Cosmid pJAR4, a novel Streptomyces-Escherichia coli shuttle vector for the cloning of Streptomyces operons

A novel shuttle cosmid vector (pJAR4), based on pK505, was constructed for the cloning of Streptomyces DNA. It is a low-copy-number vector which determines hygromycin B-resistance as a selective marker and was used to clone the puromycin biosynthesis pathway from Streptomyces alboniger. Cosmids pJAR4 and pKC505 (which determines apramycin-resistance) stably co-transform both Streptomyces lividans and Streptomyces griseofuscus.     

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.     

Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts.

Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S. alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S. alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 1H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolase and prg1, whose deduced amino acid sequence is significantly similar to that of degT, a Bacillus stearothermophilus pleiotropic regulatory gene.     

Expression in Streptomyces ambofaciens of an Escherichia coli K-12 gene which confers resistance to hygromycin B

We have constructed two plasmid vectors (pKC293 and pKC305) that can replicate in Escherichia coli K-12 and Streptomyces ambofaciens. These shuttle vectors were used to demonstrate the expression of two E. coli genes, hygromycin B (Hm) resistance and Tn5 neomycin (Nm) resistance, in S. ambofaciens.     

Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library

A strategy for gene cloning in the cyanobacterium Anacystis nidulans R2 was developed which made use of a gene library constructed in a shuttle cosmid vector. The method involved phenotypic complementation of mutants with pooled cosmid DNA. The development of the procedure and its application to the cloning of a third gene involved in nitrate reduction are described.     

A thiostrepton-inducible expression vector for use in Streptomyces spp

A shuttle expression vector containing the thiostrepton-inducible Streptomyces lividans promoter, ptipA, and the origin of transfer from plasmid RP4 was constructed. Cassettes containing a promoterless xylE gene upstream from a hyg gene were used to demonstrate thiostrepton-inducible expression from ptipA in both S. lividans and Streptomyces ambofaciens, ptipA was estimated to be induced 60-fold or more in Streptomyces ambofaciens.     

An Escherichia coli-Mycobacterium shuttle cosmid vector, pMSC1.

A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escherichia coli and Mycobacterium smegmatis. The vector was mainly derived from the lambda ori cosmid, Lawrist4, and the Mycobacterium fortuitum cryptic plasmid, pAL5000, which replicates in M. smegmatis and Mycobacterium bovis BCG. The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria. After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb. A genomic library from M. smegmatis was constructed in E. coli; clones from this library were transferred into M. smegmatis by electroporation, and back again to E. coli, without any apparent rearrangements. This vector will be useful in cloning genes encoding complex pathways in mycobacteria.     

Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces

pSAM2 is an 11-kb plasmid integrated in the Streptomyces ambofaciens ATCC23877 and ATCC15154 genomes and found additionally as a free replicon in an uv derivative. After transfer into S. ambofaciens DSM40697 (devoid of pSAM2) or into Streptomyces lividans, specific integration of pSAM2 occurred very efficiently. A 58-bp sequence (att) present in both pSAM2 (attP) and S. ambofaciens strain DSM40697 (attB) attachment regions is found at the boundaries (attL and attR) of integrated pSAM2 in S. ambofaciens strain ATCC23877. The S. lividans chromosomal integration zone contained an imperfectly conserved att sequence (attB), and the integration event of pSAM2 was located within a 49-bp sequence of attB. Only one primary functional attB sequence was present in the S. lividans or S. ambofaciens DSM40697 total DNA. The integration zone of S. lividans hybridized with the integration zone of S. ambofaciens DSM40697. The two integration zones were homologous only to the right side of the att sequence. The conserved region contained an open reading frame (ORF A) with a stop codon located 99 bp from the attB sequence in both strains. S. ambofaciens DSM40697 contained DNA sequences related to pSAM2 on the left side of the att site. The att sequence was included in a region conserved in Streptomyces antibioticus, Streptomyces actuosus, Streptomyces bikiniensis, Streptomyces coelicolor, Streptomyces glaucescens, and Streptomyces parvulus. Site-specific integration of a pSAM2 derivative was characterized in another unrelated strain, Streptomyces griseofuscus. This strain contained an imperfectly conserved 58-bp attB sequence, and the integration event took place within a 45-bp sequence of attB. Site-specific integration of pSAM2 in three nonrelated Streptomyces strains suggests the wide host range of pSAM2 integration in Streptomyces.     

