Tadao Horiuchi

Isolation and properties of bacteria capable of high rates of β-galactosidase synthesis

Abstract A procedure is described for the isolation of bacteria able to make unusually large quantities of β-galactosidase. A number of such hyper strains, as they are called, have been isolated and some have been found to make protein containing as much as 25% β-galactosidase. This enzyme in these strains appears to be identical with that in normal strains with respect to Michaelis constant, heat sensitivity, sedimentation constant, and ratio of antigenic sites to enzymic sites. The ability to make β-galactosidase at hyper rates is genetically determined in the lac region of the bacterial chromosome, and this ability can be transferred by genetic recombination.

Temperature-sensitive regulation system of prophage lambda induction

Mutants of the temperate bacteriophage λ of Escherichia coli have been isolated, which have a temperature-sensitive repression system for prophage induction. These mutants can be classified into two types: λts type I which is induced at 28°C in Penassay broth after treatment for ten minutes at 47·5°C in buffer, or at a high temperature during growth; and λts type II which is induced only when the culture is heated at a high temperature during growth. Type I and type II mutations reside in the cistron cI controlling repressor substance (immunity substance) production. The correlation between ultraviolet- and temperature-sensitivity for the induction of λts prophage has been examined. When irradiated with ultraviolet light, bacteria carrying some λts prophage were induced at lower doses than the induction dose of wild-type prophage λ . Others λts prophage retained the ultraviolet sensitivity to induction of wild-type λ . Mutants of phage λ which are more sensitive to induction by ultraviolet irradiation than the wild-type phage yet as heat-stable as the wild type were isolated. These findings indicate that although many λts prophage are induced by a small dose of ultraviolet light, the ultraviolet sensitivity is not necessarily correlated with the temperature sensitivity. Bacterial mutants which suppress the temperature-sensitive characteristic of the system of prophage λts were isolated. They also restored the ultraviolet sensitivity for the prophage induction to the level of the bacteria lysogenic for the wild-type λ . A possible mechanism of heat inactivation of repressors produced by λts prophage is discussed.

Sulfurisphaera ohwakuensis gen. nov., sp. nov., a novel extremely thermophilic acidophile of the order Sulfolobales

Three spherical thermoacidophilic archaea (strains TA-1T, TA-13, TA-14) were obtained from acidic hot springs located in Ohwaku Valley, Hakone, Japan. All the isolates are facultatively anaerobic, and grew optimally at around 85 degrees C, pH 2.0. Isolate TA-1T was characterized further. The G + C content of DNA from TA-1T is 33 mol%. Although these properties resemble those of the genus Acidianus, the sequence of the 16S rRNA gene from strain TA-1T was more similar to that of species of Stygiolobus than of Acidianus. DNA-DNA hybridization experiments also indicated that strain TA-1T is clearly distinguished phylogenetically from the members of Acidianus, Sulfolobus and Metallosphaera. On the basis of the distinct physiological and molecular properties, we describe the new strains as members of the new genus Sulfurisphaera. The type species of the genus is Sulfurisphaera ohwakuensis, and the type strain of the species is TA-1T (= IFO 15161T).

Conformational change of the lac repressor induced with the inducer

Abstract The repressor of the lac operon in Escherichia , coli undergoes a distinct conformational change upon addition of a specific inducer isopropyl-β-D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum, sedimentation coefficient and in circular dichroism spectrum. The significance of the conformational change reported here is discussed in relation to the allosteric properties of the repressor.

Lambda phage mutants insensitive to temperature-sensitive repressor: II. Genetic character of ?virC mutant

SummaryThe isolation of λ recombinant carrying virC mutation from newly isolated λ virulent carrying virL virC virR, (Horiuchi et al., 1969) was succeeded and the genetic character of virC mutation producing clear plaque was studied.λ virL virC shows weak-virulent character and produces clear plaque on λCIts lysogen but not on wild type λ lysogen. λ virC shows avirulent character and no plaque is produced on these lysogen. The virC mutation is located very closely to and on the left side of the virR region (Fig. 1) which is presumed to be the operator of the right-side operon including O and P cistrons. The genetic characters of virL, virR and virC, were compared with v1, v2, v3 mutations of classical λ vir (Jacob and Wollman, 1954) and c17 mutation of another type of λ virulent (Da Silva and Jacob, 1968). The results indicate that virL, virC or virR mutation is similar to v2, v1 or v3 mutation, respectively, and an effect of virC mutation on producing virulent character was somewhat similar to that of c17 mutation and was stronger than that of virR mutation. The length of virR regions was suggested to be smaller than one tenth of that of the CI cistron.

Studies of a thermolabile repressor

Abstract From physiological and genetic evidence, it is concluded that the repressor for β-galactosidase (EC 3.2.1.23) synthesis in a mutant bacterium, E-103, is thermolabile. The apparent inactivation of repressor is first order and irreversible; however, paradoxically, only a characteristic fraction can be inactivated at a given temperature, suggesting some heterogeneity of repressor in this strain. Following inactivation of repressor, there is a delay in reappearance of repression that is believed to be caused by the time required to establish synthesis of repressor.

Inhibition of repressor formation in the lactose system of Escherichia coli by inhibitors of protein synthesis

The synthesis of β-galactosidase has been studied in a mutant strain of Escherichia coli K12, which is induced by heat treatment in buffer to synthesize β-galactosidase upon subsequent growth at a low temperature. The re-establishment of repression after heat treatment was almost completely inhibited by chloramphenicol, puromycin or methionine starvation and to some extent by 5-methyltryptophan. These results lead us to the conclusion that protein synthesis is required for the establishment of repression. A simple explanation of these findings is that the repressor of β-galactosidase synthesis is a protein, or has a protein component. The re-establishment of repression proceeded in the absence of required uracil when RNA synthesis was reduced to 4%. This result suggests only a small amount of RNA synthesis is involved in repressor formation.

Secondary structure of the lac repressor

Studies on highly purified repressor of the lac operon in Escherichia, coli by optical rotatory dispersion and circular dichroism has led to an estimate that it contains 33–38% of α-helix and 18–27% of β-structure. Effect of urea and 2-chloroethanol on the secondary structure of the repressor was studied. Effect of sodium dodecyl sulfate on the circular dichroism of repressor is discussed in relation to dissociation of repressor into subunits.

Studies on lambda virulent mutants

SummaryLambda virC mutant, presumably an operator mutant for the operon including x, y, CII and O genes (Fig. 1), produce clearish plaque on a λ sensitive bacteria.Four revertants producing turbid plaques were isolated from λvirC and the mutational sites of which were studied. One (tw1) is located very close to and on the left side of virC34, and another (tw32) is at the almost same site of virC34. The others (tx6 and tx53) are located on the right side of virC34. λtx recombinants have been isolated and characterized. These recombinants produce very turbid plaques and the rate of the repressor formation in the presence of CIts repressor is somewhat higher than that of λ wild type. λtx develops very poorly after infection to λ sensitive cells but λCItx develops normally. λtx lysogens synthesize two to three times more λ exonuclease than the wild type λ lysogen. On a function of x region for the repressor formation and on a presence of a possible anti-repressor were discussed. The mutant tw1 might be a promotor mutation of the CI-rex operon.