# | Rank | Similarity | Title + Abs. | Year | PMID |
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 |
| 397 | 0 | 1.0000 | PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate. | 2003 | 12563033 |
| 390 | 1 | 0.9989 | A new simple method for introducing an unmarked mutation into a large gene of non-competent Gram-negative bacteria by FLP/FRT recombination. BACKGROUND: For the disruption of a target gene in molecular microbiology, unmarked mutagenesis is preferable to marked mutagenesis because the former method raises no concern about the polar effect and leaves no selection marker. In contrast to naturally competent bacteria, there is no useful method for introducing an unmarked mutation into a large gene of non-competent bacteria. Nevertheless, large genes encoding huge proteins exist in diverse bacteria and are interesting and important for physiology and potential applications. Here we present a new method for introducing an unmarked mutation into such large genes of non-competent Gram-negative bacteria. RESULTS: Two gene replacement plasmids, pJQFRT and pKFRT/FLP, were constructed to apply the FLP/FRT recombination system to introduce an unmarked mutation into a large gene of non-competent Gram-negative bacteria. In our methodology, pJQFRT and pKFRT/FLP are integrated into the upstream and the downstream regions of a target gene, respectively, through homologous recombination. The resultant mutant has antibiotic resistance markers, the sacB counter-selection marker, flp recombinase under the control of the tetR regulator, and identical FRT sites sandwiching the target gene and the markers on its chromosome. By inducing the expression of flp recombinase, the target gene is completely deleted together with the other genes derived from the integrated plasmids, resulting in the generation of an unmarked mutation. By this method, we constructed an unmarked mutant of ataA, which encodes the huge trimeric autotransporter adhesin (3,630 aa), in a non-competent Gram-negative bacterium, Acinetobacter sp. Tol 5. The unmarked ataA mutant showed the same growth rate as wild type Tol 5, but lost the adhesive properties of Tol 5, similar to the transposon-inserted mutant of ataA that we generated previously. CONCLUSIONS: The feasibility of our methodology was evidenced by the construction of an unmarked ataA mutant in the Tol 5 strain. Since FLP/FRT recombination can excise a long region of DNA exceeding 100 kb, our method has the potential to selectively disrupt much larger genes or longer regions of gene clusters than ataA. Our methodology allows the straightforward and efficient introduction of an unmarked mutation into a large gene or gene cluster of non-enterobacterial Gram-negative bacteria. | 2013 | 23594401 |
| 350 | 2 | 0.9988 | Random transposon vectors pUTTns for the markerless integration of exogenous genes into gram-negative eubacteria chromosomes. A set of random transposon vectors pUTTns that facilitates the markerless integration of new functions into the chromosome of gram-negative bacteria has been developed. The vectors, which are derived from mini-Tn5 transposons, are located on a R6K-based suicide delivery plasmid that provides the IS50(R) transposase tnp gene in cis, but they are external to the mobile element. The vectors' conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. Internal to the mini-Tn5 element is a cassette that contains a selectable antibiotic resistance marker (kanamycin, chloramphenicol, or tetracycline resistance gene), a counter-selectable marker (sacB), a 430-bp repeat of the sacB gene 3' end acted as the directly-repeated (DR) sequence, and modified multiple cloning sites (MCS). After two total rounds of transposon integration and recombination between the two DRs, only the exogenous DNA inserted into the MCS (passenger genes) and a single 430-bp scar sacBDR fragment remained in the chromosome after excision. The utility of these vectors was demonstrated by integrating the organophosphorus insecticide hydrolase gene (mpd) into the chromosome of Escherichia, Pseudomonas, Sphingomonas, and Paracoccus species. Sequential integration of another organophosphorus insecticide hydrolase gene (oph) into the previously engineered bacteria, without bringing any selectable markers, was also successful. These engineered bacteria were relatively stable. Cell viability and original degrading characteristics were not affected compared with the original recipients. This shows that the developed system is very useful for the markerless integration of exogenous genes into the chromosome of gram-negative eubacteria. | 2009 | 19778558 |
| 381 | 3 | 0.9988 | A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site. The use of Tn7-based systems for site-specific insertion of DNA into the chromosome of Gram-negative bacteria has been limited due to the lack of appropriate vectors. We therefore developed a flexible panel of Tn7 delivery vectors. In one group of vectors, the miniTn7 element, which is inserted into the chromosome, contains a multiple cloning site (MCS) and the kanamycin, streptomycin or gentamicin resistance markers. Another group of vectors intended for tagging with green fluorescent protein (GFP) carries the gfpmut3* gene controlled by the modified lac promoter PA1/04/03, several transcriptional terminators, and various resistance markers. These vectors insert Tn7 into a specific, neutral intergenic region immediately downstream of the gene encoding glucosamine-6-phosphate synthetase (GlmS) in the tested fluorescent Pseudomonas strains. The gfp-tagging vector containing a gentamicin-resistance marker is useful for tagging strains carrying a Tn5 transposon. Tn5 transposons often carry kanamycin-resistance-encoding genes and are frequently used to generate bacterial mutants and to deliver reporter constructions in gene expression studies. To demonstrate the utility of a dual marker/reporter system, the Tn7-gfp marker system was combined with a Tn5-delivered luxAB reporter system in Pseudomonas fluorescens. The system allowed detection of gfp-tagged cells in the barley rhizosphere, while expression of the Tn5-tagged locus could be determined by measuring bioluminescence. | 2001 | 11348676 |
