Cloning and expression of a gene encoding N-glycosyltransferase (ngt) from Saccarothrix aerocolonigenes ATCC39243.

In the course of our bioconversion studies on the derivatives of an indolocarbazole, J-104303, Saccharothrix aerocolonigenes ATCC39243 was found to convert J-104303, which was added into the culture medium, to its glycosylated derivative, J-109384. In order to clone the gene having the ability to convert J-104303 to J-109384, a library of Saccharothrix aerocolonigenes ATCC39243 DNA fragments was constructed using Streptomyces lividans TK21 and pIJ702 as host strain and vector, respectively. By examining more than 5,000 transformants, one was found to convert J-104303 to J-109384. Sequence analysis of the inserted DNA fragment revealed an open reading frame with 1,245 base pairs, named ngt. The transformant containing this ngt gene was also found to introduce a D-glucose moiety into 6-N-methylarcyriaflavin C. Furthermore, when ngt was introduced into Streptomyces mobaraensis BA13793, a producer of J-104303, the resulting transformant produced J-109384 directly.

Intergenus protoplast fusion between Streptomyces and Micromonospora with reference to the distribution of parental characteristics in the fusants.

The protoplasts of two strains of Micromonospora which were sensitive to kanamycin (KM(s)) and utilized raffinose (Raf+), and one strain of Streptomyces griseus which was resistant to KM (KM(r)) and did not utilize raffinose (Raf-), were prepared, mixed in the presence of polyethylene glycol (PEG) and incubated on regeneration agar plates. Recombinant colonies showing KM(r)*Raf+ were obtained at a frequency of 2 x 10(-6). Their recombinants displayed a significant exchange of taxonomic characteristics between the two genera, although the majority appeared similar to the parent Micromonospora in their morphology as well as growth at 40 degrees C. Their patterns of utilization of carbohydrates, amino acids and diammonium hydrogenphosphate were different from those of the Micromonospora. Intermediate or novel types which different from their parents in their tolerance to NaCl and sensitivity to aminoglycoside antibiotics were also observed. Out of the 31 fusants obtained, two showed antimicrobial activity against Bacillus subtilis PCI 219, without any activity against Escherichia coli K-12 or Candida albicans 3147. The active substance may be a newly formed antibiotic, different from streptomycin in S. griseus.

Mechanism of multiple aminoglycoside resistance of kasugamycin-producing Streptomyces kasugaensis MB273: involvement of two types of acetyltransferases in resistance to astromicin group antibiotics.

The biochemical basis for the multiple resistance to aminoglycoside antibiotics (AGs) of kasugamycin-producing Streptomyces kasugaensis MB273 was studied. The strain was resistant to a wide range of deoxystreptamine (DOS)-containing AGs as well as astromicin (ASTM) group antibiotics. These AGs strongly inhibited in vitro polyU-directed polyphenylalanine-synthesis using ribosomes from the strain, while they were acetylated and inactivated by the MB273 cell free extract supplemented with acetyl-CoA. It seemed thus likely that the acetyltransferase activity played a critical role for the multiple AG resistance. The acetylation was selective to AGs with 2'-NH2, suggesting the involvement of aminoglycoside 2'-N-acetyltransferase, AAC (2'). Interestingly, the acetylation of istamycin B (ISM-B; an ASTM group AG) resulted in the formation of two different products (1-N-acetyl ISM-B and 2"-N-acetyl ISM-B) at a similar ratio. In this context, an AAC (2') gene cloned as an ISM-B resistance gene from the strain MB273 directed the conversion of ISM-B to only 1-N-acetyl ISM-B. It seemed likely that two types of AACs [AAC(2') and a novel one] were involved in the mechanism of resistance to ASTM group AGs.

New antitumor substances, BE-12406A and BE-12406B, produced by a streptomycete. I. Taxonomy, fermentation, isolation, physico-chemical and biological properties.