Studies on locus expansion, library representation, and chromosome walking using an efficient method to screen cosmid libraries

We have developed an efficient screening method to search for clones in cosmid libraries prepared from human genomic DNA. Genomic, cDNA, and cosmid probes have been used to isolate homologous cosmids from human chromosomes 7, 10, 16, 17 and X as part of a search for polymorphic nucleotide sequences. This method has been successfully applied to chromosome walking experiments at the interstitial retinol-binding protein locus on chromosome 10, and may be a useful tool for investigating representation of cloned sequences in cosmid libraries. Our library was prepared in the vector c2RB (Bates and Swift, 1983), but the method is applicable to any cosmid cloning system in which the inserted DNA can be separated from the vector by restriction enzyme digestion. A cosmid library containing five human genome equivalents can be rapidly screened using three to four Southern hybridization filters. This results in substantial labor saving, particularly when screening genomes of high complexity with many different probes. Another advantage of the system is that it allows for the long-term storage of the cosmids so that they can be screened whenever necessary. As a consequence, cosmid screening can be made a routine laboratory procedure.     

醇醛脱氢酶的分离纯化及其基因文库的构建和筛选

在维生素C的发酵生产过程中,普通生酮基古龙酸菌S2(Ketogulonigenium vulgare)能产生醇醛脱氢酶,将L-山梨糖转化为VC的前体2-酮基-L-古龙酸(2-KLG)。通过超声波破碎菌体、硫酸铵分级沉淀、DEAE Sepharose Fast Flow阴离子交换层析,QSepharose High Performance柱层析等过程,从普通生酮基古龙酸菌S2发酵液中分离纯化了醇醛脱氢酶,并用该纯化酶免疫新西兰兔制备出了合格抗血清。同时,普通生酮基古龙酸菌S2基因组DNA经Sau3AⅠ部分酶切后,与黏粒载体pKC505连接,用包装蛋白进行包装,转染大肠杆菌DH5浕,构建了基因组文库。最后应用免疫酶斑点技术(Dot-ELISA)从12000个克隆子中筛选得到一个阳性克隆K719#。通过检测该基因工程菌的活性,表明K719#具有使L-山梨糖转化为2-KLG的功能,从而使醇醛脱氢酶在大肠杆菌中获得了高效表达,这为简化VC的生产工艺奠定了基础。     

链霉菌Streptomyces tenebrarius H6中与抗生素有关的糖生物合成基因的克隆

链霉菌S.tenebrarius H6产生多种氨基糖甙类抗生素,主要有阿普霉素、妥普霉素及卡那霉素B,其中阿普霉素因含有8碳糖的一种特殊结构令人注目,它的抗菌谱广,特别是对革兰氏阴性菌有较强的抗菌活性,不容易产生耐药性,对已有的耐药菌产生的氨基糖苷转移酶等失活酶仍有抵抗力.主要用于牛、猪、鸡等的大肠杆菌、沙门氏菌和支原体所引起的白痢、腹泻和肺炎等疾病.迄今有关八碳糖生物合成基因簇的研究在国内外尚无报道,在该菌株开展有关糖合成代谢基因的研究有着一定的意义.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

节杆菌BT801基因文库构建及其乙内酰脲酶基因分离与表达

L-乙内酰脲酶产生菌节杆菌 BT801的染色体DNA经Sau3A I 部分酶切后分离30kb左右的片段,与经HpaI和PstI酶切的黏粒载体Pkc505进行连接,将连接产物用包装蛋白包装,转染大肠杆菌DH5α得到10 000多个转化子,构建成节杆菌BT801的基因组文库。通过薄层层析等方法筛选得到了1个阳性克隆,通过亚克隆得到了乙内酰脲酶的完整基因,该基因能分别利用自身的启动子和T5启动子在大肠杆菌中进行表达产生有活性的蛋白。     

Resistance to macrolides, lincosamides and streptogramin type B antibiotics due to a mutation in an rRNA operon of Streptomyces ambofaciens.

