In this study, the cell surface charge of 109 strains of the bacteria isolated from a paddy soil, which showed a wide range of growth rates (0.7-15.1 h doubling time), were examined. As found in the case of grassland-soil bacteria previously (Morisaki, H., Kasahara, Y., and Hattori, T., J. Gen. Appl. Miclobiol., 39, 65-74, 1993), among the strains, fast-growing isolates showed a greater level of negative charge at the cell surface at pH 7.0 compared with the slow-growing isolates. To determine the pH-dependence, the cell surface charge was measured as a function of pH varying from 2.4 to 9.0. Under acidic conditions (below pH 4.0), the negative cell- surface charge became lower. At the most acidic condition (pH 2.4) 34 out of 40 strains of the fast-growing bacteria showed a positive surface charge. This change in the cell surface charge caused by pH-shift from neutral to acidic condition, which was considered to be because charged functional groups (weak acids such as carboxylic acid) existing or exposed at the cell surface lost their negative charge, was greater for the fast-growing isolates than that for the slow-growing ones.
Decreasing the NaCl concentration from 3 to 0.5% or lower in the growth medium caused a drastic change in the ratio of the two outer membrane proteins b and b′ with respective apparent molecular weights of about 36, 000 and 35, 000: the amounts of proteins b and b′ decreased and increased, respectively. However supplementation of the growth medium with high concentrations of sucrose did not affect the synthesis of the two proteins. Both proteins were not solubilized with 2% sodium dodecyl sulfate at 50°C for 30min and resistant to trypsin, indicating that they are porin-like proteins. Their peptide fragments created by limited proteolysis with Staphylococcus aureus V8 protease, their amino acid compositions and their antigenecity differed from each other. Only one major porin-like protein, b′, was formed in the cells grown on 0.2% NaCl-nutrient broth, and pentose and hexose diffused through the outer membrane freely but di-, tri-, tetra- and pentasaccharides did not. On the other hand, when the cells were grown in synthetic medium which substantially contain protein b as the only one major porin-like protein, pentose and hexose penetrate the outer membrane freely, di- and tri- saccharides diffused partly but tetra- and pentasaccarides did not.
Thiobacillus ferrooxidans JCM 7811 was cultivated in a basal salts medium containing thiosulfate and ferric ion (Fe3+). T. ferrooxidans grew with consuming thiosulfate. Appropriate concentrations of thiosulfate and Fe3+ were 20 and 30mM, respectively. When o-phenanthroline, which chelates ferrous ion (Fe2+), was added to the culture, Fe2+ reduced from Fe3+ was gradually accumulated. In the cultures with 25 and 50mM of o-phenanthroline, the bacterial growth and thiosulfate consumption were inhibited. These results indicated that thiosulfate was oxidized through the sulfur oxidation route with a ferric ion-reducing (FIR) system. During 5 days of cultivation, the cells grew up to 5.0×108 cells/ml, and the grown cells had the iron oxidizing activity of 2.2μmol Fe2+/mg protein; min. These values were comparable with those of the cells grown on ferrous sulfate. The cell yield against thiosulfate (Yx/s) was 250×108cells/mmol-S2O32-, which was about 20-fold higher than that against ferrous sulfate. Iron ions were cyclically utilized in the FIR system, therefore, the usage of ferrous sulfate could be reduced to obtain a desired cell mass.
Profiles of bacteriolytic enzyme production during the growth of Alteromonas sp. No. 8-R in nutrient broth were investigated by using polyacrylamide gel electrophoresis (PAGE) containing cells of Micrococcusluteus without SDS-treatment. Four bacteriolytic bands (a, b, c, and d) were detected in the culture supernatant and the cell at different stage of growth. Bacteriolytic bands a and c were detected in the culture supernatant in the decreasing phase. Band b was maintained in intracellular to the stationary phase and detected in the culture supernatant at the decreasing phase. Band d was detected in the supernatant from the logarithmic phase to the stationary phase and disappeared at the decreasing phase. The results suggest that the strain has at least four bacteriolytic enzymes and produces enzymes according to different processes during the growth in the nutrient broth.