New antitumor substances, designated BE-12406A and BE-12406B, were isolated from the culture broth of a streptomycete, strain BA 12406. The active principles were extracted from mycelium by methanol and successively purified by silica gel column chromatography and preparative TLC. BE-12406A and BE-12406B inhibited the growth of vincristine-resistant or doxorubicin-resistant P388 murine leukemia cell lines as well as their parent sensitive cell line. In in vivo experiments, BE-12406A inhibited the growth of S-180 murine ascites tumor.

New antitumor substances, BE-12406A and BE-12406B, produced by a streptomycete. II. Structure determination.

The structure of BE-12406A and BE-12406B, which were isolated from the culture broth of a streptomycete as antitumor substances, were determined by means of spectral analyses and chemical studies. The structure of BE-12406A is 1-hydroxy-10-methoxy-8-methyl-12-alpha- L-rhamnopyranosyloxy-6H-benzo[d]naphtho[1,2-b]pyran-6-one, and that of BE-12406B is 1,10-dihydroxy-8-methyl-12-alpha-L-rhamnopyranosyloxy-6H- benzo[d]naphtho[1,2-b]pyran-6-one.

A new antitumor substance BE-13793C, produced by a streptomycete. Taxonomy, fermentation, isolation, structure determination and biological activity.

A new antitumor substance, BE-13793C, which has topoisomerase inhibitory activity was isolated from the culture broth of a strain of actinomycetes. The producing strain, BA13793, isolated from a soil sample collected in Seto, Aichi Prefecture, Japan, has a resemblance to Streptoverticillium mobaraense. The active principle was extracted from the mycelium of strain BA13793 with methanol and purified by Sephadex LH-20 column chromatography. BE-13793C showed strong inhibitory activity against topoisomerases I and II and inhibited the growth of doxorubicin-resistant or vincristine-resistant P388 murine leukemia cell lines, as well as their parent P388 cell line.

A new topoisomerase-II inhibitor, BE-10988, produced by a streptomycete. I. Taxonomy, fermentation, isolation and characterization.

A new topoisomerase inhibitor, BE-10988, was isolated from the culture broth of a strain of actinomycetes. The producing strain had a close resemblance to Streptomyces fimicarius and Streptomyces xanthocidicus. The active principle was extracted from the whole broth of strain BA10988 with ethyl acetate and purified by silica gel chromatography and by HPLC. BE-10988 increased DNA-topoisomerase complex formation and inhibited the growth of both doxorubicin-resistant and vincristine-resistant P388 murine leukemia cell lines, as well as sensitive P388 cell lines.

BE-14348 substances, new specific estrogen-receptor binding inhibitors. Production, isolation, structure determination and biological properties.

Streptomyces graminofaciens BA14348, isolated from a soil sample, was found to produce new specific inhibitors of estrogen binding to its receptor. Five related substances, BE-14348A approximately E, were isolated, and their structures were determined by analyses of spectral properties. Of these substances, A was identical with the known flavanone, naringenin. On the other hand, B, C, D and E were all new compounds; the structure of B was determined to be 2(S): 3(S)-3-methyl-4',5,7-trihydroxyflavanone, C was a racemic mixture of 2(S): 3 (R) and 2(R): 3(S)-3-methyl-4',5,7-trihydroxyflavanone; D and E were 8-chloro derivatives of B and C, respectively.

The effect of kazusamycin B on the cell cycle and morphology of cultured L1210 cells.

The effect of a potent antitumor antibiotic, kazusamycin B, on the cell cycle of L1210 cells was examined. Kazusamycin B arrested synchronized L1210 cells at G1 phase. Retardation of metaphase initiation was also observed. Flow cytometric analysis of kazusamycin B-treated asynchronized cells also confirmed G1 arresting effect of kazusamycin B. In addition, an unidentified cell population with lower fluorescence intensity than G1 population was observed when the cells were exposed to the drug longer than 12 hours. Morphology of kazusamycin B-treated L1210 cells revealed that the intranuclear structure changed within 4 hours, and that abnormal condensation of nuclei coincided with the appearance of unidentified population. Kazusamycin B inhibited RNA synthesis moderately but specifically at 2 hours. However, this inhibition might be a secondary effect of the antibiotic-induced structural abnormality of the nuclei.

