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1.
2.
Conjugative transfers of drug-resistant plasmids in Shigella dysenteriae type 1 isolated from the epidemic in West Bengal were unsuccessful. The plasmids were non-transmissible in spite of having the fertility factor F, genetically labelled with kanamycin-resistant transposon Tn903 (pWS 7). A small 2.5 kb nonconjugative plasmid encoded for the streptomycin-resistant character. Cured plasmid-less strains of S. dysenteriae 1 showed resistance to sulfonamides.  相似文献   

3.
In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasmids which have been found to be widespread throughout the mycobacteria. Further experiments showed pJAZ38 to be stably inherited in the absence of selection pressure and compatible with the most commonly used mycobacterial replicon, pAL5000. In contrast to pLR7 and pMSC262, pJAZ38 was able to replicate in M. smegmatis mc(2)155, making it a useful tool for mycobacterial genetics.  相似文献   

4.
This study presents the first complete sequence of an IncU plasmid, pFBAOT6. This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G. Rhodes, G. Huys, J. Swings, P. McGann, M. Hiney, P. Smith, and R. W. Pickup, Appl. Environ. Microbiol. 66:3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity. pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed. Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid. The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium. The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria. Partitioning and maintenance genes are homologues of genes in IncP plasmids. The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102. The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS630 relative, and other transposable elements in a 43-kb region that may be a novel Tn1721-flanked composite transposon. This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria. The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.  相似文献   

5.
Abstract The potential of the antibiotic resistance transposon Tn5 for random insertion mutagenesis in Rhodopseudomonas palustris was assessed. The Tn5 containing suicide vector plasmid pSUP2021, was transferred from Escherichia coli to Rhodopseudomonas palustris and kanamycin-resistant transconjugants arose at a frequency of 2.7×10−7 per recipient. In the majority of transconjugants tested, Tn5 was found to have successfully transposed to yield a single chromosomal insertion, with the concomitant loss of the vector plasmid through segregation. Two Tn5 mutants, one defective in carotenoid synthesis, and one exhibiting a reduced anaerobic growth rate on aromatic acids, were partially characterised. This is the first study to show that Tn5 mutagenesis can be applied successfully to isolate mutants of Rhodopseudomonas palustris .  相似文献   

6.
Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.  相似文献   

7.
Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi. This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ (rrmJ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.  相似文献   

8.
Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR assays with chromosomal DNA. PCR with two primers was performed with isolated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analyzed, and the sequence was found to be identical for two E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6-kb signal was also used for detection of the fire blight pathogen in the presence of other plant-associated bacteria and in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finally, arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.  相似文献   

9.
C E Rubens  L M Heggen 《Plasmid》1988,20(2):137-142
The tetracycline resistance gene encoded within the transposon Tn916 was replaced with the gene encoding erythromycin resistance from the plasmid pVA838. The derivative transposon of Tn916 was designated Tn916 delta E and was introduced into the Streptococcus faecalis chromosome by protoplast transformation. The conjugation/transposition functions of Tn916 delta E were similar to those observed for Tn916 in S. faecalis and Tn916 delta E was capable of self-conjugation at frequencies similar to those of other S. faecalis and Group B Streptococcus. This transposon will be useful for mutagenesis studies in gram-positive organisms, especially in those species where erythromycin resistance is a more desirable selectable marker.  相似文献   

10.
K E Weaver  D B Clewell    F An 《Journal of bacteriology》1993,175(7):1900-1909
A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.  相似文献   

11.
Recent studies on the metabolic activities of genus Rhodococcus have shown rhodococci to be of important use in industrial, pharmaceutical and environmental biotechnology. The increasing economic significance of Rhodococcus encourages renewed efforts to characterize their genetic systems, as Rhodococcus genetics are still poorly understood. The goal of this study is to adapt a transposon system for use in creating random mutagenesis in Rhodococcus erythropolis. A plasmid carrying IS1415, a member of IS21 family identified from Rerythropolis, has been constructed and designated as pTNR. pTNR is a non-replicating transposon tool introduced into target cells by electroporation. During its transposition, the transposable-marker gene is separated from the open reading frames (istAB) of IS1415, which should avoid secondary transposition. Transposition of pTNR into wild-type R. erythropolis created mutagenesis with a high efficiency of 1.23x10(6)mutants per microgram plasmid DNA. However, it could also be transposed into other Rhodococcus spp. at lower frequencies in comparison with that of R. erythropolis. It has been indicated by Southern hybridization that the generated kanamycin-resistant mutants were resulted from single transposition event of pTNR. The results also revealed that the transposable-marker gene of pTNR was randomly inserted into the chromosomal DNA of R. erythropolis. The affected DNA regions carrying the transposed DNA element could be conveniently recovered for further characterization using a plasmid rescue procedure. Sequence data of the insertion sites of 40 random mutants analyzed indicated that transposition of pTNR generated 6-bp direct target duplications in 36 cases, while in the remaining four mutants; it generated 5- or 7-bp target duplications (two cases each). This study concluded that pTNR could be served as an efficient genetic tool for construction of random mutagenesis system in Rhodococcus species.  相似文献   

12.
The kanamycin resistance encoded by the neomycin phosphotransferase II gene (nptII) of transposon Tn5 is widely used in higher plant genetic transformation. The general process of plant transformation using nptII as a selectable marker gene, however, requires selecting kanamycin-resistant plants or tissues in culture. Even with the recently developed vacuum infiltration method for Arabidopsis transformation, the plant culture steps are not completely eliminated in selection for kanamycin-resistant transformants. The herbicide resistance genes, such as bar, which provides resistance to bialaphos, allow Arabidopsis transformation to become a true non-culture procedure. In this report, we assessed the feasibility of applying kanamycin as a spray in selecting for kanamycin-resistant Arabidopsis transformants grown in soil. We find that kanamycin-resistant transformants were effectively selected by spraying soil-grown Arabidopsis seedlings.  相似文献   