When bacteria of a paddy field soil were incubated on counting plates the number of colonies increased intermittently with broadening intervals. To analyze the bacterial populations, 30 to 51 bacteria were respectively isolated from the colonies appearing in each of the incubation periods: from 22 to 30h, 31 to 51h, 52 to 114h, and 115 to 265h. The isolates from each period were divided into groups I, II, III, and IV. The generation time of bacteria of each group, grown on the liquid medium of the same composition as used for counting, increased in order from group I to group IV. Frequency distribution of generation time in each group was of L-type. Morphological and physiological characteristics of the isolates differed among the types. The percentage of oligotrophic bacteria among isolates increased sharply from group I to group IV.
Sixteen strains of cellulolytic yeasts and yeast-like microorganisms were isolated from natural materials collected in Japan by enrichment technique. Seven of them belonging to ascomycetous anamorphic yeasts, an ascosporogenous yeast and an yeast-like genus Prototheca were classified into five species, Candida cellulolytica sp. nov. (1 strain), Candida fukuyamanensis sp. nov. (1 strain), Candida krusei (3 strains), Williopsissaturnus var. saturnus (1 strain) and Prototheca zopfii (1 strain). Candidacellulolytica has Q-7 as the major ubiquinone and 41.6mol% of G+C in DNA, and is assumed to have a glucan-mannan type cell wall, and C. fukuyamanensis has Q-9 as the major ubiquinone and 47.8mol% of G+C in DNA, and is assumed to have a glucan-mannan type cell wall. Descriptions of two new Candida species are given.
Female Sprague-Dawley rats, whose plasma cholesterol level had been previously increased, were fed on a diet including 22% biomass of the marine microalga Dunaliella tertiolecta. This diet was compared to two control diets, one of casein and the other of soy flour. After 14 days of feeding, the group fed the microalgal diet exhibited the largest decrease in the plasma cholesterol concentration. The plasma cholesterol level in the group fed the microalgal diet was 45.2% of that in the control group (casein diet) and 28.4% lower than the group fed the soy flour diet. These results suggest that the marine microalga Dunaliella tertiolecta has marked anti-hypercholesterolemic activity when incorporated into the diet.
Promoter libraries of Zymomonas mobilis were generated in Escherichiacoli using the CAT promoterless vector, pKK232-8. Among the 15 clones retained for further study, quantitative analysis of promoter strength revealed a 140-fold difference between the weakest and strongest promoter fragment. Nucleotide sequence determination and primer extension analysis were used to locate possible promoter-like sequences. Inspection of the DNA sequence surrounding the mRNA start-site, revealed regions similar to the E. coli sigma 70 consensus sequence (TTGACA-17bps- TATAAT), approximately 4-8bps upstream. Analogous to E. coli upstream activators, regions high in AT content (70-80%) and rich in static DNA bands, were found preceding the -35 hexamer. Only one promoter fragment clone (Z1) was found to carry an open reading frame preceded by a sequence resembling a ribosome-binding site with a free energy of SD-anti-SD binding (ΔG°) of -12.4kcal/mol.
The present study was carried out to investigate the applicability and usefulness of the protoplast isolation and regeneration method as a new technique for artificially dedikaryotizing Lentinula edodes dikaryons. When protoplasts derived from dikaryons were incubated in a regeneration agar medium at 25°C, about 11% of them individually started regeneration into hyphae within 3 days and formed visible colonies, varying in size, after 7 days of incubation. By isolating preferentially smaller colonies out of these visible colonies, it was found that neohaplonts could be obtained at the high frequencies of 40-92% in every dikaryon sampled and that the two-component nuclear types appeared. These neohaplonts showed considerable variation of mycelial growth rates. However, robust neohaplonts exhibited no apparent change in biological properties such as colony morphology, and electophoretic zymograms of esterase and malate dehydrogenase, suggesting that they might retain the original genetic traits. From these results, it is concluded that the protoplast regeneration method for dedikaryotizing L. edodes dikaryons is more useful than previous methods such as the physical procedure and chemical treatment.