Kazusamycin B, a novel antitumor antibiotic.

A novel antibiotic, kazusamycin B (C32H46O7, MW 542), was isolated from the fermentation broth of Streptomyces sp. No. 81-484 and the structure was established mainly on the basis of its physico-chemical properties. Unambiguous 13C NMR spectral analysis of kazusamycin B has been also accomplished. Kazusamycin B possesses potent cytocidal activities against L1210 (IC50 0.0018 micrograms/ml) and P388 (IC100 0.0016 micrograms/ml) leukemia cells in vitro.

Molecular cloning of a xylanase gene from Streptomyces sp. No. 36a and its expression in streptomycetes.

The gene for an extracellular xylanase from Streptomyces sp. No. 36a was cloned into Streptomyces lividans TK21 using pIJ702 as a vector plasmid. The smallest DNA fragment encoding the xylanase gene and its possible promotor was determined to be a 1.04 kb Sph I-Sac I fragment by sub-cloning studies. This xylanase gene fragment was transferred into the pSK2 series of plasmids and introduced into Streptomyces kasugaensis G3 protoplasts. The cloned xylanase gene was expressed in both S. lividans TK21 and S. kasugaensis G3, and these clones produced and secreted high yields of xylanase into the culture medium. The xylanase production was not detected when a foreign DNA fragment was inserted into the Bcl I site locating in the xylanase gene fragment.

Mutual relation of three pock-forming plasmids resident in Streptomyces noursei.

From Streptomyces noursei B3, two plasmids--designated pSCY5 and pSCY6--were isolated in addition to the known plasmid pSCY3. Strains carrying one or more of these plasmids generated pocks on a lawn of the plasmid-free strain, S. noursei KL3. The pocks elicited by pSCY3 and pSCY5 belonged to the A type; i.e., showed a clear inhibition zone, while those produced by pSCY6 were of the B type; i.e., exhibited a turbid inhibition zone. The strains carrying either pSCY3 or pSCY5, were also capable of forming pocks on a lawn of S. noursei 6T-11 harboring pSCY6, and vice versa. However, pock formation was not observed between strains harboring pSCY3 and strains carrying pSCY5. The endonuclease cleavage maps of these plasmids revealed that pSCY3 differed clearly from that of pSCY6, whereas pSCY5 was found to be a hybrid plasmid consisting of the entire pSCY3 plasmid and an 8.4 Md or longer fragment originating from pSCY6. The pocks elicited by pSCY5 were much smaller than those produced by pSCY3. Transformation experiments showed that pSCY6 elicited pocks in Streptomyces lividans as well as in S. noursei, whereas the pSCY6 transformants of S. lividans failed to produce pocks on a lawn of plasmid-free S. noursei.

Construction of plasmid vectors from Streptomyces kasugaensis plasmids, pSK1 and pSK2.

Streptomyces kasugaensis G3 was transformed by pIJ702 DNA carrying the thiostrepton-resistance gene at a frequency of 2 X 10(5) transformants/micrograms DNA, but it was found that the introduced pIJ702 was very unstable in this strain. This result led us to make useful vectors using the stable plasmids resident in S. kasugaensis. The Bcl I-fragment, containing the thiostrepton-resistance gene obtained from pIJ702, was inserted into the pSK1 and pSK2 plasmids isolated from S. kasugaensis. Two composite plasmids, pSK11-1 (8.0 Md) and pSK21-1 (4.8 Md), were isolated from the thiostrepton-resistant transformants of strain Ge. The constructed pSK11-1 consisted of the entire pSK1 molecule and the thiostrepton-resistance gene fragment. pSK21-1 consisted of the large Bcl I-fragment of pSK2 (4.1 Md) and the same thiostrepton-resistance gene. These plasmids were stably maintained in S. kasugaensis G3. Small derivatives of these composite plasmids were prepared by restriction enzyme cleavage and self-ligation, and several unique insertion sites were also constructed in these small plasmids. By analysis of the physical maps of these plasmids, the essential regions of pSK1 and pSK2 were determined from their DNA segments to be 2.5 Md in pSK11-1 and 1.9 Md in pSK21-1. pSK21-B5, one of these plasmid vectors, showed a wide host range in the genus Streptomyces and was stably maintained in all streptomycete species tested, except S. kasugaensis M338.

