Characterization of an 8.7-kilobase thiostrepton resistance-encoding plasmid (pGIF3) of Streptomyces incarnatus.

The low-copy-number 8.7-kb plasmid pGIF3 of Streptomyces incarnatus was studied after cloning in the Escherichia coli vector pBR322; a restriction map was constructed. Southern blot analysis showed that pGIF3 in S. incarnatus occurs predominantly as integrated in a larger replicon. The plasmid carries a gene for thiostrepton resistance having no homology with the known thiostrepton resistance gene from Streptomyces azureus.     

Production of l-DOPA by Aspergillus terreus

The sinefungin producing, pock forming strain Streptomyces incarnatus was shown to be thiostrepton resistant. However, it does not produce thiostrepton and structurally related antibiotics. In this strain, five low copy plasmids of variable sizes were detected with electron microscopy. The strain S. lividans TK24 became thiostrepton resistant upon transformation by one of the plasmids of S. incarnatus.     

Efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmids.

Conditions for efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmid cloning vectors were identified. Three streptomycete plasmid origins of replication function in A. orientalis, as do the apramycin resistance gene from Escherichia coli, the thiostrepton resistance gene from Streptomyces azureus, and the tyrosinase gene from Streptomyces antibioticus. A. orientalis appears to express some restriction and modification, because highest transformation frequencies (10(6)/micrograms of DNA) were obtained when plasmid pIJ702 was modified by passage in A. orientalis.     

pFJ265, a new cloning vehicle for Streptomyces

A 9.3-kb plasmid, pNM100, was isolated from Streptomyces virginiae (NRRL 15156) and characterized. Streptomyces genes for thiostrepton and neomycin resistance were cloned into pNM100 to yield a small plasmid derivative, pFJ265, that is suitable for Streptomyces gene cloning. pFJ265 is a 9.2-kb nonconjugative plasmid and has a copy number of several hundred per chromosome.     

The minimal replicon of a Streptomycete plasmid produces an ultrahigh level of plasmid DNA

A functional map of Streptomyces coelicolor plasmid SCP2* was deduced from derivatives constructed by in vitro deletions. Functions were analyzed on bifunctional shuttle plasmids that contained pBR322 for selection and replication in Escherichia coli and fragments of SCP2* for replication in Streptomyces griseofuscus C581 and strains of Streptomyces lividans. The aph gene for neomycin resistance from Streptomyces fradiae and the tsr gene for thiostrepton resistance from Streptomyces azureus were incorporated as selectable antibiotic resistance markers in streptomycetes. An 11.8-kb sequence bounded by EcoRI and KpnI restriction sites contains the information for self-transfer and normal replication of the plasmid. A 5.9-kb EcoRI-SalI fragment contains all of the information for normal replication. Partial digestion generated a 2.2-kb Sau3A fragment that is sufficient for replication but it produces ten times higher plasmid copy number than the basic replicon. pHJL400 and PHJL401 are useful shuttle vectors containing the moderate-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19. A 1.4-kb BclI-Sau3A fragment with an additional internal BclI site contains the minimal replicon but it produces 1000 times higher plasmid copy number than the basic replicon. pHJL302 is a useful shuttle vector containing the ultrahigh-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19.     

Molecular cloning of the nosiheptide resistance gene from Streptomyces actuosus ATCC 25421

An 8.5 kb BamHI DNA fragment conferring resistance to nosiheptide, a peptide antibiotic of the 'thiostrepton group', was cloned from Streptomyces actuosus ATCC 25421 in Streptomyces lividans 1326. Two BamHI fragments of S. actuosus, the 8.5 kb fragment and an additional 3.0 kb fragment, hybridized with a thiostrepton resistance gene probe (pIJ30). The 8.5 kb fragment showed a relatively low degree of homology with the thiostrepton resistance gene. The restriction map of the nosiheptide resistance gene isolated here was significantly different from the map of the thiostrepton resistance gene previously published.     

