The cholesterol oxidase gene (choA) of Streptomyces sp. was cloned into S. lividans. The cloned cells of S. lividans produced several fold more cholesterol oxidase extracellularly than did the original producing strain. We determined the entire nucleotide sequence of the choA gene and found an open reading frame encodes a mature cholesterol oxidase of 504 amino acids with a Mr of 54,913. The signal sequence extends over 42 amino acids. Regions of homology with flavoproteins were identified in the cholesterol oxidase. The promoter for choA exists about 1.3 kb upstream from choA. Thus, we sequenced the fragment between the promoter and the choA gene and we found another open reading frame (381 amino acids, Mr 41668) which has a high degree of homology to the Pseudomonas P-450 CAM and conserves the HR2 region in cytochrome P-450s. The mRNA start sites and sizes of their transcripts were determined. Here, we proposed a new operon model concerned with a sterol metabolism in Streptomyces.
A new method was developed to isolate actinomycetes from soil more selectively and more thoroughly. Elements of the procedure are (1) treatment of soil suspension with a solution containing yeast extract (YE) 6% and sodium dodecyl sulfate (SDS) 0.05%, at 40°C for 20 min, (2) subsequent dilution with water and (3) a few week’s incubation on HV agar plates containing nalidixic acid (n.a.) 20 mg/L. The YE and the heat shock at 40°C activated spore germination of a variety of actinomycete strains, the SDS acted as a germicide only on bacterial cells with rare exception, and the n.a. suppressed growth of bacterial spore formers without any effect on the actinomycetes, under the conditions used. By the treatment of soil suspension with YE and SDS, the count of actinomycete cfu per g. of various soils (10 samples) was increased by 40% and the count of bacteria was decreased to 20%, on the averages. The bacterial count was further decreased to less than 10% by addition of n.a. into the isolation medium.
Numerous streptomycete strains were isolated from soil samples collected from several extreme environments in Yunnan, China, in recent years. These strains are alkalophiles, acidophiles, thermophiles or psychrophiles. Some of them were compared with Streptomyces from common environments and 8 other genera of Actinomycetales with cell wall types I, II, III, and IV. In total, 73 strains were studied. The relationships of these strains using 66 unit characters were analysed by Ssm coefficient and clustering by UPGMA algorithm. Cluster thermophile, cluster psychrophile, cluster alkalophile and cluster acidophile were distinguished at the 67% Ssm similarity level. The authors proposed that thermophilic (growth at 65°C), psychrophilic (optimum growth at 10 to 15°C, growth at 0°C, no growth at 28°C), acidophilic (optimum growth at pH 3 to 4) and alkalophilic (optimum growth at pH 10) streptomycetes should be regarded as independent species-groups. When a numerical classification of Streptomyces is made, growth temperatures and optimum pHs for strains can be selected as diagnostically valuable characters for clustering.
This paper is an outline of initial idea and experimental process of our research which has been carried out in our laboratory of National Institute of Health, Japan, for 25 years from 1964 to 1988. The research is divided into 4 fields. 1. On the basis of DNA homology, the determination of systematic relation among 30 species of Streptomyces and the evaluation of taxonomic criteria for species identification. In this study, method for the determination of DNA homology, DNA homology of 30 species in a so-called Griseus group, their systematic relation, and evaluation of species-identifying character were described. 2. Basic technology for introduction of actinophage DNA into streptomycete protoplasts and regeneration of the protoplasts to mycelial form. 3. Involvement of plasmid genes in antibiotic production. The contents of this research were concerned in the suggestive phenomena of plasmid involvement in antibiotic production, the physical detection of the flower-shape DNA suggesting a type of plasmids, the genetical evidence of plasmid involvement in chloramphenicol production, the genetic mapping of chloramphenicol biosynthetic genes and the role of plasmid genes in its production, the isolation of ccDNA from 8 species of Streptomyces including S. kasugaensis, and the function of plasmid genes in aureothricin and kasugamycin production. 4. Development of host-vector systems in Streptomyces. Pathogenicity, survival activity of DNase and restriction enzyme activity of S. kasugaensis as a host, the preparation of mec- mutant as a more safety host, the nucleotide sequence of the mec+ gene, the cloning of extracellular xylanase gene and determination of its nucleotide sequence, the construction of secretion vectors using its xylanase gene, and the mutual relation of three pock-forming plasmids resident in S. noursei were described and discussed.
The study of differentiation in Streptomyces has been an important topic, especially in industrial microbiology, because the genus produces numerous antibiotics whose production is known to be linked to differentiation of the producing organisms. By isolating and analysing the relaxed mutants of Streptomyces spp., I have stressed the significance of the stringent response for the initiation of both differentiation and secondary metabolism of this genus. It is concluded that morphological differentiation results from a decrease in the pool of GTP, whereas antibiotic production results from a more direct function of the rel gene product (ppGpp). A-factor may render the cell sensitive to receive and respond to the specified signal molecule, presumably ppGpp (for antibiotic production), or GTP (for morphological differentiation).
Institute of Food Microbiology, Chiba University, was established in 1946 and reorganized into Research Institute for Chemobiodynamics in 1973. The institute ceased its existence in 1987. Research work on actinomycetes, nocardia and related organisms in particular, carried out in these institutes was briefly reviewed. As early as in 1963, infrared spectrometry was successfully introduced to characterize actinomycetes more precisely. Four regions in the whole cell infrared spectrum were selected and they were classified into five characteristic absorption patterns. The spectra of N. madurae, S. pelletieri, S. somaliensis and S. paraguayensis showed the spectra which were observed neither with the strains of Nocardia nor Streptomyces. Later Lechvalier et al. proposed a generic name Actinomadura to place these actinomycetes. Diagnostic studies of the agents of nocardiosis and mycetomas had also been performed. Chemical soil stabilization has been applied to a wide variety of soils and permitted the building of roadways, dams, banks, blocks and the like. Soil stabilization by acrylamide gel involves the incorporation of acrylamide in form of the monomer into naturally occurring soils and its polymerization in situ with the aid of redox catalysts. During the year 1974 to 1975, outbreaks of toxicosis characterized by equilibrium disorder were reported. It was found that the residua acrylamide monomer still present in the polymerized soil stabilizer had contaminated the wells supplying the patients’ residences with drinking water. In an attempt to develop a microbiological process for the control of the pollution, an actinomycete strain which produced a potential constitutive enzyme deaminating acrylamide monomer was isolated and named Rhodococcus amidophilus sp. nov. More lately, the susceptibility of nocardia and related organisms to chemotherapeutic agents and the mechanism of nocardial acid-fastness are being studied.