The Thiobacillus thiooxidans S3 strain was grown on modified Silverman 9K medium containing thiosulfate as a sole energy source instead of sulfur. When the pH of medium was controlled at 5.0 during the cultivation, the growth rate greatly increased to 0.067h-1 and the cell yield obtained with 1% thiosulfate was 210mg/l. When cell growth declined, placing the culture in fresh medium effectively prolonged the period of linear growth and the cell concentration reached 891mg/l in 309h of cultivation. The cells obtained in the replacement culture efficiently oxidized the sulfite.
The extracellular chitinases produced by Streptomyces sp. S-84 were purified and characterized. Two chitinases, A and B, were separated from the culture filtrate by anion-exchange chromatography and gel filtration. Both of the enzymes catalyzed the degradation of the 4-methylumbelliferyl (4-MU) glycosides of N-acetylglucosamine disaccharide and trisaccharide. Chitinase A hydrolyzed 4-MU-disaccharide more rapidly than 4-MU-trisaccharide. Chitinase B had the reverse effects. Neither enzyme cleaved 4-MU-monosaccharide. Chitinase B, which accounted for more than 99% of the total activity in the culture, was characterized in greater detail. The molecular weight estimated by SDS-PAGE was 44, 000 and the Pi was 4.8. The optimum activity occurred between pH 6.3 and 6.8. The respective Km and Vmax values of chitinase B for 4-MU-disaccharide were 14μM and 42μmol/min/mg protein, and for 4-MU-trisaccharide they were 2.7μM and 66μmol/min/mg protein. Pb2+ and p-chloromercuribenzoic acid inhibited the activity. The major product of hydrolysis of colloidal chitin by chitinase B was disaccharide with trace amounts of mono- and trisaccharide. Chitinase A had somewhat different properties. Its molecular weight, estimated by SDS-PAGE, and Pi were 41, 000 and 8.3, respectively. The optimum activity was between pH 3.4 and 4.2. The respective Km and Vmax values of chitinase A for 4-MU-disaccharide were 49μM and 0.33μmol/min/mg protein, and for 4-MU-trisaccharide they were 14μM and 0.16μmol/min/mg protein. Chitinase A produced a large amount of disaccharide and a small amount of mono-, tri- and tetrasaccharide from colloidal chitin, but the tetrasaccharide disappeared after longer incubation. The patterns of hydrolysis of p-nitrophenylchitooligosaccharide (monosaccharide to tetrasaccharide) were different in the two chitinases. Monospecific antiserum raised against chitinase B inhibited the activity of chitinase B exclusively. These results showed that the strain S-84 produced 2 distinct chitinases.
Four strains of Xenorhabdus nematophilus, one strain each of "Xenorhabduspoinarii" and "Xenorhabdus bovienii" were isolated from the nematode Steinernema spp., and identified. The chemotaxonomic characteristics were studied in these six isolates and in three authentic strains containing Xenorhabdus luminescens. In X, nematophilus, "X. poinarii" and "X. bovienii, " the major cellular fatty acids were C16:0, C16:1, C18:1, and C17-cy acids. In X. luminescens, they were C16:0, Ci-15, C18:1, and C16:1 acids. The respiratory quinone system was ubiquinone-8 in all strains. The guanine plus cytosine contents of the DNA of the strains used were 43.3 to 45.2mol%. The DNA homology values were higher than 76% among five strains of X. nematophilus, including the type strain, and a strain of "X. poinarii" For these strains, "X. bovienii" showed homology values less than 46%. The values of X. nematophilus, "X. poinarii" and "X. bovienii" to X. luminescens were about 15%. The DNA homology value between X. luminescens ATCC 29999T (type strain) and X. luminescens ATCC 29304 was 51%, suggesting the heterogeneity of the species. These results indicate the synonymy of X. nematophilus and "X. poinarii, " and verify "X. bovienii" and X, luminescens as distinct species.