| 380 | 4 | 0.9988 | Expression of a chloramphenicol-resistance determinant carried on hybrid plasmids in gram-positive and gram-negative bacteria. To analyse the control of chloramphenicol (Cm) resistance conferred by the Staphylococcus aureus plasmid pUB112, a detailed restriction map of this plasmid has been constructed, and the position and orientation of the cat gene have been determined. An MboI restriction fragment carrying the entire cat gene of pUB112 was then cloned in another S. aureus plasmid, the kanamycin (Km) resistance vector pUB110. Depending on the orientation of the incorporated cat fragment, the level of Cm resistance varied dramatically in Bacillus subtilis cells. This effect could not be eliminated by deleting parts of the vector DNA, and only the introduction of a transcription termination signal led to orientation-independent Cm resistance. One such construct was further developed to yield a shuttle vector, replicating both in Escherichia coli and B. subtilis. Using this vector the expression of incorporated genes can be determined in both Gram-positive and Gram-negative bacteria. By in vitro transcription experiments using pUB110 DNA linearized with various restriction endonucleases as template, two pUB110 promoters could be localized and their orientations determined: one promoter controls a gene whose function is unknown, the other regulates the transcription of the KmR gene. | 1984 | 6442250 |
| 349 | 5 | 0.9987 | Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. | 1990 | 2172217 |
| 351 | 6 | 0.9987 | Rapid and efficient cloning of proviral flanking fragments by kanamycin resistance gene complementation. We have developed a technique for the rapid cloning of unknown flanking regions of transgenic DNA. We complemented a truncated kanamycin resistance gene of a bacterial plasmid with a neomycin resistance gene fragment from a gene transfer vector. Optimized transformation conditions allowed us to directly select for kanamycin-resistant bacteria. We cloned numerous proviral flanking fragments from growth factor-independent cell mutants that were obtained after infection with a replication incompetent retroviral vector and identified integrations into the cyclin D2 and several unknown genomic sequences. We anticipate that our method could be adapted to various vector systems that are used to tag and identify genes and to map genomes. | 1999 | 9863001 |
| 389 | 7 | 0.9987 | Implantation of unmarked regulatory and metabolic modules in Gram-negative bacteria with specialised mini-transposon delivery vectors. Engineering of robust and safe microbial cell factories requires genetic tools somewhat different from those traditionally used for laboratory-adapted microorganisms. We took advantage of the properties of broad-host-range mini-Tn5 vectors and two regulated expression systems (LacI(Q)/P(trc) and XylS/Pm), together with FRT-flanked, excisable antibiotic resistance determinants, to generate a set of vectors for the delivery of gene(s) into the chromosome of Gram-negative bacteria. This arrangement of modular elements allows the cloning and subsequent markerless insertion of expression cargoes and leaves behind an antibiotic-sensitive host upon the action of the yeast Flp recombinase. We engineered a Pseudomonas putida KT2440 Pm::gfp strain that displayed strong fluorescence upon exposure to 3-methylbenzoate, a XylS effector, and allowed us to examine the performance of the Pm promoter at the single cell level. We also reconstructed a device for sugar transport and phosphorylation in Escherichia coli independent of the native phosphoenolpyruvate-dependent phosphotransferase system by the stable implantation of genes derived from the obligate anaerobe Zymomonas mobilis. In both cases, the information carried by the implanted genes was stably inherited in the absence of any selective pressure. Deliverable expression systems such as those described here will enhance the applicability of various Gram-negative bacteria in biocatalysis and environmental bioremediation. | 2013 | 22609234 |
