Direct selection of DNA inserts in plasmid gene of kanamycin-resistance

Slutsky, Alexander M.; Rabinovich, P. M.; Yakubov, L. Z.; Sineokaya, Irene V.; Stepanov, A. I.; Gordeyev, Vyacheslav K.

doi:10.1007/bf00425867

Cited by 6 publications

(2 citation statements)

References 4 publications

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“…For this purpose, we adapted a published method for positive selection for loss of resistance to kanamycin in Eschenchia coli (33). Strain SH26, a streptomycin-dependent, rifampin-resistant mutant of SH21, was able to grow on media containing Pm instead of streptomycin.…”

Section: Resultsmentioning

confidence: 99%

Conjugational transfer of gentamicin resistance plasmids intra- and interspecifically in Staphylococcus aureus and Staphylococcus epidermidis

McDonnell¹,

Sweeney²,

Cohen³

1983

Antimicrob Agents Chemother

127

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We have previously reported the transfer of gentamicin resistance (Gmr) plasmids in a mixed culture inter-and intraspecifically between strains of Staphylococcus aureus and Staphylococcus epidermidis isolated at Michael Reese Hospital (Jaffe et al., Antimicrob. Agents Chemother. 21:773-779, 1982). We have now shown that representatives of these plasmids were transferred between apparently nonlysogenic strains of S. aureus either in mixed culture in broth or by filter-mating on agar medium. The mechanism of transfer appeared to be conjugation. A transferable Gmr plasmid (pSH8) mobilized or cotransferred a tetracycline R-plasmid and a chloramphenicol R-plasmid that were not independently transferable. The transfer of Gmr plasmids was accompanied by a high incidence of deletion mutations with varied loss of plasmid resistance determinants and, with some mutants, loss of the ability to effect self-transfer. Restriction endonuclease digestion of pSH8 and its deletion mutants made it possible to assign the property of self-transfer to a specific segment of the pSH8 genome and provided the basis for a physical and genetic map of that plasmid. Similar Gmr plasmids from S. aureus strains isolated in locations remote from Michael Reese Hospital had resistance determinants and transfer properties comparable to those of pSH8. Our observations provide evidence for the conjugal transfer of some staphylococcal plasmids, apparently independent of the presence of phage. This mechanism may be of significance in the intra-and interspecific dissemination of resistance to aminoglycosides and other antibiotics in Staphylococcus spp.We have reported the recovery of five classes of closely related gentamicin resistance (Gm) plasmids from Staphylococcus aureus and Staphylococcus epidermidis (13,14). Each plasmid class was defined by its spectrum of antibiotic resistance markers, molecular mass, and restriction endonuclease digestion pattern. Apparently identical Gmr plasmids of each class were present in epidemiologically related isolates of S. aureus and S. epidermidis (35). Similar results have recently been reported by others (2). Evidence of identity between tetracycline resistance plasmids in isolates of S. aureus and S. epidermidis has also been published recently (10,12,34). These findings support the hypothesis that the transfer of plasmids between S. aureus and S. epidermidis occurs in nature.Transduction has been favored as the principal mechanism of genetic exchange between S.

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Section: Resultsmentioning

confidence: 99%

Conjugational transfer of gentamicin resistance plasmids intra- and interspecifically in Staphylococcus aureus and Staphylococcus epidermidis

McDonnell¹,

Sweeney²,

Cohen³

1983

Antimicrob Agents Chemother

127

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“…pRL443, a Km s derivative of conjugal plasmid RP4, was constructed according to the method of Slutzky et al (65). A paromomycin (PM)-dependent derivative of E. coli DH1, SKA003, was isolated by plating the strain on 200 g of PM per ml and propagating it in Luria broth plus 100 g of PM per ml.…”