Streptomyces ambofaciens produces spiramycin, a macrolide antibiotic and expresses an inducible resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS). From a mutant of S.ambofaciens exhibiting a constitutive MLS resistance phenotype a resistance determinant was cloned on a low copy number vector (pIJ61) through its expression in Streptomyces lividans. Further characterization has shown that this determinant corresponded to a mutant rRNA operon with a mutation in the 23S rRNA gene. In different organisms, mutations leading to MLS resistance have been located at a position corresponding to the adenine 2058 of Escherichia coli 23S rRNA. In the 23S rRNA from S.ambofaciens a similar position for the mutation has been postulated and DNA sequencing of this region has shown an adenine to guanine transition at a position corresponding to 2058. S.ambofaciens possesses four rRNA operons which we have cloned. In Streptomyces, contrary to other bacteria, a mutation in one among several rRNA operons confers a selectable MLS resistance phenotype. Possible reasons for this difference are discussed.     

Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120.

Approximately 140 mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on media lacking fixed nitrogen (Fix-) were isolated after mutagenesis with diethyl sulfate and penicillin enrichment. A large cosmid library of wild-type Anabaena sp. strain PCC 7120 DNA was constructed in a mini-RK-2 shuttle vector, and seven mutants representing several morphologically abnormal heterocyst phenotypes were complemented. One of these mutants, 216, failed to differentiate heterocysts. All of these mutants except 216 reduced acetylene under anaerobic conditions, indicating that they are not defective in nitrogen fixation per se. Several cosmids were isolated from each complemented mutant and in most cases showed similar restriction patterns. Comparisons of the complementing cosmids from mutant 216 and two other phenotypically distinct mutants by restriction enzyme analysis identified a common region. This region, when present in either a cosmid or a 9.5-kb NheI subclone, is capable of efficiently complementing all three mutants. A 2.4-kb subclone of this region complements mutant 216 only.     

A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant

A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn901 mutant was isolated. Chromosomal DNA fragments were cloned in the Escherichia coli plasmid vector pACYC184. A recombinant plasmid carrying the inactivated met::Tn901 gene was selected after transformation to E. coli. The cloned met::Tn901 DNA fragment was used as a probe to select the corresponding A. nidulans R-2 wild-type met gene from a gene library prepared in E. coli, using the newly constructed shuttle cosmid vector pPUC29. When transformed into A. nidulans Met- mutants, this cloned gene allowed the mutants to grow prototrophically.     

Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen.

Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.     

Physical mapping of the Mycoplasma pneumoniae genome.

In order to study the genome organization of Mycoplasma pneumoniae a cosmid library of M. pneumoniae DNA was established using a newly designed cosmid vector (pcosRW2). From this library 32 overlapping clones were isolated covering a contiguous 720 kbp DNA segment representing about 90% of the genome assuming a genome size of about 800 kbp.     

Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa.

A hemolysin determinant was cloned from Pseudomonas aeruginosa PA103 by inserting Sau3a-generated DNA fragments between the BamHI sites of the lambda replacement vector WL47.1. A 9.5-kilobase HindIII fragment encoding the hemolysin was subcloned from this phage and inserted into the plasmid vector pHC79 to generate the recombinant plasmid pKC95. Escherichia coli K-12 strains harboring pKC95 exhibited zones of hemolysis after several days of growth on blood agar plates. Hemolysis was shown to be due to phospholipase C activity by using the chromogenic substrate p-nitrophenylphosphorylcholine. Deletion mutants of pKC95 were isolated, and polypeptides expressed from these plasmids were examined by using the E. coli minicell system. A polypeptide of 78,000 daltons was associated with phospholipase C activity. The hemolytic activity was cell associated when expressed in E. coli.