The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.     

A Pair of Ligation-Independent Escherichia coli Expression Vectors for Rapid Addition of a Polyhistidine Affinity Tag to the N- or C-Termini of Recombinant Proteins

6×His tag is one of the most widely used affinity fusion tags that facilitates detection and purification of recombinant proteins. However, the location of this tag within a particular type of protein may influence the expression, solubility, and bioactivity of the protein, and the optimal location needs to be determined experimentally. To provide a tool for rapid generation of 6× His tags at the N- or C-terminus of any recombinant protein, we have constructed a pair of Escherichia coli expression vectors—pLIC-NHis and pLIC-CHis—based on the pET30a vector, for ligation-independent cloning (LIC). Construction of this new pair of LIC vectors was accomplished by replacement of the multiple cloning site of pET30a with two specifically designed LIC cloning sites. A target gene derived by PCR with a pair of predesigned primers can be inserted into the LIC site of pLIC-NHis for expression of recombinant proteins fused with the N-terminal sequence MHHHHHHG or into that of pLIC-CHis for expression of recombinant proteins with the C-terminal sequence THHHHHH. Successful expression of two normal mammalian prion proteins and five bacterial proteins in E. coli using this pair of LIC vectors reveals that these vectors are valuable tools for the production of recombinant His-tagged proteins in E. coli.     

Positive-selection and ligation-independent cloning vectors for large scale <Emphasis Type="Italic">in Planta</Emphasis> expression for plant functional genomics

Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligationindependent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3′ to 5′ exonuclease activity in the presence of only one dNTP (dGTP for the inserts and dCTP for the vector). To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance (R) genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants.     

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

Phenotypic Expression of PCR-Generated Random Mutations in a Pseudomonas putida Gene after Its Introduction into an Acinetobacter Chromosome by Natural Transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37°C. Of recombinant clones that failed to express xylE at 37°C, about 10% expressed the gene when grown at 22°C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

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.     

Construction and use of integrative vectors to express foreign genes in mycobacteria

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide’vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18kDa gene and expression in M. smegmatis was obtained.     

Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.     

Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.     

Cloning and characterization of a DNA repair gene inHaemophilus influenzae

Using a high-efficiency DNA cloning vector pJ1–8, a DNA repair geneuvr1 has been self-cloned in bacteriumHaemophilus influenzae. Chimeric plasmid pKuvrl, carrying wild type allele ofuvr1 gene and flanking DNA sequences, specifically complements auvr1 gene mutation in the bacterial chromosome. Auvr1_} mutation could be transferred from chromosome byin vivo recombination to pKuvr1 and isolated and designated as plasmid pKuvrl₋. Plasmid pKuvrl carries a 11.3 kb chromosomal DNA insert which was scanned for the presence of any other DNA repair genes by a novel method of directed mutagenesis. Preliminary analysis of the 3 new mutants isolated by this method supports the notion that the insert contains more than one gene concerned with ultraviolet radiation-sensitivity.     

USER Friendly Cloning Coupled with Chitin-Based Natural Transformation Enables Rapid Mutagenesis of Vibrio vulnificus

Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch''s postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.Vibrio vulnificus is a halophilic bacterium present naturally in estuarine waters and often contaminates oysters and other shellfish (for a review, see reference 15). V. vulnificus is an opportunistic pathogen of humans, causing primary septicemia and wound infection in susceptible individuals, and is the leading cause of reported seafood-related deaths in the United States. In susceptible humans, V. vulnificus causes a rapid, fulminating disease process resulting in extensive tissue damage. Mortality rates for susceptible individuals who develop fulminating primary septicemia are greater than 50% (17). Skin infections can lead to severe cellulitis, necrotizing fasciitis, and myositis requiring surgical debridement of infected tissues or amputation of the limb (4, 29, 42). Therapeutic intervention is often difficult since death can occur in less than 24 h after contact with the bacteria. In a mouse model of infection, V. vulnificus replicates extremely rapidly in host tissues (40, 41) and kills host cells including neutrophils (41).Over 20 years of genetic analysis, only a few virulence factors have been identified and confirmed by using the molecular version of Koch''s postulates (15). Among the confirmed virulence factors are capsular polysaccharide (49), acquisition of iron (34, 52), type IV pilus (37), RTX toxins (21, 25, 30), and flagella (22, 26). Despite these advances, the full spectrum of virulence factors responsible for the rapid and destructive disease process has not been elucidated. A major hindrance to the molecular genetic analysis of V. vulnificus is the fact that the bacteria cannot be effectively electroporated, nor can the bacteria be chemically transformed. Therefore, the only effective means of introducing plasmid DNA is by conjugation. This limitation severely restricts the availability of plasmids for creating and complementing mutations for molecular genetic analysis.A classical method to create mutants of V. vulnificus is the use of suicide vectors containing transposons, such as TnphoA (31), mini-Tn5phoA (12), mini-Tn5Km2 (12), mini-Tn5lacZ1 (12), mini-Tn10/kan (23), and Himar (1). However, transposon mutagenesis can cause polar mutations and truncated genes. The most definitive genetic analyses can be performed by deleting the genes of interest. The standard method for deleting genes is to clone the flanking upstream and downstream sequences into an allelic exchange suicide vector and introducing the plasmid into the target strain. The suicide vector integrates into the target genome via one of the flanking DNA sequences by a single-crossover event. This process is selected using antibiotic resistance encoded on the vector. The single-crossover strain is then grown without selection to allow for a second crossover that excises the allelic exchange vector. The excision event is enriched using a counterselectable marker encoded on the allelic exchange vector. A commonly used counterselectable marker is sucrose sensitivity, encoded by the sacB gene (13). If the second crossover is in the same flanking sequence as the first, the wild-type genotype is restored; however, if the second crossover is in the opposite flanking sequence, the targeted gene is deleted.This method of mutagenesis is problematic for V. vulnificus. First, one of the easiest ways to clone PCR amplicons is by TA topoisomerase-mediated ligation (TOPO TA cloning) with commercially available vectors such as pCR2.1 TOPO (Invitrogen, Carlsbad, CA). Unfortunately, these vectors are not mobilizable; hence, they cannot be used directly with V. vulnificus. Furthermore, there are no available TA topoisomerase allelic exchange vectors. Site-specific recombination vectors such as Gateway (Invitrogen) have facilitated PCR-mediated cloning and gene expression; however, none of the relevant vectors is mobilizable. Therefore, essentially all cloning for expression in or mutagenesis of V. vulnificus requires multiple subcloning steps. Second, sucrose sensitivity counterselection is not very efficient with V. vulnificus, as it is time-consuming and involves the screening of large numbers of colonies to find truly sucrose-resistant colonies. In some cases, sacB counterselection has completely failed. Another method for introducing mutations into target strains is the lambda phage red recombinase system (11). However, this procedure involves introducing additional helper plasmids into the target strain, and mobilizable helper plasmids have not been made. Finally, no generalized transducing phages and no methods for transformation have been described for V. vulnificus. Therefore, if one desires to move mutations between strains, the mutations must be cloned into suicide vectors, and the inefficient two-step allelic exchange process must be repeated for each strain.We describe here the adaption of USER (uracil-specific excision reagent) friendly cloning (3, 14) into allelic exchange and expression vectors that are commonly used with V. vulnificus. USER friendly cloning enables the creation of 3′ overhangs in PCR amplicons by use of deoxyuridine in PCR primers and treatment with USER enzyme mix (uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII). The overhangs are complementary to overhangs created in vectors. The method is very adaptable, with immense leeway in choosing the DNA sequences of the overhangs independent of restriction enzyme cleavage sites. USER friendly cloning methods and vectors enable the rapid creation of upstream-downstream clones to delete genes or loci of interest. To alleviate inefficiencies of subcloning into V. vulnificus vectors, we created an array of USER friendly cloning allelic exchange and expression vectors that should be useful to many investigators.Meibom et al. (32) recently described the ability of Vibrio cholerae to become naturally transformable during growth in the presence of chitin, as either chitohexose or crab shell fragments. We adapted their crab shell system and determined that V. vulnificus can also become naturally transformable during growth on chitin. The bacteria take up linear DNA; hence, as long as selectable markers are used, allelic exchange mutagenesis can be accomplished without the need for inefficient counterselection involving sucrose. We used these methods to create antibiotic resistance-marked deletions in V. vulnificus of the two loci encoding flagellins, flaCDE and flaFBA, and the genes encoding the following functions: the flagellar motor motAB; a critical element of signal transduction for chemotaxis, cheY-3; an RTX toxin, rtxA1; a putative RtxA-modifying enzyme, rtxC; and a regulator of fatty acid metabolism, fadR. These genetic tools and methods will facilitate the molecular genetic analysis of V. vulnificus as well as those of other gram-negative bacteria.     

