mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites: example Burkholderia mallei ATCC 23344

The mini-Tn7 vectors are universally applicable in gram-negative bacteria and thereby facilitate the manipulation of many organisms for which few genetic systems are available. These vectors, when provided with only the Tn7 site-specific transposition machinery, insert site and orientation specifically in the bacterial chromosome at an attTn7 site downstream of the essential glmS gene. A few bacteria, including Burkholderia spp., contain multiple glmS genes and therefore several attTn7 sites. Here we provide a protocol for application of the mini-Tn7 system in B. mallei as an example of bacteria with multiple glmS sites. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into B. mallei by conjugation, followed by selection of insertion-containing strains; and last, PCR verification of mini-Tn7 insertions. B. mallei possesses two glmS genes on chromosome 1 and Tn7 transposes to both sites, although transposition to attTn7-1 associated with glmS1 occurs in more than 90% of the clones examined. Transposition is efficient and the whole procedure from start to verification of insertion events can be done in less than 5 d. This first chromosome integration system in B. mallei provides an important contribution to the genetic tools emerging for Burkholderia spp. Vectors are available for gene complementation and expression, and gene fusion analyses.     

A Simple Method for Multiple Modification of the Escherichia coli K-12 Chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by λ Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.     

Mini transposon vector mediated foreign gene expression in Mesorhizobium huakuii subsp. rengei

Among the transposable elements, mini-Tn5 transposon vector has proven to be of greater utility for insertion mutagenesis of variety of Gram negative bacteria. The mini-Tn5 vector containing promoter less egfp gene and gentamycin resistant gene was used for the present study. The transposon vector was introduced to M. huakuii from E. coli S17 by conjugation. The conjugants were screened for stable expression of egfp both in free-living and in nodules of Astragalus sinicus. The result showed that the conjugant #3 showed stable expression of green fluorescent both in free-living and bacteroid stage. The visualization of sym plasmid of wild strain and conjugants showed that conjugant #3 had a fragmentation of large sized plasmid into two but without affecting the nodulating ability. These results clearly indicated that mini-Tn5 vectors (Transposon vectors) the best alternate tools for plasmid vectors for integration of foreign genes in chromosomal DNA or symbiotic plasmid and expression, both in free-living and bacteroid stage of Rhizobium.     

Effects of Insert Size on Transposition Efficiency of the Sleeping Beauty Transposon in Mouse Cells

Transposon vectors are widely used in prokaryotic and lower eukaryotic systems. However, they were not available for use in vertebrate animals until the recent reconstitution of a synthetic fish transposon, Sleeping Beauty (SB). The reacquisition of transposability of the SB transposase fostered great enthusiasm for using transposon vectors as tools in vertebrate animals, particularly for gene transfer to facilitate accelerated integration of transgenes into chromosomes. Here, we report the effects of insert sizes on transposition efficiency of SB. A significant effect of insert size on efficiency of transposition by SB was found. The SB transposase enhanced the integration efficiency effectively for SB transposon up to approximately 5.6 kb, but lost its ability to enhance the integration efficiency when the transposon size was increased to 9.1 kb. This result indicates that the SB transposon system is highly applicable for transferring small genes, but may not be applicable for transferring very large genes. Received October 20, 2000; accepted December 15, 2000.     

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.     

mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa

Broad host-range mini-Tn7 vectors facilitate integration of single-copy genes into bacterial chromosomes at a neutral, naturally evolved site. Here we present a protocol for employing the mini-Tn7 system in bacteria with single attTn7 sites, using the example Pseudomonas aeruginosa. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. aeruginosa by either transformation or conjugation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. From start to verification of the insertion events, the procedure takes as little as 4 d and is very efficient, yielding several thousand transformants per microgram of input DNA or conjugation mixture. In contrast to existing chromosome integration systems, which are mostly based on species-specific phage or more-or-less randomly integrating transposons, the mini-Tn7 system is characterized by its ready adaptability to various bacterial hosts, its site specificity and its efficiency. Vectors have been developed for gene complementation, construction of gene fusions, regulated gene expression and reporter gene tagging.     

Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosomes deleted mutant NM4 of <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> MSR-1

A magnetosome deleted mutant NM4 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 transposon mutagenesis, and a 5045-bp fragment flanking mini-Tn5 in NM4 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved six putative open reading frames (ORFs); the mini-Tn5 was inserted into ORF4. Functional complementary test indicated that the 5045-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The protein encoded by ORF4 had 25% of identity with the chemotaxis protein CheYIII of Caulobacter crescentus CB15, and the protein encoded by ORF4 contained a conserved signal receiver domain that can receive the signal from the sensor partner of the bacterial two-component systems. It was suggested that the protein encoded by ORF4 may take part in the signal transduction relating to biosynthesis of magnetosomes.     

