Vector systems allowing efficient autonomous or integrative gene cloning in Micromonospora sp. strain 40027

Vector systems allowing autonomous or site-specific integrative gene cloning were developed for Micromonospora sp. strain 40027, a producer of the antibiotic fortimicin A. The autonomous system depends on the discovery of a low-copy-number, self-transmissible covalently closed circular plasmid, pJTU112 (ca. 14.1 kb), which was shown to be present in the progenitor strain in both integrated and autonomous states. The copy numbers of both wild-type pJTU112 and three derivatives of it can be amplified at least sixfold by addition of streptomycin to the culture medium. The integrative system was developed by the use of a pBR322-derived Escherichia coli plasmid vector, pSET152, mediated by the attP site of the Streptomyces phage PhiC31. Both vectors can be transferred by conjugation from E. coli into Micromonospora sp. strain 40027. The heterologous cloning and expression of the dnd gene cluster originating from Streptomyces lividans 1326 into Micromonospora sp. strain 40027 demonstrated the use of the two systems.     

Isolation and Characterization of Micromonospora Phage ΦHAU8 and Development into a Phasmid

ΦHAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The ΦHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. ΦHAU8 was most stable at 4°C in Difco nutrient broth within a pH range of 6 to 12. ΦHAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca²⁺ and 30 mM Mg²⁺. A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a λ cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that ΦHAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.     

Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis.

Summary We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employs the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pM0116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.     

链霉菌质粒pSET152电转化稀有放线菌小单孢菌的研究

利用链霉菌(Streptomyces)噬菌体ΦC31所构建的整合型载体pSET152作为供体质粒,分别以小单孢菌(Micromonospora)40027菌株的萌发孢子和新鲜菌丝体作为受体菌,在不同的电场强度下进行电转化实验,结果表明:以小单孢菌40027菌株萌发孢子为受体菌,未获得电转化子;以小单孢菌40027菌株新鲜菌丝体为受体菌,获得了电转化子。电场强度为13kV/cm时可获得最高转化效率。Southern杂交结果表明:质粒pSET152可通过菌丝体电转化法导入小单孢菌40027菌株,并整合到小单孢菌40027菌株的染色体上,暗示链霉菌噬菌体ΦC31的整合酶基因和整合位点在异源宿主小单孢菌40027菌株中仍具有相同的功能。质粒稳定性检测实验表明:质粒pSET152可稳定地存在于小单孢菌40027菌株中。     

A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.     

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.     

Characterization of the Minimal Replicon of a Cryptic Deinococcus radiodurans SARK Plasmid and Development of Versatile Escherichia coli-D. radiodurans Shuttle Vectors

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.     

Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria

Streptomyces cholesterol oxidase was produced in Escherichia coli by a modification of the cholesterol oxidase gene (choA′) in which the native codons for the precursor NH₂-terminal region and the ribosome binding site were substituted for those favored by E. coli. The choA′ gene was expressed under the control of the lac or tac promoter in a multiple copy plasmid vector, although no expression of the native choA gene from Streptomyces was observed in E. coli. E. coli cells carrying the plasmid, pCo117, produced 2-fold more cholesterol oxidase intracellularly during 18-h culture than did the producing strain of Streptomyces sp. SA-COO cultured for 4 d. The NH₂-terminal amino acid sequence of cholesterol oxidase produced by E. coli appeared to be processed between Ala²⁰ and Ala²¹ of the precursor enzyme, while the Streptomyces enzyme was processed between Ala⁴² and Asp⁴³. Based on the facts that the cholesterol oxidase was stable, could be assayed rapidly, and no endogenous cholesterol oxidase activity was found in any enteric bacteria, we developed two widely applicable, new promoter-probe vectors posessing the choA′ gene, multiple cloning sites, and either a low or high copy number plasmid. Since these plasmids can replicate in enteric bacteria, the new plasmid vectors have a great potential for use in enteric bacteria without the isolation of Cho⁻ mutants.     

Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete λP_R or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage λ Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding β-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.     

Ultra-Low Background DNA Cloning System

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an “ultra-low background DNA cloning system” on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp^r). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp^r 5′ UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp^r 3′ UTR. This cassette allowed conversion of the Amp^r-containing vector into the yeast/E. coli shuttle vector through use of the Amp^r sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific “origins of replication” to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.     

A Rapid and Efficient Method for Cloning Genes of Type II Restriction-Modification Systems by Use of a Killer Plasmid

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.     

Isolation of Methyl Parathion-Degrading Strain M6 and Cloning of the Methyl Parathion Hydrolase Gene

A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp. strain M6 was able to hydrolyze methyl parathion to p-nitrophenol. A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the mpd gene was determined. The gene could be effectively expressed in Esherichia coli.     

A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

We describe the construction of a series of vectors suitable for gene cloning in the Cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.     

Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10⁴ positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.     

A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10⁸ to 10⁹ per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the β-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.     

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cm^r, Tc^r and Em^r determinants that are expressed in E. coli. Only the Em^r determinant is expressed in S. sanguis. Both the Cm^r and the Tc^r of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345–353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.     

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.     

Cloning and maintenance of the housefly sodium channel gene using low copy number vector and two sequential host strains

In spite of the long history of recombinant DNA technology, some genes have not been successfully cloned in Escherichia coli. This is probably due to the toxic effects of the expressed foreign gene product on E. coli. In initial attempts to clone the full-length Vssc1 voltage-sensitive sodium channel α-subunit gene from houseflies, we used one of the most popular vectors and hosts but were unable to retrieve any intact clone. By using two vectors with different copy numbers and two alternate E. coli host strains, we found that the combined use of a low copy number vector (pALTER-1) and an E. coli host strain that suppresses plasmid replication (ABLE-K) is essential to obtain intact full-length Vssc1 clone. However, since the ABLE-K strain was not a suitable host for the long-term maintenance of Vssc1 gene due to its recombination-positive genotype, it was necessary to transfer the Vssc1 plasmid from the primary host to a secondary host with a recombination-minus genotype (Stbl2) to minimize the chances of deletion or rearrangement. We believe that this cloning strategy, with a low copy number vector and the sequential use of two E. coli strains, will be also applicable for the cloning of other toxic genes.