Autogenous transduction of phi 11de in Staphylococcus aureus: transfer and genetic properties

Dyer, David W.; Rock, Marcus; Iandolo, John J.

doi:10.1128/jb.158.2.689-695.1984

Cited by 8 publications

(5 citation statements)

References 25 publications

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“…ate stringency. The membrane was dried at 80°C for 10 min and then autoradiographed as previously described (10). It is important to note that plasmid vectors pBR322 and pHV33 share no homology (with the wash stringency used here) with the genomes of S. aureus PS54 and PS54C or bacteriophages L54a or L54b.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of bacteriophage conversion of lipase activity in Staphylococcus aureus

Lee¹,

Iandolo²

1985

J Bacteriol

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Staphylococcus aureus PS54 harbors two temperate bacteriophages and manifests no lipase activity on egg yolk agar. Curing of one of the resident prophages (L54a) restores lipase activity. To study the mechanism of bacteriophage conversion, the prophage was cured, and the gene encoding lipase activity was cloned into pOR322 in Escherichia coli on a 2.9-kilobase DNA fragment of the chromosome. The fragment was subcloned into a shuttle vector and subsequently transformed into S. aureus and BaciUus subfilis. Lipase activity was expressed in all three genetic backgrounds. Transformation and transductional data indicated that conversion is due to insertiona1l inactivation of the lipase gene. Hybridization analysis with probes made from converting-phage DNA and from the cloned fragment confirmed that the phage insertion site resides within the terminal 0.8 kilobase of the insert.

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Section: Methodsmentioning

confidence: 99%

Mechanism of bacteriophage conversion of lipase activity in Staphylococcus aureus

Lee¹,

Iandolo²

1985

J Bacteriol

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“…The purified phage was disrupted by suspending the bands in 2% sodium dodecyl sulfate and extracting twice for 15 min at 37°C with equal volumes of redistilled phenol equilibrated with 0.1 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). The resulting DNA solution was then extracted twice with chloroform-isoamyl alcohol (24:1) and ethanol precipitated (4).…”

Section: Methodsmentioning

confidence: 99%

“…Hybridization analysis. Agarose gel electrophoresis and the procedures for Southern blot transfer and hybridization have been described by us (4). In certain instances, the double-blot procedure of Smith and Summers (25) was used to make mirror image blots of the same gel.…”

Section: Methodsmentioning

confidence: 99%

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Generation of transducing particles in Staphylococcus aureus

Dyer¹,

Rock²,

Lee³

et al. 1985

J Bacteriol

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Transduction of plasmid pC194 and bacteriophage 4llde varied inversely with the multiplicity of infection. As the multiplicity of infection decreased from 10-' to 10-5 PFU/CFU, the transduction frequency of pC194 increased 104-fold; the transduction frequency of 4llde increased 300-fold with a 100-fold decrease in multiplicity of infection. Physical and genetic analysis of the transduced DNA showed that pC194 resided in the phage particle as a random, circularly permuted linear concatemer. In DNA prepared from phage that cotransduced pC194 and 411de, pC194 resided in the transducing phage primarily as a linear multimer of 15.8 kilobases, or about 5.4 pC194 monomers. The pC194 multimer was randomly inserted into the 411 genome.Small staphylococcal plasmids are not transduced by generalized transduction, as are chromosomal determinants (2,27). This was first shown by Grubb et al. (6,7), who demonstrated cotransduction of staphylococcal streptomycin resistance and tetracycline resistance plasmids. Later, Stiffler et al. (26) and Iordanescu (10) extended this observation to include a variety of other small plasmids. These investigations highlight a dilemma that arises because cotransduction of plasmid DNA in Staphylococcus spp. often occurs at frequencies higher than predicted for a random packaging model. Transduction of a single plasmid species occurs at frequencies of about 10-5 to 10-8 per PFU; frequencies vary depending upon the plasmid used and the transducing phage (2). Other unselected plasmids are often cotransduced at between 1 and 10% of the single transduction frequency (9), even though the predicted cotransduction rates for randomly packaged plasmids should be the product of the probabilities of the single transductional events. This high rate leads to the suggestion that cotransducing plasmids are not randomly assorted, but associate in some specific way. Iordanescu (10) suggested that some staphylococcal plasmids become transiently associated during the formation of the transducing particle. Furthermore, this transient association is limited to specific pairs that share short regions of homology (14). Cotransduced plasmids generally resolve into individual replicons in the recipient cell, but occasionally (ca. 1%) the plasmid phenotypes remain associated as stable plasmid cointegrates that arise from site-and orientation-specific recombination (14). Plasmid cointegrates are stable in subsequent transductions or transformations and do not under normal circumstances dissociate into the component replicons (9).Examination of these transient transductional associations has been complicated by the assumption that these events occur at relatively low frequencies in a transducing phage population. However, we have utilized the finding that plasmid transduction occurs at higher rates at very low multiplicity of infection (MOI) (19,20) to directly analyze the transducing DNA in the phage particles. * Corresponding author. t Contribution no. 85-82-J from the Kansas Agricultural Experiment Station, Manhattan. t ...

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“…This phage, 44lde, which arose by in vivo recombination between 411 and the penicillin and erythromycin resistance (Pcr, Emr) plasmid p1258, acts more as a specialized transducing phage in being able to transfer the integrated Emr determinant at high frequencies; 4411de replicates as a plasmid through the presence of the p1258 replicon and is defective as a virus (345). More recently, it has been shown that 4llde is able to transduce the Cmr plasmid pC194, but not the neomycinkanamycin resistance (Nmr Kmr) plasmid pUB110 (112), and that particles able to cotransduce Cmr (pC194) and Emr (4llde) contained DNA composed of a linear multimer of approximately five copies of pC194 inserted randomly into the 411 genome (111).…”

Section: Staphylococcal Bacteriophages and Transductionmentioning

confidence: 99%