High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon

Summary A DNA fragment of the broad host range plasmid RP4 carrying the cis-acting DNA recognition site for conjugative DNA transfer between bacterial cells (Mobsite) was cloned into the kanamycin-neomycin resistance transposon Tn5. Using conventrional transposon mutagenesis techniques the new transposon, called Tn5-Mob, can easily be inserted into the host DNA of gram-negative bacteria. A host replicon carrying Tn5-Mob is then mobilizable into any other gram-negative species if the transfer functions of plasmid RP4 are provided in trans. The potential of Tn5-Mob was demonstrated by mobilizing Rhizobium meliloti plasmids as well as the E. coli chromosome at high frequencies.     

Construction of an engineered strain capable of degrading two isomeric nitrophenols via a sacB- and gfp-based markerless integration system

In this study, a gfp-based novel markerless allelic exchange integration system was developed. By employing gfp gene and sacB gene as counter-selectable markers, an ortho-nitrophenol degradation operon (onpABC gene cluster) was successfully inserted into the chromosome of meta-nitrophenol utilizer Cupriavidus necator JMP134. Through two rounds of recombination, the engineered strain (strain JMP134-ONP) was directly selected from the plate by fluorescence screening and has the ability to degrade both ortho-nitrophenol and meta-nitrophenol, simultaneously. This relatively simple and efficient method can be used as an alternative strategy of allelic exchange insertion for the application of metabolic engineering in various bacterial strains, complementary to existing gene knock-in procedures.     

Conjugal Transfer of Plasmid DNA fromEscherichia colito Enterococci: A Method to Make Insertion Mutations

Shuttle vector pAT18 was transferred by conjugation fromEscherichia coliS17-1 toEnterococcus faecalisOG1RF andEnterococcus faeciumSE34. Transfer was mediated by the transfer functions of plasmid RK2 inE. coliS17-1 and the origin of conjugal transfer (oriT) located on pAT18. TheoriTsequence was then inserted into two plasmids to generate vectors pTEX5235 and pTEX5236. These two vectors cannot replicate in gram-positive bacteria and can be used to make insertion mutants in gram-positive bacteria. An internal sequence from an autolysin gene ofE. faecalisOG1RF was cloned into pTEX5235 and transferred by conjugation fromE. coliS17-1 toE. faecalisOG1RF. The plasmid was found to integrate into the chromosome of OG1RF by a single crossover event, resulting in a disrupted autolysin gene. A cosmid carrying the pyrimidine gene cluster fromE. faecalis,with a transposon insertion inpyrC,was also transferred fromE. coliS17-1 toE. faecalisOG1RF. After selection for the transposon, it was found to have recombined into the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. This resulted in apyrCknockout mutant showing an auxotrophic phenotype.     

利用转座子将parDE与pCPP430体内重组提高生防工程菌的遗传稳定性

带有梨火疫欧氏杆菌 (Erwiniaamylovora)完整的hrp基因簇的重组粘粒pCPP43 0转化到植物的附生菌成团泛菌 (Pantoeaagglomerans) 3 0 8R中后可以使成团泛菌获得在烟草等植物上引发过敏反应和诱导植物抗病性的能力。pCPP43 0携带着约 40kb的梨火疫欧氏杆菌的染色体DNA ,在成团泛菌 3 0 8R中不能稳定遗传。本文首先把广宿主范围质粒RK2的控制质粒稳定性的parDE片段插入到转座载体pUT mini Tn5Km的唯一克隆位点NotI上 ,然后利用Tn5的转座特性 ,将parDE体内重组到pCPP43 0上 ,经过在无抗生素选择压下反复传代和过敏反应活性测定 ,筛选到了遗传稳定性显著提高的重组生防工程菌。     

Translating Sleeping Beauty transposition into cellular therapies: Victories and challenges

Recent results confirm that long‐term expression of therapeutic transgenes can be achieved by using a transposon‐based system in primary stem cells and in vivo. Transposable elements are natural DNA transfer vehicles that are capable of efficient genomic insertion. The latest generation, Sleeping Beauty transposon‐based hyperactive vector (SB100X), is able to address the basic problem of non‐viral approaches – that is, low efficiency of stable gene transfer. The combination of transposon‐based non‐viral gene transfer with the latest improvements of non‐viral delivery techniques could provide a long‐term therapeutic effect without compromising biosafety. The new challenges of pre‐clinical research will focus on further refinement of the technology in large animal models and improving the safety profile of SB vectors by target‐selected transgene integration into genomic “safe harbors.” The first clinical application of the SB system will help to validate the safety of this approach.     

Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns

Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.     

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.

Summary A new procedure is described to recombine plasmid-bornelacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flankinglacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncatedlacZ andaadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carryinglacZ andaadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy inPseudomonas putida the regulatory elements controlling expression of the Xy1S-activatedPm promoter of the TOL catabolic plasmid pWWO. We show here that thePm promoter undergoes a Xy1S-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.     

