Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22

Schmidt, Christine; Schmieger, Horst

doi:10.1007/bf00334103

Cited by 41 publications

(30 citation statements)

References 23 publications

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“…pRSM14 was found to carry cyslH on a 4.9-kb insert, and pRSM15 carried the entire cysJIH region on a 7.8-kb fragment which overlapped both ends of the pRSM14 insert. The restriction map of this region (not shown) corresponded to that previously found for the cysJIH region cloned from S. typhimurium LT7 (J. Ostrowski and N. M. Kredich (20) and demonstrated by Schmidt and Schmieger for plasmids containing cloned sequences of P22 (22). In agreement with these investigators, we found that P22HT lysates from the wild type carrying either pBR322 (4.4 kb), pRSM3 (7.1 kb), or pRSM15 (12.3 kb) gave fragments could account for this discrepancy by giving large inserts derived from fragments too small to constitute a complete gene.…”

Section: Methodssupporting

confidence: 75%

Isolation of Salmonella typhimurium cys genes by transduction with a library of recombinant plasmids packaged in bacteriophage P22HT capsids

Monroe

Kredich

1988

J Bacteriol

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We have prepared a library of Salmoneila typhimurium genonic fragments cloned in pBR322 and packaged in P22HT capsids. Plasmids carrying 24 of 26 specific genes searched for were isolated by transduction at frequencies of 1 to 344 per 106 plasmid transductants. All 11 known genes of the cysteine regulon were isolated from this library, indcluding cysK, which we had previously been unable to clone in a recombinant plasmid with an Escherichia coli host. This library provides a simple and rapid method for isolating most S. typhimurium genes by using S. typhimurium itself as a host and should be particularly useful for cloning genes that might be deleterious to E. coli.Genes from enteric bacteria are often cloned in Escherichia. coli hosts owing to high transformation efficiencies and the availability of strains with a large variety of mutant markers. Selection for specific genes is mnade possible by a high degree of genetic relatedness between species, which usually allows a gene product from one to replace the function of its counterpart in another. Exceptions to this rule might be expected for proteins that interact with other species-specific macromolecules, such as those found in mtiembranes or cell walls, and for components of DNA restriction-modification systems (1). Another possible exception is our inability to subclone the Salmonella typhimurium cysK gene in pBR322 by selecting for Cys+ in a cysK E. coli host, even though it was easily obtained in a X phage vector (10). As a result, we investigated the possibility of cloning cysK and other cys genes as well in pBR322 by using S. typhimurium itself as a host. Poor transformation efficiencies of cys mutants led us to create a plasmid library of S. typhimurium DNA fragments packaged in P22HT capsids. We describe the construction of this library and document its content and the ease with which it can be screened for specific genes by transduction. MATERIALS AND METHODSStrains and culture media. All strains were derivatives of S. typhirnuriumn LT2. LB5000 is metA22 metE551 trpD2 leu hsdLT hsdSA hsdSB and is m+ for all three modification systems; LB5010 is a galE derivative of LB5000. Both strains Were obtained from L. Bullas (3). DW378 is trpC109 cysK1772 cysMl770 (11), and SB2309 is trpB223 A(cysK ptsHI41) (5). Other auxotrophic strains used for screening our phage P22 plasmid library were from P. Hartman or K. E. Sanderson, Salmonella Genetic Stock Centre, University of Calgary. Phage P22HT int3 was obtained from M.Stem. The pBR322 derivative pRSM3 was constructed in this laboratory by R. Monroe and carries the cysB region of S. typhimurium LT2 on a 2.7-kilobase (kb) SafI fragment that appears to be identical to that isolated from the LT7 strain (14).Medium E of Vogel and Bonner (25) (19) was used as the rich medium and was supplemented with 0.1 mM L-cystine for the growth of cysteine auxotrophs. Solid medium contained 1.5% agar and either ampicillin (25 ,ug/ml) or tetracycline (12.5 ,ug/ml) when required. Transformation. The method of Hanahan (9) was modifie...

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Section: Methodssupporting

confidence: 75%

Isolation of Salmonella typhimurium cys genes by transduction with a library of recombinant plasmids packaged in bacteriophage P22HT capsids

Monroe

Kredich

1988

J Bacteriol

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“…Lytic Yersinia phages from environmental sources have been shown to perform generalized transduction of small plasmids (19). Plasmid transduction with phage P22 was also reported either by homologous recombination between phage DNA and plasmid DNA that harbored P22 sequences (cointegrate formation), or when a pac-like sequence was present on a plasmid that can be packaged in a headful mechanism from concatemeric DNA (40). It seems feasible to develop PY100 phage derivatives that can be used for transduction.…”

Section: Resultsmentioning

confidence: 99%

Broad-Host-Range Yersinia Phage PY100: Genome Sequence, Proteome Analysis of Virions, and DNA Packaging Strategy

et al. 2008

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PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage Aa⌽23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.

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“…Two ml of the phage lysate was mixed with 5.4 ml of phage dilution buffer (50 mM Tris-HCI (7.5), 0.15 M NaCI, 5 mM MgS0 4 ) containing CsCl (0 = l.740), and the density was finally adjusted to 1.500. Centrifugation was done in a Hitachi RBS swing rotor at 19000 rpm for 27 hours (4°C).…”

Section: Plasmids With Inserted Cohesive End Sites (Cos) Of Phages Sumentioning

confidence: 99%

Transduction of a Plasmid with an Inserted R4 Phage DNA Fragment inStreptomyces lividans

Morino

Takahashi

1997

Bioscience, Biotechnology, and Biochemistry

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Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22

Cited by 41 publications

References 23 publications

Isolation of Salmonella typhimurium cys genes by transduction with a library of recombinant plasmids packaged in bacteriophage P22HT capsids

Isolation of Salmonella typhimurium cys genes by transduction with a library of recombinant plasmids packaged in bacteriophage P22HT capsids

Broad-Host-Range Yersinia Phage PY100: Genome Sequence, Proteome Analysis of Virions, and DNA Packaging Strategy

Transduction of a Plasmid with an Inserted R4 Phage DNA Fragment inStreptomyces lividans

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