Section: Methodsmentioning

confidence: 99%

Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120

et al. 1997

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The efficiency of conjugal transfer of plasmids from Escherichia coli to the cyanobacterium Anabaena sp. strain PCC 7120 was quantitated as a function of the number of restriction sites for the restriction enzymes carried by the recipient. In addition to the previously recognized isoschizomers of AvaI and AvaII, PCC 7120 was found to possess an isoschizomer of AvaIII. Plasmids modified in E. coli with methylases that protect in vitro against restriction by the three enzymes were transferred with high efficiency, nearly independent of the number of restriction sites on the plasmid. Plasmids left unprotected against one of the three restriction enzymes were transferred with lower efficiencies. For low numbers of sites, the efficiency of conjugal transfer decreased as an exponential function of the number of unprotected sites. The methods presented may be used to increase the efficiency of conjugal transfer into restriction-competent bacteria.Since first recognized, restriction in vivo has been most closely identified with the reduction of plating efficiency of bacteriophage (7, 10). Although conjugal transfer of unmodified DNA also was shown to be sensitive to restriction in Escherichia coli (10), the inability of certain restriction enzymes to act on single-stranded DNA (49, 56) has led to the view that conjugally transferred single-stranded DNA (37) should be immune from restriction (5, 69). Accordingly, there are many examples in which conjugal transfer is unaffected by restriction barriers that prevent the infection of unmodified phage or the transformation of unmodified DNA (5,20,36,41,55,66). However, there are also reports of restriction in nonenteric bacteria indeed limiting conjugal efficiency (18,44,64,79).Regardless of the biological importance of restriction, the resulting inefficiency of DNA transfer is a frequently encountered barrier against introducing DNA into bacteria of experimental interest. While restriction may reduce the frequency of DNA transfer below levels of detectability (40,42,79), it is also possible that some introduced DNA with unmodified restriction sites may escape destruction. With this in mind, it is often of practical importance to know quantitatively the degree to which each restriction site impairs DNA transfer into restriction-competent recipients.The effect of restriction sites on the efficiency of DNA transfer has been quantitated in several studies using plasmids or phage with a known number of sites. Such studies have measured the effect of restriction on phage infection (47, 48, 54), phage transfection (35), transformation (17,51,71), and electroporation (8,34,43). Reduction of conjugal transfer efficiency by restriction has not been systematically studied, although there have been some quantitative reports (30,70 (14,18,42). We report here the construction of plasmids carrying methylases that protect against one or more restriction activities of Anabaena sp. strain PCC 7120, including a third restriction activity reported here for the first time. Using these plasmids, ...

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Use of phenotypic suppression for direct selection of loss of aminoglycoside antibiotic resistance determinants

Hector

1984

Mol Gen Genet

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A strain of Escherichia coli containing a conditional drug dependent arginine auxotrophy was used to select for the loss of plasmid and/or transposon encoded kanamycin (Km) or streptomycin (Sm) resistance determinants. Because these determinants inactivate the corresponding drug thus eliminating drug suppression, loss of the drug-resistance determinants was selected directly by growth on minimal media plates containing sub-lethal dosages of the drug. This method was used to select loss of Km or Sm resistance determinants due to loss of plasmids, transposition from plasmid to chromosome, and education of transposons from the chromosome. Drug suppression was compared to phage PRD1 resistance in selecting for loss of plasmid vehicles during transposition and was found to be 10-1,000 times more efficient. Eighty percent of the eductant clones had undergone imprecise eductions suggesting that this method may be useful in selecting stable deletion mutants. An antibiotic suppressible strain of Pseudomonas stutzeri was obtained implying a broad utility of this selection procedure.

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Direct selection of DNA inserts in plasmid gene of kanamycin-resistance

Abstract: In this paper we propose a method of direct selection for hybrid DNA molecules in paromomycin-dependent E. coli cells.

Cited by 6 publications

References 4 publications

Conjugational transfer of gentamicin resistance plasmids intra- and interspecifically in Staphylococcus aureus and Staphylococcus epidermidis

Conjugational transfer of gentamicin resistance plasmids intra- and interspecifically in Staphylococcus aureus and Staphylococcus epidermidis

Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120

Use of phenotypic suppression for direct selection of loss of aminoglycoside antibiotic resistance determinants

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