# | Rank | Similarity | Title + Abs. | Year | PMID |
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 |
| 537 | 0 | 1.0000 | Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria. To combine the features of the omega interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposon, called Omegon-Km. The Omegon-Km transposon is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS1. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the omega interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene (alph A) which is expressed well in many Gram-negative bacteria. The IS1 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transposition. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain in which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gram-negative soil and water bacteria (Rhizobium leguminosarum, Paracoccus denitrificans) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event. | 1989 | 2546859 |
| 380 | 1 | 0.9990 | 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 |
| 429 | 2 | 0.9989 | 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 |
| 390 | 3 | 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 |
| 379 | 4 | 0.9988 | 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 | 5 | 0.9988 | 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 |
| 381 | 6 | 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 |
| 349 | 7 | 0.9988 | 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 |
| 353 | 8 | 0.9988 | 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 | 9 | 0.9988 | 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 |
| 377 | 10 | 0.9988 | 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 |
| 350 | 11 | 0.9987 | 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 |
| 391 | 12 | 0.9987 | New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Three types of new variants of the broad-host-range transposon Tn5 are described. (i) Tn5-mob derivatives with the new selective resistance (R) markers GmR, SpR and TcR facilitate the efficient mobilization of replicons within a wide range of Gram-negative bacteria. (ii) Promoter probe transposons carry the promoterless reporter genes lacZ, nptII, or luc, and NmR, GmR or TcR as selective markers. These transposons can be used to generate transcriptional fusions upon insertion, thus facilitating accurate determinations of gene expression. (iii) Tn5-P-out derivatives carry the npt- or tac-promoter reading out from the transposon, and TcR, NmR or GmR genes. These variants allow the constitutive expression of downstream genes. The new Tn5 variants are available on mobilizable Escherichia coli vectors suitable as suicidal carriers for transposon mutagenesis of non-E. coli recipients and some on a phage lambda mutant to be used for transposon mutagenesis in E. coli. | 1989 | 2551782 |
| 428 | 13 | 0.9986 | Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin. | 1986 | 2438417 |
| 286 | 14 | 0.9986 | Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells? The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tcr region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed. | 1984 | 6323922 |
| 263 | 15 | 0.9986 | 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 |
| 451 | 16 | 0.9986 | Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes. Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The A. baumannii xerC and xerD genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported the resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when the cells were grown in a low osmolarity growth medium. Binding experiments showed that the partially purified A. baumannii XerC and XerD proteins (XerC(Ab) and XerD(Ab)) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in the covalent attachment of DNA to a recombinase, probably XerC(Ab), indicating that the first step in the recombination reaction took place. The results described show that XerC(Ab) and XerD(Ab) are functional proteins and support the hypothesis that they participate in horizontal dissemination of resistant genes among bacteria. | 2020 | 32668667 |
| 376 | 17 | 0.9986 | Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis. Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection. | 2001 | 11728719 |
| 382 | 18 | 0.9985 | Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r), that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km(r), and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence. | 2001 | 11292758 |
| 289 | 19 | 0.9985 | 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 |