Analysis of plasmid pMZ1 from Micromonospora zionensis

Plasmid pMZ1, isolated from Micromonospora zionensis, was also able to replicate by the rolling circle mechanism in Micromonospora melanosporea and Streptomyces lividans. Southern hybridisation experiments with probe prepared from pMZ1 and immobilised M. zionensis DNA fragments separated on pulsed-field gel electrophoresis, indicated that the plasmid is present in the progenitor strain in both integrated and autonomous states. Thiostrepton resistant derivatives of pMZ1 plasmid, pMZS25 and pMZS34, were used to study conjugal transfer in M. melanosporea and S. lividans. A 3.4 kb NcoI-MluI fragment from pMZ1 cloned in pIJ702 (plasmid pIJNM3) was shown to be sufficient to promote plasmid transfer and pock formation in S. lividans.     

Unstable genetic determinant of A-factor biosynthesis in streptomycin-producing organisms: cloning and characterization.

We cloned a DNA fragment directing synthesis of A-factor from the total cellular DNA of streptomycin-producing Streptomyces bikiniensis on the plasmid vector pIJ385 . Introduction of the recombinant plasmid ( pAFB1 ) into A-factor-deficient S. bikiniensis and Streptomyces griseus mutants led to A-factor production in the host cells, as a result of which streptomycin production, streptomycin resistance, and spore formation of these mutants were simultaneously restored. The plasmid pAFB1 also complemented both afsA and afsB mutations of Streptomyces coelicolor A3(2). These results indicated that the cloned DNA fragment contained the genetic determinant of A-factor biosynthesis. The cloned fragment, when carried on a multicopy vector plasmid, induced production of a large amount of A-factor in several Streptomyces hosts. In Southern blot DNA/DNA hybridization analyses with a trimmed 5-kilobase fragment containing the intact A-factor determinant as probe, total cellular DNA from A-factor-deficient mutants gave no positive hybridization. The DNA blot experiment also showed a wide distribution of sequences homologous to the S. bikiniensis A-factor determinant among most, but not all, A-factor-producing actinomycetes with a varying extent of homology and the absence of these sequences from most A-factor nonproducers .     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.     

圈卷产色链霉菌分化关键基因——saw1的克隆及酶切分析

链霉菌是现代生物学研究中一种重要的微生物,它有两个突出的特征:其一,有无与伦比的合成次生代谢产物的能力,世界上所知数千种抗生素的70％由其产生。其二,有一个复杂的发育分化的生命周期,是微生物分化研究的一个最好的模式材料。链霉菌分化主要为形态分化和生理分化,两者彼此独立又相互关联,构成复杂的分化调控网络,研究分化基因的调控不但有重要的理论意义,而且可用于控制抗生素的生物合成,因此弄清合成途径的分子机制,也有潜在的应用价值。圈卷产色链霉菌是从我国东北土     

Cloning and characterization of the carbapenem biosynthetic genes from Streptomyces fulvoviridis

Carbapenem non-producing mutants were isolated from Streptomyces fulvoviridis and divided into six cosynthesis groups. By using one of the mutants as the host and plasmid pIJ385 as the vector, we cloned carbapenem biosynthetic genes from the parental S. fulvoviridis strain. A cloned 6-kb DNA fragment complemented the defects of three mutants each of which had a mutation in different genes. Southern blot hybridization using the cloned 6-kb fragment as probe showed the presence of the nucleotide sequences homologous to the probe in other carbapenem-producing Streptomyces spp. In addition, Streptomyces griseus, a carbapenem non-producer, possessed the sequence homologous to the probe and showed co-synthesis phenomena with some of the carbapenem non-producing mutants of S. fulvoviridis.     

Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans.

A-factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone), an autoregulating factor originally found in Streptomyces griseus, is involved in streptomycin biosynthesis and cell differentiation in this organism. A-factor production is widely distributed among actinomycetes, including Streptomyces coelicolor A3(2) and Streptomyces lividans. A chromosomal pleiotropic regulatory gene of S. coelicolor A3(2) controlling biosynthesis of A-factor and red pigments was cloned with a spontaneous A-factor-deficient strain of S. lividans HH21 and plasmid pIJ41 as a host-vector system. The restriction endonuclease KpnI-digested chromosomal fragments were ligated into the plasmid vector and introduced by transformation into the protoplasts of strain HH21. Three red transformants thus selected were found to produce A-factor and to carry a plasmid with the same molecular weight, and a 6.4-megadalton fragment was inserted in the KpnI site of pIJ41. By restriction endonuclease mapping and subcloning, a restriction fragment (1.2 megadaltons, approximately 2,000 base pairs) bearing the gene which causes concomitant production of A-factor and red pigments was determined. The red pigments were identified by thin-layer chromatography and spectroscopy to be actinorhodin and prodigiosin, both of which are the antibiotics produced by S. coelicolor A3(2). The cloned fragment was introduced into the A-factor-negative mutants (afs) of S. coelicolor A3(2) by using pIJ702 as the vector, where it complemented one of these mutations, afsB, characterized by simultaneous loss of A-factor and red pigment production. We conclude that the cloned gene pleiotropically and positively controls the biosynthesis of A-factor, actinorhodin, and prodigiosin.     