Extraction, cloning and physical maps of plasmid DNAs from Streptomyces noursei.

Three plasmids, pSCY 2, pSCY 3 and pSCY 4, were detected in Streptomyces noursei B3, a producer of cycloheximide and nystatin. pSCY 3 and pSCY 4 had molecular sizes of 15.1 megadaltons (Md) and 3.2 Md, respectively. When covalently closed circular (ccc) DNAs were extracted from the mycelia during the course of cultivation, the yield of ccc DNA extracted per mycelium reached the highest value at the starting point of the exponential growth phase and decreased rapidly during exponential phase; the yield increased gradually in stationary phase. The smallest plasmid, pSCY 4, could not be detected after 42 hours of cultivation. To purify and characterize DNA of the major plasmid pSCY 3, it was cloned into Escherichia coli using pACYC 184 as a vector, and the physical maps of the composite plasmids were constructed.

Basic techniques for DNA cloning and conditions required for streptomycetes as a host.

To develop a host-vector system in streptomycetes for DNA cloning, we examined the technical problems encountered and the conditions required for use of Streptomyces kasugaensis MB273 as the host. Basic techniques, such as plasmid DNA isolation, regeneration of mycelia from protoplasts and elimination of plasmids from cells were investigated. These techniques were found to be useful for many streptomycetes. Strain M518, a derivative of S. kasugaensis MB273, was found to have the following useful characteristics as a host. The plasmids of MB273 were easily cured by regeneration of mycelia from protoplasts. The protoplasts prepared from M518 regenerated mycelia at high frequency using an improved method and were efficiently transformed by plasmid DNA. The extra and intra cellular DNase activities were very weak, and no restriction endonuclease activity was detected. The sensitivity to various antibiotics was determined. This strain did not show any pathogenicity in mice nor suvival in the digestive organs of rats. MB273 and its derivatives died rather quickly in natural soil. M518 still forms aerial mycelial conidia. These results indicate that S. kasugaensis M518, derived from MB273, has useful characteristics as a host for DNA cloning. The techniques thus developed were found to be useful in other streptomycetes.

Function of plasmids in the production of aureothricin. I. Elimination of plasmids and alteration of phenotypes caused by protoplast regeneration in Streptomyces kasugaensis.

The spontaneous mutant 18a derived from Streptomyces kasugaensis MB273 exhibited pleiotropic effect such as loss of aerial mycelium formation, aureothricin (AT) production, and of citrullin biosynthesis, as well as changes in plasmid; the mutant required cystine for production of aureothricin. An improved method of protoplast regeneration was applied to S. kasugaensis MB 273-18a and a regeneration efficiency of 90% or more was obtained. Sixty to ninety percent of the colonies regenerated from the 18a protoplasts exhibited reversion of the pleiotropic mutation in 18a. Moreover, of 13 regenerated strains which showed these drastic phenotypic variations, it was found that their plasmid types varied. These types could be divided into two groups; the RI type (5 strains) which contained a large amount of pSK2, a small amount of pSK3 and no pSK1, and the RII type (8 strains) in which no closed-circular DNA was detected. From these results, the following conclusions were obtained. First, plasmid curing in RII type strains and also the variation of plasmid copy in the RI type strains occurred as the result of protoplast regeneration. Second, the structural genes for biosynthesis of AT probably exist on chromosome. Third, regeneration of 18a protoplasts causes the reversion of pleiotropic mutation with high frequency. A working hypothesis was proposed to explain these complex phenomena.