| 289 | 8 | 0.9987 | A genetic system that reports transient activation of genes in Bacillus. Site-specific recombination is a powerful tool for precise excision of DNA fragments. We used this characteristic to construct a genetic system to report the transient activation of a promoter by promoting the stable acquisition of an antibiotic resistance marker by the bacterium. The system is composed of two compatible plasmid derivatives from Gram-positive bacteria. One of the plasmids allows the insertion of promoters upstream from tnpI, which encodes the site-specific recombinase of Tn4430. The second plasmid carries two selectable resistance genes: one is flanked by two site-specific recombination sequences and is lost following recombination; in contrast, the other resistance gene becomes functional after the site-specific recombination event. By inserting conditionally controlled promoters (the xylose-inducible xylA promoter or the plcA promoter whose expression is dependent on the growth medium) upstream of tnpI, we demonstrated that our genetic system responds to signals inducing transcription by conferring a new resistance phenotype to the host bacteria. Thus, this system can be used to identify genes which are transiently or conditionally expressed. | 1997 | 9427554 |
| 353 | 9 | 0.9987 | Genome modifications and cloning using a conjugally transferable recombineering system. The genetic modification of primary bacterial disease isolates is challenging due to the lack of highly efficient genetic tools. Herein we describe the development of a modified PCR-based, λ Red-mediated recombineering system for efficient deletion of genes in Gram-negative bacteria. A series of conjugally transferrable plasmids were constructed by cloning an oriT sequence and different antibiotic resistance genes into recombinogenic plasmid pKD46. Using this system we deleted ten different genes from the genomes of Edwardsiella ictaluri and Aeromonas hydrophila. A temperature sensitive and conjugally transferable flp recombinase plasmid was developed to generate markerless gene deletion mutants. We also developed an efficient cloning system to capture larger bacterial genetic elements and clone them into a conjugally transferrable plasmid for facile transferring to Gram-negative bacteria. This system should be applicable in diverse Gram-negative bacteria to modify and complement genomic elements in bacteria that cannot be manipulated using available genetic tools. | 2015 | 28352570 |
| 352 | 10 | 0.9987 | Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines). | 1990 | 2172216 |
| 429 | 11 | 0.9986 | An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria. We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcus faecalis. Since pAT112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by alpha-complementation in E. coli carrying the delta M15 deletion of lacZ alpha. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria. | 1991 | 1657722 |
| 379 | 12 | 0.9986 | Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2. The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning. Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon. By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli. One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae. | 1980 | 7012838 |
| 378 | 13 | 0.9986 | Construction and use of a self-cloning promoter probe vector for gram-negative bacteria. Transposon Tn5 has been used extensively for the genetic analysis of Gram- bacteria. We describe here the construction and use of a Tn5 derivative which contains the ColE1 origin of DNA replication, thereby allowing the cloning of DNA adjacent to the Tn without the need for construction of genomic libraries. The Tn is derived from Tn5-B21 [Simon et al., Gene 80 (1989) 161-169] and contains a promoter-probe lacZ gene and genes encoding resistance to tetracycline and beta-lactams. It is housed within a mobilisable suicide plasmid which can be transferred to a wide range of Gram- bacteria. The Tn was tested using pyoverdine siderophore-synthesis genes (pvd) from Pseudomonas aeruginosa. The simple cloning procedure allowed 15.9 kb of pvd-associated DNA to be cloned; in addition, the lacZ reporter gene allowed the transcription of pvd genes to be studied. The bacteria were resistant to carbenicillin only if the Tn (and hence the beta-lactamase-encoding gene) was downstream from an active promoter. | 1993 | 8386128 |
| 377 | 14 | 0.9986 | Construction of improved plasmid vectors for promoter characterization in Pseudomonas aeruginosa and other gram-negative bacteria. We report the construction of two broad host range promoter-probe plasmid vectors for rapid analysis of promoters in Gram-negative bacteria. The new vectors, pME4507 and pME4510, carry carbenicillin and gentamycin resistance genes, respectively, and are small sized (4 kb) with a flexible multiple cloning site to facilitate directional cloning of putative promoter elements. The vectors allow rapid plate-based screening for promoter activities, using beta-galactosidase as the reporter enzyme. In the absence of an inserted promoter fragment, they display very low background activity, making them a useful tool for analysis of low expression level promoters. | 1998 | 9851050 |
| 263 | 15 | 0.9985 | Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria. BACKGROUND: In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. RESULTS: The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion. CONCLUSION: We isolated and characterized a S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome. | 2005 | 15651989 |