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G

A 4.9-kb DNA fragment containing the bla gene for the extracellular beta-lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme.     

Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans

Summary Streptomyces glaucescens GLA000 contains the integrated 15 kb DNA element pIJ408 which, during mating of the parent strain with S. lividans, can be transferred into recipient cells. In S. lividans cells, pIJ408 was found in an autonomously replicating form and in a chromosomally integrated state. In the majority of the S. lividans transconjugants studied, a deletion derivative pIJ408. 1 (12.4 kb) occurred. The deletion form was found in some strains only as a free plasmid, in others it was also chromosomally integrated. The integration region of pIJ408 was subcloned and precisely mapped by hybridization, restriction and sequencing analyses. The DNA junction fragments of the integrated plasmid in S. glaucescens, as well as the DNA fragment containing the attachment site of the S. lividans chromosome, were also cloned, submitted to detailed restriction analysis and sequenced. The attachment site of pIJ408 (attP) and the junctions of its integrated form with the chromosomal DNA in S. glaucescens (attL and attR) contain an identical 43 bp sequence. The chromosomal attachment site in S. lividans (attB) differs from the S. glaucescens att sequence by a single base substitution. The similarities between attachment sites of SLP1, pMEA100, pSAM2 and pIJ408 are discussed.     

An RNA species involved in Escherichia coli ribonuclease P activity. Gene cloning and effect on transfer RnA synthesis in vivo

The isolation and characterization of a plasmid capable of complementing the temperature-sensitive transfer RNA biosynthetic mutation rnpA49 (ribonuclease P) is described. The DNA segment responsible for complementation codes for an RNA species, approximately 340 bases long. Hybridization-selection experiments indicated that all rnp mutants were deficient in the production of the complementing RNA at high temperature; these defects were not due to the accumulation of a precursor form of this RNA. Examination of tRNA species synthesized in vivo indicated that the plasmid clone did not completely relieve the deficiency in RNase P activity of rnpA49 since some tRNA precursors still accumulated in strain A49 containing the plasmid. At least one other tRNA precursor was no longer detectable in plasmid-containing rnpA49 cells.     

Plasmid transformation of Streptomyces venezuelae: modified procedures used to introduce the gene(s) for p-aminobenzoate synthase

Sucrose was unsuitable as an osmotic stabilizer in buffer solutions and media used for transformation of Streptomyces venezuelae ISP5230. Its replacement with NaCl, together with other modifications in the procedure, allowed efficient formation and regeneration of protoplasts but did not support transformation of S. venezuelae ISP5230 by vectors pIJ41 and pIJ941. With pIJ702, transformants with a low plasmid-copy-number and altered growth characteristics were obtained. Both pIJ702 and pIJ941, but not pIJ41, transformed S. venezuelae 13s; when pIJ941 was used, the plasmid in 18 of 20 transformants contained a deletion in the region reported to code for replication and transfer. The modified plasmid transformed S. venezuelae ISP5230 efficiently and was used to introduce a fragment of DNA from the pab locus of the wild-type into a Cml-1 mutant of ISP5230 blocked in chloramphenicol formation. Transformants that overproduced p-aminobenzoic acid were obtained but they remained blocked in chloramphenicol production; thus, the cloned pab fragment did not contain genes able to complement the cml-1 mutation. The results also suggest that the Cml-1 phenotype is not due to a defective reaction common to the biosynthesis of p-aminobenzoic acid and chloramphenicol.     

The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily

Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype. By selecting cells of the supersensitive strain for normal sensitivity after transformation with a wild-type yeast genomic library, a complementing 10-kb DNA fragment was isolated, a 3.4-kb subfragment of which was sufficient for complementation. DNA sequence analysis revealed that the complementing fragment comprised the recently sequenced SGE1 gene, a partial multicopy suppressor of gal11 mutations. The supersensitive strain was found to be a sge1 null mutant. Overexpression of SGE1 on a high-copy-number plasmid increased the resistance of the supersensitive strain. Disruption of SGE1 in a wild-type strain increased the sensitivity of the strain. These features of the SGE1 phenotype, as well as sequence homologies of SGE1 at the amino acid level, confirm that the Sge1 protein is a member of the drug-resistance protein family within the major facilitator superfamily (MFS).     