13.
Summary Parameters influencing the stable transformation of Sorghum bicolor protoplasts with a chimeric neomycin phosphotransferase II (NPT II) gene by electroporation were investigated. The mean number of kanamycin-resistant calli produced increased in direct proportion to the concentration of DNA used for transformation. Linearization of the plasmid doubled the mean number of kanamycin-resistant calli produced, while the addition of carrier DNA had no effect. The copy number (1–4) of integrated genes was low compared with that frequently reported for PEG-mediated transformation. Two strategies for transforming protoplasts with a nonselectable, -glucuronidase (GUS) gene were compared. One utilized a plasmid containing a CaMV 35S-NPT II gene covalently linked to a CaMV 35S-GUS gene, and the other strategy utilized the two genes on separate plasmids. DNA from all 77 kanamycin-resistant calli analyzed contained restriction fragments hybridizing to the NPT II probe; approximately 70% of the clones from all transformation treatments contained a 1.7-kb EcoRI/HindIII restriction fragment corresponding to the full-length gene. Of the kanamycin-resistant calli, 38–63% (depending on the transformation treatment) contained GUS-hybridizing fragments, and 8–19% contained the full-length gene. The addition of NPT II and GUS genes on a single plasmid or on separate plasmids did not appear to lead to an appreciable difference in the frequency of cointegration of these genes, although an increased proportion of the plasmid bearing the nonselectable (GUS) gene appeared to favor its cointegration.  相似文献   

14.
ColE1 copy number mutants.   总被引:1,自引:1,他引:0       下载免费PDF全文
  相似文献   

15.
Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd- mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd- phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10 insertions were also found to be unstable in PAO1.  相似文献   

16.
Relationships among some R plasmids found in Haemophilus influenzae.   总被引:17,自引:1,他引:16       下载免费PDF全文
Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 X 10(6) daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSF007 isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSF007 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSF007 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UB2832. We conclude either that the strains we studied received R factors of the same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a commom resident plasmid of H. influenzae.  相似文献   

17.
Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.  相似文献   

18.
In order to study functional gene expression in Streptomyces coelicolor, a mini-transposon encoding the apramycin resistance gene aac(3)IV within its inverted repeat (IR) boundaries was constructed based on IS204, which was previously identified in the genome of Nocardia asteroides YP21. The mini-transposon and IS204 transposase gene were then put on a kanamycin-resistant conjugative plasmid pDZY101 that can only replicate in Escherichia coli. After mating with S. coelicolor A3(2) M145, resistant colonies arose efficiently on both apramycin and kanamycin plates. Plasmid rescue indicated that entire plasmids were inserted into the M145 genome with cleavage at an inverted repeat junction formed by the right inverted repeat (IRR) and the last 18 bp of the transposase gene, while the left inverted repeat (IRL) was untouched. Southern blot analysis of the mutants using an aac(3)IV gene probe showed that transposition of plasmid pDZY101 was genetically stable, with a single-copy insertion within the S. coelicolor M145 genome. Several mutagenesis libraries of S. coelicolor M145 were constructed using plasmid pDZY101 derivatives and the transposon insertion site was determined. The correlation between novel mutant phenotypes and previously uncharacterized genes was established and these transposon locations were widely scattered around the genome.  相似文献   

19.
Broad-host-range IncP and IncQ plasmids have been transferred to the aerobic magnetic bacterium Aquaspirillum sp. strain AMB-1. Conjugal matings with Escherichia coli S17-1 allowed high-frequency transfer of the RK2 derivative pRK415 (4.5 x 10(-3) transconjugant per recipient cell) and the RSF1010 derivative pKT230 (3.0 x 10(-3) transconjugant per recipient). These plasmids successfully formed autonomous replicons in transconjugants and could be isolated and transformed back into E. coli, illustrating their potential as shuttle vectors. A mobilizable plasmid containing transposon Tn5 was transferred to Aquaspirillum sp. strain AMB-1 and also to the obligately microaerophilic magnetic bacterium Aquaspirillum magnetotacticum MS-1. Five nonmagnetic kanamycin-resistant mutants of Aquaspirillum sp. strain AMB-1 in which Tn5 was shown to be integrated into the chromosome were obtained. Different genomic fragments containing the mutagenized regions were cloned into E. coli. Two genomic fragments were restriction mapped, and the site of Tn5 insertion was determined. They were shown to be identical, although derived from independent transposon insertions. One of these clones was found to hybridize strongly to regions of the A. magnetotacticum MS-1 chromosome. This is the first report of gene transfer in a magnetic bacterium.  相似文献   

20.
Abstract Lactobacillus plantarum ATCC 8014 was transformed with pTV1 by electroporation using a modification of a procedure described for Escherichia coli . The plasmid pTV1 which contains the pE194 replicon from Staphylococcus aureus and transposon Tn917 from Streptococcus faecalis was shown to replicate as a high copy number plasmid in L. plantarum , and the two encoded antibiotic resistance traits were expressed. Tn917 transposed with a high frequency into plasmid DNA of L. plantarum as shown by restriction enzyme analysis and Southern hybridization studies. There are no previous reports on transposition in the lactobacilli. This system may prove to be an important tool in further work on the genetics of these organisms.  相似文献   

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