Apr 11, 1978 - system recognizes unglucosylatedhydroxymethylcytosine residues in phage DNA, while the absence of a functional rB restriction function ...
JOURNAL OF VIROLOGY, Sept. 1978, p. 815-818 0022-538X/78/0027-0815$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 27, No. 3
Printed in U.S.A.
Isolation of a Bacterial Host Selective for Bacteriophage T4 Containing Cytosine in Its DNA JUDITH RUNNELS AND LARRY SNYDER* Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 Received for publication 11 April 1978
An Escherichia coli B strain, B834 galU56, has been isolated which supports growth of bacteriophage T4 with cytosine in its DNA while restricting growth of T4 with hydroxymethylcytosine. This host is partially deficient in uridine diphosphoglucose as determined by the ability of DNA isolated from T4 grown on it to accept glucose in an in vitro assay. In this mutant an intact rgl restriction system recognizes unglucosylated hydroxymethylcytosine residues in phage DNA, while the absence of a functional rB restriction function prevents degradation of unmodified DNA containing cytosine.
Because of the myriad of genetic data available regarding bacteriophage T4, the DNA of this phage is ideal for use in cloning studies. One drawback, however, is the inclusion of glucosylated hydroxymethylcytosine (HMC) in the DNA which renders it immune to the action of most restriction nucleases. During infection, HMC allows T4 DNA to escape cleavage by the Escherichia coli rm (restriction modification) system which recognizes unmodified foreign DNA. Another host restriction system, rgl, is believed to specifically destroy DNA with unglucosylated HMC. The sole source of glucose found in the phage DNA is uridine diphosphoglucose (UDPG), which is synthesized by the host (3). Therefore, infection of a UDPG pyrophosphorylase-deficient host yields T4 with DNA which has less than the normal amount of glucose and, therefore, is subject to restriction by the rgl system upon infection of another E. coli host. Phage unglucosylated due to the absence of the host UDPG donor are designated T*4 (5). The transfer of glucose from UDPG to HMC requires two phage-specific enzymes, the HMCa- and HMC-fi-glucosyl transferases (6), the products of the agt and /3gt genes, respectively (4). DNA from agt ,/gt mutants also lacks glucose. T4 has been made which possesses cytosine rather than HMC in its DNA (10). These virus strains contain mutations in four genes: gene 56
(deoxycytidine-triphosphatase); denA (endo II,
a double-stranded-DNA-specific nuclease which degrades the host chromosome); denB, (endo IV, a single-stranded-DNA-specific endonuclease which recognizes and destroys T4 progeny DNA molecules which contain cytosine); and alc (a gene whose function prevents true late 81E
transcription from a template containing cytosine) (10). Because each of these mutations confers a selective disadvantage on most E. coli strains and reversion of any one of them lowers the cytosine content of the DNA, it was desirable to create a bacterial strain which would select against revertants. It was reasoned that since UDPG is the donor molecule used in T-even phage DNA glucosylation, a host UDPG pyrophosphorylase deficiency (galU-) in an rgl+ genetic background would accomplish this result. Such a host must also lack the normal r component of the rm restriction system which restricts T4 with cytosine (10). T4 with HMC would be restricted because glucosylation of HMC residues would not occur, and rgl restriction nucleases would recognize these unglucosylated HMC residues, destroying the DNA. We have isolated such a strain from E. coli B834 (11) by using mutagenesis with nitrosoguanidine (1) followed by the tet selection procedure of Mathews (7). To demonstrate that the defect in the mutant (B834 galU56) is, in fact, a UDPG pyrophosphorylase deficiency, some in vitro and in vivo assays were performed. The degree to which the phage DNA can be glucosylated by T4 ,B-glucosyl transferase in vitro is a measure of the degree to which the DNA has been underglucosylated in vivo, as this enzyme will glucosylate all available HMC residues (6). Phage DNA was isolated from a number of sources and tested for its ability to accept [14C]glucose from [14C]UDPG by using a crude extract of T4+-infected E. coli B as the source of fB-glucosyltransferase (9). The DNA isolated from T4 grown on E. coli B834 galU56 accepts glucose (Table 1). These data demonstrate that E. coli B834 galU56 is UDPG pyrophosphorylase deficient, although less so than E. coli
NOTES 816 TABLE 1. Extent of glucosylation of phage DNA after growth on E. coli B834 galU56' Source of accepting DNA
Pertinent genotype
% Glucose
accepted 100l 54.3 89.9
T4 agt- fgtT4 fgtT*4 derived from T4/W4597 W4597 infection 0o T4 glucosylated T4/W3110 DNA 53.3 T4/B834 galU56 T*4 derived from B834 galU56 infection ob T4 glucosylated T4/B834 DNA a The extent of glucosylation was determined using in vitro ,B-glucosyltransferase assay. Phage which served as sources of accepting DNA were CsCl purified, the DNA was extracted, and DNA concentration was determined by measuring the absorbance at 260 nm of a dilution of a weighed volume. A crude extract of E. coli B infected with T4+ served as the source of ,B-glucosyltransferase. Incubation mixtures consisted of 20 ,ul of ["4C]UDPG (2.0 x 10-8 mol, 0.04,uCi present in final reaction mixture), 50 ,ul of,-glucosyltransferase, 50 Il of a 0.06-mg/ml acceptor DNA solution, and 80 ,ld of 0.1 M potassium phosphate buffer, ph 7.8 (9). Incubation was at 30°C for 60 min. Denatured calf thymus DNA was added as a carrier, and immediately 5% trichloroacetic acid was added to stop the reaction. Samples were filtered and counted. In each case, the amount of radioactivity in assays performed without acceptor DNA was subtracted from the total radioactivity before percentages were determined. This background radioactivity made up
