Streptomyces cholesterol oxidase, an enzyme on the first step of microbial catabolism of cholesterol, was overproduced in a secretory fashion in Streptomyces lividans with a multi-copy shuttle vector. The gene coding for cholesterol oxidase (choA) was modified and overexpressed in Escherichia coli. The modified choA gene was also used to develop promoter-probe vectors to use in enteric bacteria. Expression of the choA gene in lactic acid bacteria might facilitate the degradation of cholesterol in dairy food. The Streptomyces cholesterol oxidase was unexpectedly found to constitute a novel class of insecticidal proteins that may be useful for controlling pests that are resistant to Bacillus thuringiensis toxins. Furthermore, the Arthrobacter simplex gene (ksdD) encoding 3-ketosteroid-Δ1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned and overexpressed in S. lividans using a multi-copy shuttle vector, leading to an about 100-fold overproduction of the enzyme compared to the natural producer. Nucleotide sequence analysis revealed that ksdD is clustered with two more genes possibly involved in steroid catabolism. Upstream of ksdD, a gene (ksdR) encoding a hypothetical regulatory protein that shows similarities to several regulators was found. Adjoining downstream to ksdD in an apparent translational coupling is a gene (ksdI) coding for a protein that would display strong similarities to 3-ketosteroid-Δ5-isomerase. Further sequence analysis downstream of ksdI revealed three ORFs. The deduced protein product of one of these showed significant similarities to the KsdD protein. Thus, genes involved in steroid metabolism seem to be clustered in bacteria.
pSN22, an 11 kb multicopy conjugative plasmid from Streptomyces nigrifaciens, promotes chromosome recombination in Streptomyces lividans. Five genes have been identified to be involved in plasmid transfer and pock formation: traB is essential for plasmid transfer; traA for pock formation; spdA and spdB are concerned with pock size; and traR, which corresponds to a kor gene in a kil-kor system, encodes a repressor of traR itself and the traA-traB-spdB (tra) operon. Studies on the interaction of TraR with promoter regions suggest that the negative regulation of transfer-related genes by TraR is achieved by two mechanisms, i.e. promoter hiding and roadblock. The predicted ATPase activity and the membrane localization of TraB suggest that the protein plays a direct role in ATP-driven DNA translocation. TraB is also thought to be involved in intra- and intermycelial transfers of pSN22.
The genes for hrdA and hrdD were isolated from a streptomycin-producing Streptomyces griseus 2247, using a probe for the ‘rpoD box’. The nucleotide sequence of the hrdA region revealed the presence of two open reading frames (ORF). One of them consisting of 1083 bp encodes a HrdA protein of 363 amino acids (Mr 40574). The predicted protein (Mr 69212) of the other ORF in the hrdA region is highly homologous to ATP-dependent transporters. The nucleotide sequence of the hrdD region was also determined. The ORF of 999 bp for hrdD encodes a putative protein of 333 amino acids with a Mr 37196 (HrdD). The deduced amino acid sequences of HrdA and HrdD possess the subregions which are conserved in the rpoD (σ70) family of eubacteria. These hrd genes of S. griseus encode the group 2 σ factors of rpoD family.
Taxonomy of a soil isolate, strain AL-23456 which produces a hematopoietic cytokine inducer TAN-1511, was studied. The organism had branched substrate mycelia and spherical sporangia including nonmotile spores on the tips of sporangiophores arising from the aerial mycelia. The chemotaxonomic characteristics of this organism are as follows: the cell wall chemotype is type III (meso-diaminopimelic acid), the whole-cell sugar pattern is type B, the fatty acid pattern is type 3c, major menaquinones are MK-9(H2) and MK-9(H4), phospholipid type is type PIV, the N-acyl type of muramic acid in the cell wall is the acetyl type. These morphological and chemotaxonomic features suggested that the isolate belongs to the genus Streptosporangium. The isolate was compared with taxonomically most similar strain, Streptosporangium amethystogenes IFO 13986 by examining cultural and physiological properties, and a DNA-DNA hybridization technique. From the results, we propose a new subspecies, S. amethystogenes subsp. fukuiense, for the isolate. The type strain is strain AL-23456 (=IFO 15365).
Kitasatosporia setae KM-6054T produces both filamentous mycelia and submerged spores (SS) in the submerged culture with shaking. SS were not produced in a chemically defined medium, MMT, but were produced when the medium was added with yeast extract or casamino acids. In MMT supplemented with casamino acids (MMT-CA), SS began to elongate within 1 hour incubation and grew up to form long filamentous mycelia after 6 hours. Spore chains were produced at the tips of the hyphal branches after 10 hours, and then short spore chains were fragmented to yield SS after 15 hours. In feeding experiments, the strain produced SS in MMT when casamino acids were added within 4 hours after inoculation, but not when added after 6 hours. In MMT-CA, additional feeding of casamino acids did not influence the initiation time of SS formation. In a nutritional shift-down experiment, the strain formed SS when casamino acids were removed from MMT-CA after 8 hours of cultivation, but not when removed within 6 hours. The strain starts to form cells committing SS already after about 8 hours under nutrient rich condition. Cells that commit SS formation started to be formed within about 8 hours incubation under nutrient-rich conditions. It seems thus likely that the SS formation of K. setae KM-6054T is not regulated with nutritional starvation, but by a clock mechanism.