Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa

Author:

Darzins A1,Casadaban M J1

Affiliation:

1. Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.

Abstract

Small bacteriophage D3112 transposable elements deleted for most of the phage-lytic functions while retaining the sites required for transposition and packaging were constructed to facilitate genetic studies in Pseudomonas aeruginosa. These mini-D derivatives were constructed with the terminal 1.85 kilobases (kb) of the phage left end and 1.4 kb of the phage right end and either the Tn5 kanamycin resistance or the pSC101 (pBR322) tetracycline resistance determinant. Thermally induced lysates of strains lysogenic for both a mini-D element and D3112 cts (temperature-sensitive repressor) transduced P. aeruginosa PAO recipients to drug resistance at frequencies of between 10(-4) and 10(-5)/PFU of the helper phage. As for the parent plaque-forming D3112 phage, the mini-D171 element could insert itself into many different sites in the chromosome but the frequency of insertion into particular genes varied widely. Among 1,000 insertions, none resulted in auxotrophy but 10 resulted in pigment production. Insertions were also selected in a cloning plasmid with a transduction scheme. At least eight different insertion sites were found to have been used among 10 individual insertions. Transductants harboring these mini-D elements were immune to infection by D3112, since they contained the D3112 repressor gene in the left 1.85-kb terminal fragment. Chromosomal genes were transduced in a generalized fashion 100 to 1,000 times more frequently by the mini-D-D3112 cts lysates than by the D3112 cts phage alone. Mini-D171-D3112 cts lysates also yielded some transductants that retained the drug resistance marker of the mini-D element and which were unstable for the chromosomal transduced marker. This is consistent with the miniduction properties of Mu whereby transduced genes are flanked by two mini-D elements in the same orientation.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

Reference55 articles.

1. Wide distribution of transposable phages in natural populations of Pseeudomonas aerutiginosa;Akhverdyan V. Z.;Genetika,1984

2. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomnonas putida. Construction of broad host-range, controlled expression vectors;Bagdasarian M. M.;Gene,1983

3. Permanent insertion of foreign genes into the chromosomes of soil bacteria;Barry G. F.;Bio/Technology,1986

4. Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulfonamides;Barth P. T.;J. Bacteriol.,1974

5. Nucleotide sequence and exact location of the neomycin phosphotransferase gene from transposon Tn 5;Beck E.;Gene,1982

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