Cloning and expression of an extracellular-agarase gene from Streptomyces coelicolor A3(2) in Streptomyces lividans 66

An extracellular agarase gene was cloned from Streptomyces coelicolor A3(2) strain M130 into S. lividans 66 using the multicopy plasmid vector pIJ702. Various deletion derivatives of the initial clone (pMT605) were obtained by in vitro and in vivo methods. This allowed the gene to be localised to a 1.9-kb segment of DNA. The agarase enzyme was overproduced (up to 500 times) and exported efficiently into the medium. The agarase protein was identified as a 28-kDal band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE); in the case of one derivative, pMT608, this band accounted for nearly 50% of the total extracellular protein. Differences in agarase production between the deletion derivatives correlated well with plasmid stability.     

可溶性白细胞介素6受体基因在链霉菌的克隆表达研究

以分离提取的ＨｅＬａ细胞总ＲＮＡ为模板,通过ＲＴ－ＰＣＲ反应扩增得到了１０１７ｂｐ的人可溶性白细胞介素６受体（ｓＩＬ－６Ｒ）ｃＤＮＡ片段,将扩增片段克隆到ｐＵＣ１９质粒中进行序列测定。结果证明该片的序列与文献报道的ｓＩＬ－６ＲｃＤＮＡ的序列完全一致,将ｓＩＬ－６ＲｃＤＮＡ与链霉素信号肽ｍｅｌＣｌ的编码序列融合后得到的融合基因ｍｅｌ／ｓＩＬ－６Ｒ克隆到链霉菌质粒ｐＬＪ４５９中,构建成重组表达质粒ｐＬ     

Cloning of grisin resistance gene gsr and the study of its functioning in Streptomyces griseus Kr. strain

S. griseus Kr. is a commercial strain producing grisin, an antibiotic of the streptothricin group used as a feed additive. It was shown earlier that genetic instability of the strain was very high which was evident from a high frequency of nonreverting Grn- Grns mutants. With densitographic analysis of chromosomal DNA electrophoregrams and DNA-DNA hybridization it was revealed that the molecular basis of the genetic instability of the S. griseus strain was deletion of a DNA fragment about 20 kb in size containing a grisin resistance gene. The resistance gene designated as gsr was cloned to S. lividans TK 64 within the plasmid vector pIJ699. The restriction map of a cloned DNA fragment with a gsr gene was constructed and its similarity to that of a nat gene resistant to norseothricin, another streptothricin was observed. Introduction of a gsr gene within the multicopy plasmid pIJ699 into S. griseus 212, a highly productive strain synthesiing the antibiotic, led to an increase in its resistance and productivity. Proceeding from the preliminary data on possible linkage of a gsr gene and grisin biosynthesis genes, it appeared possible to use the cloned gene as a molecular probe in cloning the biosynthesis genes.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Analysis of the nourseothricin-resistance gene (nat) of Streptomyces noursei

A gene (nat) conferring resistance to the streptothricin antibiotic nourseothricin (Nc) was cloned from the producer Streptomyces noursei into Streptomyces lividans on the vector pIJ702 to form pNAT1. The nat gene was localized on a 1-kb SalI-MboI fragment, which also carries the nat promoter. Divergent promoter activity from the nat promoter region was identified on the cloned fragment using promoter probe plasmids pIJ486 and pIJ487. The nat gene is not expressed from its own promoter in Escherichia coli as shown by its failure to promote cat expression in promoter-less plasmid pBB100 and by the expression of NcR in only one orientation, when cloned in pUC19. In S. lividans 7A, harbouring plasmid pNAT1, an Nc-acetylating activity (NAT) was associated with the cloned resistance gene. The substrate specificity of NAT correlated well with the substrate range of the acetyltransferase in S. noursei and Tn1825-determined streptothricin resistance in Gram-negative bacteria. Moreover, an extract of S. lividans carrying pNAT1 showed specific serological cross-reactivity with an extract of E. coli carrying Tn1825.     

Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was Isolated as an Intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd30₁. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPH1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex plants in two such constructs BSL 12–1 and BSL 12–3.     

麦迪霉素产生菌具有启动功能的DNA片段的克隆和分析

利用启动子探针质粒载体pIJ486从麦迪霉素产生菌总DNA中克隆得到了一段具有启动功能的DNA片段.通过限制性酶酶切分析,测定插入DNA片段大小为2.3kb.又利用载体pIJ486和pIJ487的新霉素抗性结构基因上游有多酶切点方向相反的性质,分析了插入片段在两个不同方向上的启动能力.结果表明,在两个方向上均有启动功能,但强弱相差六倍.其中在XbaI-HindIII方向上具有较强的启动能力,在变铅青链霉菌中新霉素抗性水平可达20mg/ml以上.进一步对插入片段的三个BamHI小片段进行分析的结果表明,较强启动子区域集中在BamHI-BamHI 0.79kb DNA片段上.