐rich, while the surface membrane of the spore showed a lower mobility because there are fewer sugars on the outer membrane. The spores survive in high temperatures because the surface membrane of the spore is tight and has relatively few sugars. Dehydration causes the rigidity of the spores. On the other hand, the vegetative cells are sugar‐ and water‐rich, which makes them more fluid. The difference between the vegetative cells and spores is the glycosylation of their surface membranes. Copyright

Disulphide linkage in mouse ST6Gal-I: determination of linkage positions and mutant analysis

All cloned sialyltransferases from vertebrates are classified into four subfamilies and are characterized as having type II transmembrane topology. The catalytic domain has highly conserved motifs known as sialylmotifs. Besides sialylmotifs, each family has several unique conserved cysteine (Cys) residues mainly in the catalytic domain. The number and loci of conserved amino acids, however, differ with each subfamily, suggesting that the conserved Cys-residues and/or disulphide linkages they make may contribute to linkage specificity. Using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF)-mass spectrometry, the present study performed disulphide linkage analysis on soluble mouse ST6Gal-I, which has six Cys-residues. Results confirmed that there were no free Cys-residues, and all six residues contributed to disulphide linkage formation, C(139)-C(403), C(181)-C(332) and C(350)-C(361). Study of single amino acid-substituted mutants revealed that the disulphide linkage C(181)-C(332) was necessary for molecular expression of the enzyme, and that the disulphide linkage C(350)-C(361) was necessary for enzyme activity. The remaining disulphide linkage C(139)-C(403) was not necessary for enzyme expression or for activity, including substrate specificity. Crystallographic study of pig ST3Gal I has recently been reported. Interestingly, the loci of disulphide linkages in ST6Gal-I differ from those in ST3Gal I, suggesting that the linkage specificity of sialyltransferase may results from significant structural differences, including the loci of disulphide linkages.

Determination of the water necessary for survival of Bacillus subtilis vegetative cells and spores

Abstract To determine the minimal amount of water necessary for survival in Bacillus subtilis, bound and free water were measured by time domain reflectometry (TDR) and the gel—sol phase transition of bound water was measured by differential thermal analysis—thermogravimetry (DTA—TG). Both the vegetative and the spore form of Bacillus subtilis were dried at different temperatures. From the measurements, the relationship between survival of the bacteria and water can be summarized as follows. (1) The vegetative form of bacteria was still 100% viable even when 80% of bound water was removed. (2) The spore form contained only 20% of bound water, which was easily and quickly removed by drying, and resulted in loss of viability. (3) Phase transition between gel and sol phases of bound water occurred repeatedly in the dried vegetative 100%-viable bacteria, which still had 20% bound water. The bacteria were still viable after such measurements. (4) In the spore form, phase transition was not detected.

Illustrations of the value of calorimetry in biology

Calorimetry was used to measure heat generated by both living microorganisms and macroorganisms. This seemingly simple measurement can give an evaluation of the state of an organism and can differentiate between rest, sleep, under anaesthesia, activity and inactivity. The great advantage of calorimetry is that the measurement is not harmful and does not cause any damaging effect on the samples.