A partially constitutive mutant strain for penicillin amidase production was derived from the parent strain Bacillus megaterium ATCC 14945 by treatment with UV light. The mutant (B. megaterium KFCC 10029) showed two phenotypical changes in the mode of penicillim amidase production and in the size of cell chains. While the parent strain produced penicillin amidase only in the presence of an inducer, phenylacetic acid, the mutant strain could produce the enzyme without the inducer and the enzyme titer increased three to four times as much as that in the presence of the inducer. The mutant appeared as isolated single cells or short chains on the nutrient agar medium, whereas the parent strain usually appeared as long chains of cells. The composition of media was optimized including the inducer concentration. After finding the optimal sets of operating conditions with regard to the pH adjustment and the inducer addition time in a submerged culture, we were able to increase the enzyme productivity 7-8 times that without pH control. Although the correlation between two phenotypical changes is not yet clear, the mutant strain can be used as a potent producer of penicillin amidase.
The chemical composition of bacterial protoplasmic membrane was studied using Clostridium saccharoperbutylacetonicum ATCC 13564. The membrane was prepared from the autoplasts. The purity of prepared membrane was certified by chemical analysis for the existence of diaminopimelic acid due to contaminated cell wall debris and of adenosinetriphosphatase (ATPase) activity in its preparation, and also by electron microscopic observation for the existence of contaminated cell wall debris. The membrane composition was made up of lipid (26%), protein (65%), carbohydrate (2%) and nucleic acid (2%). The lipid consisted of neutral lipid (26%), glycolipid (26%) and phospholipid (48%). The main composition of phospholipid was phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The membrane contained 22 kinds of protei s of different molecular weights.
In addition to the usual bipolar budding, the multilateral budding that developed at different lateral sites on a single cell was observed in 25 strains belonging to the apiculate yeast genera Hanseniaspora, Nadsonia, Saccharomycodes, Wickerhamia, and Kloeckera. Effects of several culture conditions on the formation of this multilateral budding were also examined. The rates of this multilateral budding were about 0.001 to 4.0%, but the rates varied with kinds of media and yeast species, and several yeasts showed multilateral budding at pH 6.0-6.5. Conidiogenesis on lateral sites, as well as bipolar budding, was of the percurrent type by basipetal succession. In addition to lemon-shaped cells, cells of various shapes resulted from this multilateral budding. Thus this multilateral budding can be supplemented as one of the diagnostic characters of the above apiculate yeast genera.
The aerobic carbon monoxide-oxidizing bacterium Comamonas compransoris is noticeable by containing a low molecular weight catalase with hyperbolic substrate saturation kinetics and high specific activity. The catalase has been purified from extracts of pyruvate-grown cells. The molecular weight is 150, 000, it consists of two identical subunits of 75, 000. The isoelectric point is 4.86. The absorption spectra of the native enzyme and of the reduced pyridine hematochrome resemble the corresponding spectra of bovine liver catalase. The catalase from Comamonas compransoris apparently contains two heme IX-groups per molecule. The substrate (H2O2)-saturation curve shows Michaelis-Menten kinetics with a Km of 1.93mM and a turn-over number of 1.4× 106/min. The pH optimum is 6.5, the dependence on temperature is weak. Imidazole increases the catalatic activity. The catalase peroxidizes pyrogallol, catechol and guajacol. In an open system with continuous substrate flow the enzyme is quickly inactivated.
A new menaquinone homologue was isolated from a nocardioform bacterium, Actinomadura madurae. The isolated compound had ultraviolet and infrared absorption spectra very similar to those of menaquinone. The mass spectrum of the compound showed the molecular ion peak at m/z 790 with the coexistence of two intense fragment ion peaks at m/z 225 and 187. However, two weak fragment ion peaks were not found at m/z 721(M-69) and 653 (M-69-68) due to the successive loss of isoprene units. The 1H-NMR spectrum represented the signals of the following groups: 4×φ-H, δ 8.02-7.60; 6×-CH=C-, δ 5.04; Q-CH2-, δ 3.32 (doublet, J=6.7Hz); Q-CH3, δ 2.16; 10×=C-CH2-, δ 1.98; =C-CH3 (trans, 1st unit from quinone ring), δ 1.78; =C-CH3 (cis, 9th unit), δ 1.66; 5×=C-CH3 (trans, internal and 9th units), δ 1.57; 3×-CH2-CH2-CH-CH2-, δ 1.28; 3×-C-CH3, δ 0.90 (φ, benzene ring; Q, quinone ring). The chromenyl acetate was synthesized from the menaquinone, and was subjected to ozonolysis. The ozonolysis product had a mass spectrum showing ion peaks at m/z 464 (M+), 449 (M-15), 422 (M-42), 407 (M-15-42), 267, and 225. The results obtained indicated that the isolated menaquinone is formulated as 2-methyl-3-II, III, VIII-hexahydromultiprenyl9-1, 4-naphthoquinone.
Fundamental investigations were made on the oxidation of pyrite and consequent dissolution of iron by pure and mixed cultures of Thiobacillusferrooxidans and Thiobacillus thiooxidans. The release of iron from pyrite was remarkably enhanced with large inocula above ca. 109 cells per flask (or 1% pulp density) of 2-day (active) cultures of T. ferrooxidans, but not with inoculum of 108 cells or less. Furthermore, a phenomenon was observed that the enhanced oxidation of pyrite always proceeded with the coexistence of a 108 cells or less inoculum of T. ferrooxidans and T. thiooxidans incapable of oxidizing pyrite. During the bacterial oxidation of pyrite, high iron oxidation ratios (Fe3+/total Fe) above 90% were maintained, coincident with the enhanced release of iron from pyrite. Contrarily, in the absence of T. ferrooxidans, a major portion of iron was in the ferrous form and iron release was not promoted. Thus, it was thought that T. ferrooxidans contributes to the oxidation of pyrite through the regeneration reaction of ferric iron.
A germination mutant of Bacillus subtilis 168, deficient in response to glucose (GLC), was isolated. The mutation, tentatively named gerK, was mapped between aroI and dal. Spores of this mutant germinated normally in L-alanine (ALA) but failed to do so in L-asparagine+GLC+fructose (FRU)+K+. Deficiency of the mutant in response to GLC was evidenced by the fact that GLC was without effect in; (i) stimulating ALA-initiated germination and (ii) enhancing reversion by FRU of D-alanine inhibition. Furthermore, introduction of a gerA (temperature-sensitive) mutation into a gerK mutant by transduction gave rise to a double mutant that did not respond to ALA at 43° even in the presence of GLC. No appreciable difference in glucose dehydrogenase activity was detected between wild-type and mutant spores.
The Co-Q system was investigated in thirty-one strains of the species of the genus Trichosporon and related organisms. Three Co-Q systems comprised of Q-8, Q-9, and Q-10 were recognized in these organisms. The Q-8 system was found only in T. behrendii, a synonym of Pichiaburtonii [Endomycopsis burtonii]. The Q-9 system was distributed in T. aculeatum, T. capitatum, T. cutaneum, T. fermentans, T. penicillatum, T. pullulans, T. brassicae, T. sericeum [Endomyces ovetensis], and Geotrichumcandidum. The Q-10 system was restricted in some strains of T. cutaneum. The taxonomic significance of the Co-Q system in the genus Trichosporon and related organisms was discussed.