Chromosomal complementation using tn7 transposon vectors in enterobacteriaceae

Genetic complementation in many bacteria is commonly achieved by reintroducing functional copies of the mutated or deleted genes on a recombinant plasmid. Chromosomal integration systems using the Tn7 transposon have the advantage of providing a stable single-copy integration that does not require selective pressure. Previous Tn7 systems have been developed, although none have been shown to work effectively in a variety of enterobacteria. We have developed several mini-Tn7 and transposase vectors to provide a more versatile system. Transposition of Tn7 at the chromosomal attTn7 site was achieved by a classical conjugation approach, wherein the donor strain harbored the mini-Tn7 vector and the recipient strain possessed the transposase vector. This approach was efficient for five different pathogenic enterobacterial species. Thus, this system provides a useful tool for single-copy complementation at an episomal site for research in bacterial genetics and microbial pathogenesis. Furthermore, these vectors could also be used for the introduction of foreign genes for use in biotechnology applications, vaccine development, or gene expression and gene fusion constructs.     

Bi-functional gfp- and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies

The gfp gene, encoding the green fluorescent protein, was combined with the gusA gene, coding for the beta-glucuronidase enzyme, in mini-Tn5 transposon derivatives for use in Gram-negative bacteria. These mini-Tn5 elements allow simultaneously monitoring of gene expression and localization of the marked bacteria. Introduction of the resultant mini-Tn5 transposons into Rhizobium etli, Azospirillum brasilense and Pseudomonas stutzeri allowed us to visualise the interaction of these bacteria with their host plant. The dual-marker mini-Tn5 transposons constitute a powerful new tool for studying gene expression and ecology of bacteria in the environment and during the interaction with plants.     

Mini-Tn7 vectors as genetic tools for gene cloning at a single copy number in an industrially important and phytopathogenic bacteria, Xanthomonas spp.

Transposon mini-Tn 7 vectors insert into the chromosome of several Gram-negative bacteria in a site-specific manner. Here, we showed the application of mini-Tn 7 as single copy site-specific integration vector system for Xanthomonas campestris pv. campestris . The transposition of the mini-Tn 7 into the bacterial genome was detected at a Tn 7 attachment ( att Tn 7 ) site located downstream of glmS1 . Furthermore, using a newly constructed vector pBBR1FLP2 containing the flipase (FLP) recombinase for site-specific excision of the sequence between the FLP recombinase target (FRT) sites, and a sacB counter selection marker, an unmarked mini-Tn 7 insertion mutant was created. Mini-Tn 7 insertion did not affect bacterial virulence on the tested plant. The mini-Tn 7 and FLP–FRT systems also work well in Xanthomonas oryzae pv. oryzae .     

Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil

Pseudomonas putida strain BNF1 was isolated to degrade aromatic hydrocarbons efficiently and use phenol as a main carbon and energy source to support its growth. Catechol 2,3-dioxygenase was found to be the responsible key enzyme for the biodegradation of aromatic hydrocarbons. Catechol 2,3-dioxygenase gene was cloned from plasmid DNA of P. putida strain BNF1. The nucleotide base sequence of a 924 bp segment encoding the catechol 2,3-dioxygenase (C23O) was determined. This segment showed an open reading frame, which encoded a polypeptide of 307 amino acids. C23O gene was inserted into NotI-cut transposon vector pUT/mini-Tn5 (Km^r) to get a novel transposon vector pUT/mini-Tn5-C23O. With the helper plasmid PRK2013, the transposon vector pUT/mini-Tn5-C23O was introduced into one alkanes degrading strain Acinetobacter sp. BS3 by triparental conjugation, and then the C23O gene was integrated into the chromosome of Acinetobacter sp. BS3. And the recombinant BS3-C23O, which could express catechol 2,3-dioxygenase protein, was obtained. The recombinant BS3-C23O was able to degrade various aromatic hydrocarbons and n-alkanes. Broad substrate specificity, high enzyme activity, and the favorable stability suggest that the BS3-C23O was a potential candidate used for the biodegradation of crude oil.     

Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

Pseudomonas putida has emerged as a promising host for the production of chemicals and materials thanks to its metabolic versatility and cellular robustness. In particular, P. putida KT2440 has been officially classified as a generally recognized as safe (GRAS) strain, which makes it suitable for the production of compounds that humans directly consume, including secondary metabolites of high importance. Although various tools and strategies have been developed to facilitate metabolic engineering of P. putida, modification of large genes/clusters essential for heterologous expression of natural products with large biosynthetic gene clusters (BGCs) has not been straightforward. Recently, we reported a RecET-based markerless recombineering system for engineering P. putida and demonstrated deletion of multiple regions as large as 101.7 kb throughout the chromosome by single rounds of recombineering. In addition, development of a donor plasmid system allowed successful markerless integration of heterologous BGCs to P. putida chromosome using the recombineering system with examples of – but not limited to – integrating multiple heterologous BGCs as large as 7.4 kb to the chromosome of P. putida KT2440. In response to the increasing interest in our markerless recombineering system, here we provide detailed protocols for markerless gene knockout and integration for the genome engineering of P. putida and related species of high industrial importance.     

Transposon-mediated insertion of R factor into bacterial chromosome

Summary Insertion of transposon Tn1 into the E. coli JC411 chromosome results in a sharp increase of plasmid RP4 integration frequency. This effect is absent in JC1553 recA cells. The RP4 integration with the chromosome is probably accomplished via recA-dependent recombination between transposon Tn1 inserted into the chromosome and the same transposon in the RP4 plasmid.