The partial sequences of 18S and 26S rRNAs were studied in eighteen strains of Phaffia, Cryptococcus, Candida, and Filobasidiella species. The positions determined were 1451 through 1618 (168 bases) of 18S rRNA, and 492 through 625 (134 bases), and 1686 through 1835 (150 bases) of 26S rRNA. These three determinations showed that the genus Cryptococcus is phylogenetically heterogeneous: the maximum homologies of 26S rRNA were 65-94% in the positions 492 through 625 and 69-93% in the positions 1686 through 1835, and the base differences of 18S rRNA were 0-13 in the positions 1451 through 1618. Filobasidiella neoformans, a teleomorph of the genus Cryptococcus, had partial sequences somewhat different from the species examined of the genus Cryptococcus (maximum homologies, 74-84% and 74-86%; base differences, 4-12, respectively). Candidahumicola was included in the cluster of the genus Cryptococcus. However, the base differences of the species were not so small (maximum homologies, 69-77% and 72-84%; base differences, 5-13). The genus Phaffia constituted its own cluster separate from the species of Cryptococcus, Candida, and Filobasidiella (maximum homologies, 61-70% and 71-85%; base differences, 6-16, respectively). So the genus Phaffia is retained as an independent genus.
Three strains of endospore forming, thermophilic and aerobic bacteria tentatively named JF1, JF2 and D strains were isolated from Chinese traditional koji. These strains were gram positive and belonged to the genus Bacillus. The optimal growth temperature was 55 to 65°C; and the optimal pH, 6 to 8. They produced extracellular thermostable α-amylase in media containing lactose, galactose, raffinose, ethanol or other carbohydrates. It is suggested that strains JF and D are two new species of the Bacillus genus.
Nine strains of Arxula, Stephanoascus, Endomyces, Yarrowia, Dipodascus, and Geotrichum were examined regarding the partial sequences of 18S and 26S rRNAs. In the positions 471 through 627 of 26S rRNA, the two strains examined of the genera Arxula and Stephanoascus had 99% and 98% maximum homologies, respectively. The two species of the genus Arxula, A. terrestris and A. adeninivorans had 80% maximum homology. The maximum homologies were 81-84% between the genera Arxula and Stephanoascus. Arxula, Endomyces, Yarrowia, Dipodascus, Geotrichum, and Saccharomyces species constituted their own clusters at 49-76% maximum homologies. In the positions 1685 through 1835 of 26S rRNA, the two strains examined of the genera Arxula and Stephanoascus had 2 and 0 base difference, respectively. The two species of the genus Arxula had 7-9 base differences. The base differences were 4-8 between the genera Arxula and Stephanoascus. Arxula, Endomyces, Yarrowia, Dipodascus, Geotrichum, and Saccharomyces species constituted their own clusters at 13-25 base differences. In the positions 1451 through 1618 of 18S rRNA, the two strains examined of the genera Arxula and Stephanoascus had no base difference. The two species of the genus Arxula had 2 base differences. The base differences were 2-4 between the genera Arxula and Stephanoascus. Arxula, Endomyces, Yarrowia, Dipodascus, Geotrichum, and Saccharomyces species constituted their own clusters at 5-9 base differences. The data obtained are discussed from the taxonomic and phylogenetic points of view.
Twelve strains of the Q10-equipped species of the teleomorphic yeast genus Rhodosporidium and the anamorphic yeast genus Rhodotorula were examined for the partial sequences of 18S and 26S rRNAs. In the positions 492 through 625 of 26S rRNA, the strains of R. kratochvilovae, R. paludigenum, R. diobovatum, R. sphaerocarpum, and Rh. graminis were linked at 71% or more maximum homologies to the type species of the genus Rhodosporidium (R. toruloides). Rhodosporidium dacryoidum constituted its own separate cluster (66% maximum homology). In the positions 1451 through 1618 of 18S rRNA, the strains of the above-mentioned species were divided into two clusters. The first cluster included R. kratochvilovae, R. paludigenum, R. diobovatum, R. sphaerocarpum, and Rh. graminis (within 0 or 2 base differences), and the second cluster included the only species, R. dacryoidum (11 base differences). These results indicate that R. dacryoidum should be classified in a separate new genus.