Mutagenesis by insertion of drug resistance transposon Tn7 into a vibrio species

Thomson, Jennifer A.; Hendson, M.; Magnes, R M

doi:10.1128/jb.148.1.374-378.1981

Cited by 26 publications

(8 citation statements)

References 19 publications

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“…The chromosomal insertion site and the termini of Tn7 have been sequenced (7). There is evidence that there are also preferred sites or "hot spots" for the efficient insertion of Tn7 into the chromosomes of other bacterial genera (8,9,10,11). The high transposition frequency of Tn7…”

Section: Introductionmentioning

confidence: 99%

Tn7 transposition: a multigene process, Identification of a regulatory gene product

Smith¹,

Jones

1986

Nucl Acids Res

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Tn7 transposition is abolished by the deletion of a 2.2kb HindIII fragment from the central region of the transposon. Transposition is restored when the fragment is present in trans. When this fragment is present in trans with wild-type Tn7, transposition frequencies are stimulated 10-100-fold. The DNA sequence of this fragment has been determined and found to contain one long open reading frame coding for a protein of molecular weight 61 187. We have visualised this protein using a DNA-directed prokaryotic transcription-translation system. This gene may fill a regulatory role in the mechanism of Tn7 transposition. When present in trans the 2.2kb HindIII fragment alleviates a transcriptional block of promoter activity detected in Tn7.

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Section: Introductionmentioning

confidence: 99%

Tn7 transposition: a multigene process, Identification of a regulatory gene product

Smith¹,

Jones

1986

Nucl Acids Res

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“…All or some of these possibilities have been realized in the enterobacteria Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium (1,2,12,15,23,24,29,33) and in other bacteria such as Caulobacter crescentus (18), Pseudomonas aeruginosa (26,27), Rhizobium leguminosarum (3, 5), R. meliloti (17,28), Vibrio cholerae (21), and a Vibrio sp. (36).…”

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confidence: 99%

Tn 5 -Induced Mutations in the Enterobacterial Phytopathogen Erwinia chrysanthemi

Chatterjee

Thurn

Feese

1983

Appl Environ Microbiol

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Escherichia coli (2492/pJB4JI) matings with Erwinia chrysanthemi produced kanamycin resistant (Kmr) transconjugants, a majority of which were gentamicin sensitive (Gm'). A small proportion (about 0.8%) of the Kmr Gm' clones were either auxotrophic or failed to catabolize galacturonate (Gtu-). The R plasmid (pJB4JI) DNA was detected in the parent E. coli strain and in a Kmr Gmr transconjugant, but not in Kmr Gm' E. chrysanthemi strains carrying TnS-induced mutations. In Hfr crosses, Kmr (TnS) was found linked with most mutations. A majority (>95%) of prototrophic recombinants were Kmi, except for Leu+ and Arg+ recombinants which were 30 to 50% Km'. Spontaneous revertants were obtained for all markers except car, gtu, lys, thr, and trp. Prototrophic revertants, with the exception of Met+, Leu+, or His' clones, were Kms. We conclude from both genetic and physical data that TnS transposed from pJB4JI into different sites on the chromosome of E. chrysanthemi.

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“…1% of the transductants were mutant in lateral flagella. Since a variety of mutant phenotypes was observed, the transposition of mini-Mu (Tetr) was not limited to a few sites or "hot spots", as has been reported for another transposon (23). Restriction analysis of several Laf mutants by the Southern blot technique demonstrated the actual insertion of mini-Mu DNA into the V. parahaemolyticus genome.…”

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confidence: 90%

Transposon mutagenesis of marine Vibrio spp

Belas¹,

Mileham²,

Simon³

et al. 1984

J Bacteriol

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MATERIALS AND METHODSReagents, enzymes, and DNA analysis. Antibiotics were purchased from Sigma Chemical Co., St. Louis, Mo., and were used at the following concentrations: tetracycline, 20 ,ug/ml for Escherichia coli and 10 ,ug/ml for Vibrio spp.; ampicillin, 80 ,ug/ml; chloramphenicol,20 ,ug/ml; kanamycin, 80 ,ug/ml. Restriction endonucleases were purchased from New England BioLabs, Inc., Boston, Mass. T4 DNA ligase was purchased from Bethesda Research Laboratories, Rockville, Md. Conditions for restriction and ligation of DNA followed the instructions of the manufacturer. ONPG (oto the arms of TnS-132 (pAl3105) was obtained from Alan Boyd, University of California, San Diego.Mutagenesis with Tn5. Transposon TnS-132 inserted into the genome of transducing phage P1 (P1 clrlOOCM) was obtained as a lysogen of E. coli C600 from D. Berg, Washington University. This transposon was constructed to encode tetracycline resistance in place of the wild-type kanamycin resistance (4, 16). Wild-type TnS could not be used, since Vibrio strains show considerable natural resistance to kanamycin. P1 lysates were prepared by temperature induction (5). The recipient bacterium, V. harveyi BB7 (from R. Belas, Agouron Institute), was grown in LM broth (10 g of tryptone [Difco], 5 g of yeast extract [Difco], and 20 g of NaCl per liter) to 5 x 108 cells per ml. CaCl2 was added to 25 mM, and P1 clrlOOCM::TnS-132 was added to a multiplicity of infection of 0.1. Cells were incubated at 30°C without shaking for 30 min to allow phage adsorption. The cell suspension was then diluted with 5 volumes of LM broth and incubated for 2 h at 30°C with shaking to permit expression of the tetracycline resistance phenotype. To select tetracy-on August 4, 2020 by guest

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Mutagenesis by insertion of drug resistance transposon Tn7 into a vibrio species

Cited by 26 publications

References 19 publications

Tn7 transposition: a multigene process, Identification of a regulatory gene product

Tn7 transposition: a multigene process, Identification of a regulatory gene product

Tn 5 -Induced Mutations in the Enterobacterial Phytopathogen Erwinia chrysanthemi

Transposon mutagenesis of marine Vibrio spp

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