Genetic instability in Streptomyces niveus plasmid pSN2: in vivo formation of deletion derivatives

A plasmid designated pSN2 (molecular size 32.0 kb) was isolated from the wild type of Streptomyces niveus ATCC 19793. To permit phenotypic identification of pSN2 the 1.9 kb BclI fragment was replaced in vitro by the 1.1 kb BclI fragment of pIJ702 carrying the thiostrepton resistance (tsr) gene to form the plasmid pSN3. pSN3 transforms S. lividans to thiostrepton resistance at high frequency and is stably maintained. However, when used to transform S. niveus pSN3 was unstable and produced a 5.5 kb thiostrepton resistant deletion derivative pLG5. pLG5 is also stable and expresses thiostrepton resistance in S. lividans but on transformation of S. niveus was unstable and produced a further thiostrepton resistant derivative, pLG10, of 6.5 kb. pLG5 and pLG10 like pSN3 transform S. lividans at high frequency and produce pocks. DNA hybridizations with a probe derived from pLG5 confirm that pLG5 is derived from DNA sequences present on pSN2 and pSN3.Abbreviations SDS sodium dodecyl sulphate - PEG polyethylene glycol     

大肠杆菌-链霉菌高效接合载体的构建及其应用

以链霉菌质粒SCP2^*的衍生质粒pHJL400为基础,构建了能够在大肠杆菌到链霉菌之间进行高效接合转移的质粒DGH112。pGH112含有在大肠杆菌和链霉菌中复制起始位点,以及分别在大肠杆菌和链霉菌中进行筛选的抗性标记。用pGH112转化Escherichia coli ET12567(pUZ8002)后,与天蓝链霉菌(Streptomyces coelicolor A3(2))、除虫链霉菌(Streptomyces avermitilis)、变铅青链霉菌(Streptomyces lividans TK54)、毒三素链霉菌(Streptomyces toxytricini NRRL15443)、委内瑞拉链霉菌(Streptomyces．vertezuelae ISP5230)和红色糖多孢菌(Saccharopolypora erythraea)进行接合,发现本构建的pGH112与pKC1139相比,接合转移效率较高,稳定性好,而且宿主范围较广。把组成型启动子ermE^*与绿色荧光蛋白基因(gfp)克隆到本构建的pGH112,通过接合转移到链霉菌中,gfp获得表达,证明其可以用作基因接合转移的有效工具载体,这为研究链霉菌的基因功能创造了有利条件。     

Streptomyces cloning vector derived from Streptomyces lavendulae-grasserius mini-plasmid pSLG33

Abstract The cryptic multicopy plasmid designated pSLG33 (2.65 kb) was isolated from the vegetative mycelium of Streptomyces lavendulae-grasserius RIA 746 and physically characterized. pRS410 vector (5.4 kb) was constructed by insertion of aph and tsr genes coding for neomycin and thiostrepton resistance, respectively, in a non-essential part of the plasmid molecule. The pRS410 is compatible with multicopy Streptomyces plasmid vectors derived from pIJ101 plasmid.     

Shuttle vectors for cloning recombinant DNA in Escherichia coli and Streptomyces griseofuscus C581.

The replicon of the Streptomyces plasmid SCP2 was located on a 5.9-kilobase EcoRI-SalI restriction fragment. The SCP2 replicon was combined with Escherichia coli plasmid pBR322 and genes specifying neomycin resistance and thiostrepton resistance in streptomycetes to construct shuttle vectors that are useful for cloning in E. coli and streptomycetes.     

透明颤菌血红蛋白在肉桂地链霉菌中的表达对基因细胞生长及抗生素合成的影响

肉桂地链霉菌（S.cinnamonensis)是莫能菌素（Monensin)的产生菌,大肠杆菌－链霉菌穿梭表达载体pHZ1252中的透明颤菌血红蛋白基因（vhb)位于硫链丝菌素诱导启动子ＰtipA之下,它在肉桂地链霉菌中的结构不稳定,,发生了重组缺失,缺失的片段包括大肠杆菌质粒部分vhb基因。但来自阿维链霉菌(S.avermitilis)中缺失了大肠杆菌质粒部分却保留了完整的vhb基因及tipA启动子的pHZ1252,可在肉桂地链霉菌中稳定复制,不再发生缺失,经硫链丝菌素诱导表达出了有生物活性的ＶＨb蛋白,摇瓶发酵实验证明,ＶＨb蛋白在氧限条件下可明显促进肉桂地链霉菌的菌体生长和抗生素合成。     

Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction

Restriction endonuclease cleavage maps of five DNA fragments carrying genes for neomycin phosphotransferase and neomycin acetyltransferase (from Streptomyces fradiae), viomycin phosphotransferase (from S. vinaceus), and ribosomal methylases determining resistance to thiostrepton (from S. azureus) and MLS antibiotics (from S. erythreus) are described, together with a map for the SLP1.2 Streptomyces plasmid used to isolate the fragments. Construction of a versatile Streptomyces cloning vector (pIJ61) is reported. pIJ61 carries neomycin phosphotransferase and thiostrepton resistance genes and has unique BamHI and PstI sites which will allow clone recognition by insertional inactivation of neomycin resistance; cloning sites for several other endonucleases are also present. pIJ28, a shuttle vector for Streptomyces and E. coli, carries neomycin resistance and the SLP1.2 and pBR322 replicons.     

