Transfection of Lysostaphin-treated Cells of
            <i>Staphylococcus aureus</i>

Riggs, H. G.; Rosenblum, E. D.

doi:10.1128/jvi.3.1.33-37.1969

Cited by 11 publications

(8 citation statements)

References 11 publications

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“…Nomura et al (11) performed transformation in hypertonic broth containing low levels of penicillin and streptomycin. Riggs and Rosenblum (16) reported transfection of two strains of S. aureus with phage deoxyribonucleic acid (DNA) in hypertonic medium only when the cell walls of the recipients had been partially disrupted with lysostaphin. A system for transfection of S. aureus with intact cell walls was recently developed, with 0.1 M CaCl2 in distilled water used as the incubation medium for uptake of phage DNA (18).…”

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confidence: 99%

Transformation of Chromosomal and Plasmid Characters in Staphylococcus aureus

1972

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Transformation of Chromosomal and Plasmid Characters in Staphylococcus aureus

1972

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“…Calcium was added to a final concentration of 0.004 M. The mixtures of phages and bacteria were allowed to stand at 37 C for 10 min to allow adsorption of phages and were then further incubated with shaking at 37 C until lysis occurred. The lysates were purified from bacteria and cell debris by centrifugation at 16,300 x g for 15 min. The phages were pelleted at 107,000 x g for 2 hr.…”

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Transfection of Staphylococcus aureus with Bacteriophage Deoxyribonucleic Acid

1972

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Staphylococcus aureus cells of strain 8325 (N) are competent for phage deoxyribonucleic acid (DNA) when harvested in the early exponential growth phase. Phenotypic expression of the competence requires divalent cations, and calcium ions are most effective. Treatment of phage DNA with deoxyribonuclease completely destroys infectivity and heat-denaturated DNA is not infectious. The highest frequency of transfection is around 104 plaque-forming units per jig of DNA.

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“…The basic obstacle in effecting transformation appears to be the obtaining of high-molecular-weight, biologically active deoxyribonucleic acid (DNA) which would be resistant to the action of staphylococcal deoxyribonuclease, thus permitting the DNA to effect transformation. Riggs and Rosenblum (11) reported that two strains of Staphylococcus aureus were transfected with DNA obtained from staphylococcal phages in hypertonic media.…”

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Induction of Hemolysin Synthesis by Transformation in Staphylococcus aureus

et al. 1971

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A nonhemolytic strain of Staphylococcus aureus was transformed with deoxyribonucleic acid extracted from two hemolytic strains of S. aureus. In each case the hemolysin pattern after transformation was identical to that of the donor strain. However, bacteriophage type, serotypes, and other biological properties of the recipient strain remained unaffected.Information on transformation in staphylococci has been limited to the studies of Pakula (8) and Dobrzanski (3) who investigated intergeneric activities between streptococci and staphylococci.It was suggested by other workers (4, 15) that genetic transformation does not occur either intraor interspecifically in staphylococci. The basic obstacle in effecting transformation appears to be the obtaining of high-molecular-weight, biologically active deoxyribonucleic acid (DNA) which would be resistant to the action of staphylococcal deoxyribonuclease, thus permitting the DNA to effect transformation. Riggs and Rosenblum (11) reported that two strains of Staphylococcus aureus were transfected with DNA obtained from staphylococcal phages in hypertonic media.The investigations mentioned above led us to study transformation in S. aureus; it appears from this study that transformation is achieved by, and may be recognized by, the acquired ability to synthesize beta or delta hemolysins, or both (9).S. aureus strains 48, 406-10, and 8094 were obtained from human lesions. The patterns of bacteriophage typing (1), the serotypes (7, 10), and the enzymatic properties of these strains are recorded in Table 1. Strain 8094 was used as the recipient, and before transformation it could not be induced to produce any hemolysins (14). Several colonies of strain 8094 were picked and grown to concentrations of 109 cells and then tested for hemolytic activity. Several colonies were then picked again, and the procedure was repeated for over 10 serial subculturings and testings. No hemolysin activity was ever demonstrated.Cells of the two donor strains, 48 and 406-10, were grown aerobically in Trypticase Soy Broth (TSB) for 36 hr with constant shaking at 37 C. The cells were collected by centrifugation at 10,000 x g for 30 min and washed twice in EDTA-saline (0.1 M ethylenediaminetetraacetic acid in 0.1 M NaCl solution). DNA was prepared by using a modification of the methods described by Marmur (5,6) and Saito and Miura (12). The washed cells were suspended in 0.1 M triethanolamine-glycine buffer (pH 9.0) containing 1% sodium lauryl sulfate and 0.1 M NaCl. The cells were lysed by repeated freezing in an acetone-dry ice mixture and rapid thawing in a water bath at 60 C. Ribonuclease I (Worthington Biochemical Corp., lot 2-4632) and ribonuclease T1 (Sankyo Co., Tokyo, Japan; lot 8M-020) were employed to remove residual ribonucleic acid (RNA) from the product (50 gg of ribonuclease 1, 30 1ug of ribonuclease T1, and 500 ,ug of DNA per ml). After deproteinizing, the "spool" of DNA was washed with 70% (v/v) ethanol and dissolved in 0.15 M NaCl solution containing 0.015 M sodium citrate. The DNA wa...

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Transfection of Lysostaphin-treated Cells of Staphylococcus aureus

Cited by 11 publications

References 11 publications

Transformation of Chromosomal and Plasmid Characters in Staphylococcus aureus

Transformation of Chromosomal and Plasmid Characters in Staphylococcus aureus

Transfection of Staphylococcus aureus with Bacteriophage Deoxyribonucleic Acid

Induction of Hemolysin Synthesis by Transformation in Staphylococcus aureus

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