卡介苗阿拉伯糖苷转移酶embC基因敲除株的构建和初步鉴定

目的采用基因敲除技术构建了卡介苗embC基因缺失株。方法从卡介苗基因组中扩增出embC基因,定向插入自杀质粒p2NIL中,切除embC基因中约1000bp片段使其失活,再定向插入标记片段,筛选鉴定阳性克隆,电穿孔转入卡介苗,筛选重组菌株。结果PCR和酶切鉴定证明构建成功用于基因打靶的置换型自杀质粒,并筛选成功获得重组卡介苗。结论获得了卡介苗embC基因敲除株,为进一步研究对卡介苗免疫活性的影响奠定了基础。     

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa

Background

Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.

Methods and Principal Findings

To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.

Conclusions

We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.     

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.     

Characterization of Tn5-Induced Mutants of Xenorhabdus nematophilus ATCC 19061

A negative-selection vector, pHX1, was constructed for use in transposon mutagenesis of Xenorhabdus nematophilus ATCC 19061. pHX1 contains the Bacillus subtilis levansucrase gene which confers sucrose sensitivity. In addition, various Tn5-containing plasmids with different replication origins were transferred by conjugation from Escherichia coli into X. nematophilus ATCC 19061, and one of these plasmids, pGS9, yields Tn5 insertion mutants of X. nematophilus ATCC 19061. By using these two delivery vehicles, more than 250 putative Tn5 insertion mutants of X. nematophilus ATCC 19061 were isolated and were then characterized. Mutants that were altered in bromothymol blue adsorption, ability to lyse sheep erythrocytes, production of antibiotics on a variety of media, and virulence for Galleria mellonella were found.     

Construction of new cloning vectors that employ the phytoene synthase encoding gene for color screening of cloned DNA inserts in Thermus thermophilus

Strains of Thermus thermophilus produce unique carotenoids called thermozeaxanthins and their colonies are light-yellow pigmented. Here, we developed a new cloning system allowing for the rapid and convenient detection of recombinants by color screening based on carotenoid production in T. thermophilus. We constructed two cloning vectors that overexpress the crtB gene encoding a phytoene synthase under the strong promoter of the slpA gene. Phytoene synthase is one of essential enzymes for the production of carotenoids. We also isolated a carotenoid-overproducing mutant that formed orange colonies. Because disruption of crtB in the carotenoid-overproducing mutant resulted in white colonies, we used the disruptant as a host strain. Whereas transformants carrying a new cloning vector, pTRK1-PRslpA-crtBcas, grew into unusual red-pigmented colonies probably because of the extreme accumulation of thermozeaxanthins, those carrying the vector with a foreign DNA inserts formed white colonies. Thus, recombinants can be detected easily by color screening (red/white screening) in T. thermophilus. This cloning system requires no additional chromogenic substrate in the medium. We also constructed a promoter-probe vector, pTRK1-crtBmcs-PP, employing the open reading frame of crtB with multiple cloning sites. Using this vector, a series of colony-color phenotypes is observed probably depending on promoter activities of foreign DNA inserts, which enables the rapid probing of promoters. These vectors are useful to simplify cloning procedures and to identify the promoters of different strengths in T. thermophilus.     

Deletions in the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA₁₂-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.