阿维链霉菌孢子色素生物合成对阿维菌素产量的影响

以野生型阿维链霉茵NRRL8165为出发菌株,用PCR方法克隆孢子色素基因簇直系同源基因(whiEa)侧翼片段,并构建基因置换载体pHL643.将pilL643跨属接合转移进入阿维链霉菌NRRL8165,通过置换载体和染色体之间的同源双交换,对染色体上的whiEa基因簇进行置换,得到3株阿泊拉霉素抗性、硫链丝菌素敏感的重组菌株,均表现为孢子色素合成缺陷.通过Southern杂交分析,证明whiEa基因簇被置换.通过摇瓶发酵和HPLC检测,发现whiEa基因簇置换菌株所产阿维菌素产量明显提高,表明孢子色素与阿维菌素生物合成之间可能有竞争底物的现象.     

Development of pLR591, a Streptomyces-Escherichia coli positive selection shuttle vector

The Escherichia coli positive selection vector pEcoR251 was ligated with the broad host range, high copy number Streptomyces plasmid pIJ702 to produce pLR591, a Streptomyces-E. coli positive selection shuttle vector. The EcoRI and thiostrepton resistance genes of pLR591 were expressed in E. coli and Streptomyces lividans respectively. The positive selection shuttle vector pLR591 facilitates the construction in E. coli of genomic libraries which can be screened in Streptomyces strains.     

Characterization of an autonomously replicating region from the Streptomyces lividans chromosome.

The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker. pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability. pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S. lividans 66 and hybridized with DNA from several different Streptomyces strains. Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S. coelicolor A3(2) chromosome. DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters. The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA. In contrast, in S. lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences.

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.     

Cloning vectors derived from Streptomyces niveus plasmid pSN2

Two high copy number, broad host range, general purpose cloning vectors, pLG5 and pLG10, derived from the unstable Streptomyces niveus plasmid pSN2 are described. pLG5 (5.5 kb) and pLG10 (6.5 kb) both carry the thiostrepton resistance (TsrR) and lethal zygosis (Ltz+) markers and have single cloning sites within a non-essential region and the tsr gene. pLG505 (7.4 kb) was constructed by cloning the viomycin resistance (vph) gene into the single BamHI site of pLG5 to give a further vector with insertion and replacement sites which inactivate either the TsrR or VioR functions.     

Cloning of a DNA fragment from Streptomyces griseus which directs streptomycin phosphotransferase activity

DNA from Streptomyces griseus ATCC 12475 was partially digested with Sau3A and fragments were ligated into BglII-cleaved pIJ702. When the ligation mixture was used to transform protoplasts of Streptomyces lividans TK54, two transformants resistant to both thiostrepton and streptomycin were isolated. The hybrid plasmids pBV3 and pBV4 which they contained, carrying inserts of sizes 4.45 and 11.55 kbp respectively, each retransformed S. lividans to streptomycin resistance at high efficiency. Both plasmids hybridized to restriction digests of S. griseus chromosomal DNA in Southern blot experiments. In vitro deletion and sub-cloning experiments showed the sequence conferring streptomycin resistance to lie within a segment of 1.95 kbp. Extracts of TK54(pBV3) and TK54(pBV4) contained a streptomycin phosphotransferase similar to that in extracts of S. griseus. Streptomycin phosphotransferase activity appeared in extracts of S. griseus, TK54(pBV3) and TK54(pBV4) within 2 d of inoculation. When pBV3 and pBV4 were retransformed into S. griseus with selection for thiostrepton resistance, plasmid DNA of sizes corresponding to the incoming plasmids was found in the transformants. In these transformants the phosphotransferase appeared at 1.5 rather than 2 d, and reached a level over twice that of the original S. griseus strain.