Restriction of infectious bacteriophage fd DNA's and an assay for in vitro host-controlled restriction and modification.

Abstract

Arber2 has shown that bacteriophage fd, which contains single-stranded circular DNA (Marvin and Schaller12) is restricted by E. coli B and E. coli (P1) +. This report presents detailed data on the restriction of both the infectious, singlestranded fd-phage DNA and the infectious, double-stranded fd-replicative form (RF)18 DNA. It also describes an infectivity assay for in vitro host-controlled restriction and modification. This assay has been used to find restriction enzymes in fractionated extracts of E. coli (Linn and Arber11). Materials and Methods.-Bacterial strains: E. coli F+meth-gal-rK -mK-18 E. coli F+ metK-leuK-rB+mB + threoB+, E. coli F+met-gal-(P1) +, and E. coli B gal-met-rB -mB+ were kindly provided by Prof. Werner Arber (for derivation of strains, see Arber2). Phage strains: Bacteriophage fd was donated by Prof. Werner Arber; a phage mutant which is only partially restricted by E. coli B (Smith, Arber, and Kuehnlein'7) was the gift of Urs Kuehnlein.Purification and characterization of phage and phage DNA's: The 6X174 phage was purified to the first high-speed centrifugation step described by Rueckert, Zillig, and Huber."1 The fd phage was harvested from the medium of Fraser and Jerrel7 and purified by polyethylene glycol precipitation and CsCl density-gradient centrifugation. The spectrum of the purified phage coincided with the spectrum reported by HoffmannBerling, Marvin and Duerwald.8 Phage DNA was extracted as described by Benzinger, Delius, Jaenisch, and Hofschneider.3 Following exhaustive dialysis against 0.01 M Tris buffer, pH 8, containing 1 mM EDTA, the DNA was further purified by CsCl density gradient centrifugation.The fd RF was isolated according to slight modifications of the procedure of Jansz, Pouwels, and Schiphorst.'0 The filtered RF was sedimented on linear 5-20% sucrose gradients and fractions were collected. Electron micrographs of these fractions (kindly prepared by Dr. H. Delius) showed that more than 80% of the DNA was circular. The fast-sedimenting RF fractions were mostly twisted circles; the slower-sedimenting fractions were mostly open circles.'4 Contamination by single-stranded phage DNA was ruled out as described below. Infectious DNA assays: Lysozyme-EDTA spheroplasts were prepared according to the following modifications of the procedure of Ray, Bscheider, and Hofschneider:13 the harvested cells were resuspended in storage medium (plus 10 ,g/ml of required amino acids) at a titer of 1 X 1010 to 2 X 1010 cells/ml. They were then aerated vigorously for 30 min at 370 (Bscheider, personal communication). The bacteria were treated with lysozyme-EDTA in the presence of Povite albumin for 1 min as described by Ray et al."3 Following addition of MgSO4, the spheroplasts were incubated without aeration for another 30 min at 370 (Bscheider, personal communication). After the spheroplasts had been chilled in an ice bath, they were ready for testing with infectious fd DNA's. The titer of infectious DNA with these preparations remained constant for at least 5 days and did not va...

Restriction of a Transducing Bacteriophage in a Strain of Proteus mirabilis

“…Particular physiological conditions of cells may ca'use weakness in restriction of phages (Bertani & Weigle, I953; Uetake, Toyama & Hagiwara, 1964;Holloway, 1965;Rolfe & Holloway, 1968) and Luria & Human (1952) encountered this condition with the use of stationary phase cells. Benzinger (1968) explains the weaker restriction of phage fd infectious DNA by spheroplasts compared to that of intact fd phage in cells ofE. coli as due to the greater age of spheroplasts.…”

“…Takano et al (I968) found the restricting enzyme of fi-R factors in Escheriehia coli K 12 for phage A to be retained by spheroplasts and the DNA's of bacteriophage fd are restricted by spheroplasts of E. coli r~ + and E. coli (v I) strains (Benzinger, 1968 ). Molholt & Fraser 0965) and Schell & Glover (1966) conclude that a surface-localized enzyme plays an essential role in the host-controlled restriction of phage/~.…”

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

“…The rest might be explained if the single-stranded state of DNA transferred by conjugation affords special protection to incoming DNA, but this is a difficult notion to accept. Even when single-stranded DNA is introduced directly, by transformation, it is equally as sensitive to restriction by EcoB as is double-stranded DNA (6), despite the fact that EcoB, like many other endonucleases (49,56), has little or no measurable activity towards single-stranded DNA in vitro (38).…”

Transfection of Enterobacteriaceae and its applications.

“…Neither has the effect of endonuclease II on transfection been tested. The Kspecific and B-specific restriction endonucleases inactivate unmodified transfecting DNA nearly as strongly as unmodified DNA injected from viable phage (26,30,88). Finally, the weak single-strand specific endonuclease encoded by the recBC genes (131) has no detectable effect on the infectivity of circular, single-stranded 4X174 DNA (32).…”