| 440 | 16 | 0.9985 | Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria. Previous studies had localised the gene (mmr) for resistance to methylenomycin A (Mm) to a 2.5-kb PstI fragment in the middle of a cluster of Mm biosynthetic genes from the Streptomyces coelicolor plasmid SCP1. In this paper, the gene has been more precisely located by sub-cloning, and the nucleotide sequence of the whole fragment has been determined. The predicted mmr-specified protein (Mr 49238) would be hydrophobic, with some homology at the amino acid level to tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria. Comparisons of hydropathy plots of the amino acid sequences reinforces the idea that the proteins are similar. It is suggested that Mm resistance may be conferred by a membrane protein, perhaps controlling efflux of the antibiotic. No significant homology was detected by hybridisation analysis between mmr and a cloned oxytetracycline (OTc)-resistance gene (tetB) of the OTc producer Streptomyces rimosus, and no cross-resistance was conferred by these genes. Sequences on both sides of mmr appear to encode proteins. The direction of translation in each case would be opposite to that of mmr translation. This suggests that mmr is transcribed as a monocistronic mRNA from a bidirectional promoter. An extensive inverted repeat sequence between the stop codons of mmr and the converging gene may function as a bidirectional transcription terminator. | 1987 | 2828187 |
| 445 | 17 | 0.9985 | Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm. We describe an in vivo expression technology (IVET)-like approach, which uses antibiotic resistance for selection, to identify Shigella flexneri genes specifically activated in bacteria resident in host cell cytoplasm. This procedure required construction of a promoter-trap vector containing a synthetic operon between the promoterless chloramphenicol acetyl transferase (cat) and lacZ genes and construction of a library of plasmids carrying transcriptional fusions between S. flexneri genomic fragments and the cat-lacZ operon. Clones exhibiting low levels (<10 micro g ml-1) of chloramphenicol (Cm) resistance on laboratory media were analysed for their ability to induce a cytophatic effect--plaque--on a cell monolayer, in the presence of Cm. These clones were assumed to carry a plasmid in which the cloned fragment acted as a promoter/gene which is poorly expressed under laboratory conditions. Therefore, only strains harbouring fusion-plasmids in which the cloned promoter was specifically activated within host cytoplasm could survive within the cell monolayer in the presence of Cm and give a positive result in the plaque assay. Pai (plaque assay induced) clones, selected following this procedure, were analysed for intracellular (i) beta-galactosidase activity, (ii) proliferation in the presence of Cm, and (iii) Cm resistance. Sequence analysis of Pai plasmids revealed genes encoding proteins of three functional classes: external layer recycling, adaptation to microaerophilic environment and gene regulation. Sequences encoding unknown functions were also trapped and selected by this new IVET-based protocol. | 2002 | 12390353 |
| 259 | 18 | 0.9985 | Dual-Plasmid Mini-Tn5 System to Stably Integrate Multicopy of Target Genes in Escherichia coli. The efficiency of valuable metabolite production by engineered microorganisms underscores the importance of stable and controllable gene expression. While plasmid-based methods offer flexibility, integrating genes into host chromosomes can establish stability without selection pressure. However, achieving site-directed multicopy integration presents challenges, including site selection and stability. We introduced a stable multicopy integration method by using a novel dual-plasmid mini-Tn5 system to insert genes into Escherichia coli's genome. The gene of interest was combined with a removable antibiotic resistance gene. After the selection of bacteria with inserted genes, the antibiotic resistance gene was removed. Optimizations yielded an integration efficiency of approximately 5.5 × 10(-3) per recipient cell in a single round. Six rounds of integration resulted in 19 and 5 copies of the egfp gene in the RecA(+) strain MG1655 and the RecA(-) strain XL1-Blue MRF', respectively. Additionally, we integrated a polyhydroxybutyrate (PHB) synthesis gene cluster into E. coli MG1655, yielding an 8-copy integration strain producing more PHB than strains with the cluster on a high-copy plasmid. The method was efficient in generating gene insertions in various E. coli strains, and the inserted genes were stable after extended culture. This stable, high-copy integration tool offers potential for diverse applications in synthetic biology. | 2024 | 39418641 |
| 432 | 19 | 0.9985 | Repressor gene finO in plasmids R100 and F: constitutive transfer of plasmid F is caused by insertion of IS3 into F finO. Fertility factor F confers bacterial conjugation, a process which involves at least 20 tra genes. Resistance plasmids such as R100, R6-5, and R1 have homology with F in the tra region. Conjugal transfer of these plasmids is, however, repressed, while transfer of F is constitutive. Repression of R transfer is due to the existence of the two genes, called finO and finP; constitutive transfer of F is believed to be due to a lack of finO in F. In this paper, we report the identification and DNA sequence of the finO gene of R100, encoding a protein of 21,265 daltons. We show that F does actually encode finO, but the gene has been inactivated by insertion of IS3. Lederberg and Tatum (Nature [London] 158:558, 1946), who discovered sexuality in bacteria, may have had an Escherichia coli K-12 strain harboring such an finO F factor, which facilitated the generation of recombinant progeny useful for genetic analysis of bacteria and established the foundation for molecular genetics. | 1987 | 3027040 |