Aug 25, 1972 - the enzyme uridine diphosphoglucose pyrophosphorylase and thus produce .... NOTES tions in Escherichia coli that affect uridine diphosphate.
Vol. 11, No. 1 Printed in U.S.A.
JOURNAL OF VIROLOGY, Jan. 1973, p. 150-152 Copyright © 1973 American Society for Microbiology
T-Even Bacteriophage-Tolerant Mutants of Escherichia coli B III. Nature of the tet Defect MARTINEZ J. HEWLETT AND CHRISTOPHER K. MATHEWS Department of Biochemistry, College of Medicine, University of Arizona, Tucson, Arizona 85724 Received for publication 25 August 1972
T-even phage-tolerant (tet) mutants of Escherichia coli B are shown to lack the enzyme uridine diphosphoglucose pyrophosphorylase and thus produce nonglucosylated progeny phage deoxyribonucleic acid.
lated progeny phage deoxyribonucleic acid (DNA). E. coli B/40 (UDPG pyrophosphorylase negative) and Krgl (permissive for nonglucosylated T2, T4, or T6) were kindly supplied by H. Revel (California Institute of Technology). E. coli B, B/40, tet-1, and tet-2 in the exponential phase of growth in nutrient broth were infected with phage T6 at an input multiplicity of 5. Table 1 shows that all but the E. coli B infection produce progeny which are seen only when the plating bacteria are restrictionless (e.g., Krgl), suggesting defective DNA glucosylation. The tet mutants (along with B and B/40) were assayed for UDPG pyrophosphorylase activity by a coupled enzyme system (1, 4). The data in Table 2 demonstrate that the tet mutants are defective in this activity. Mixing experiments (data not shown) eliminated the possibility of inhibitors in the cell extracts. As a further check of the nonglucosylating phenotype, E. coli B, B/40, tet-1, and tet-2 were infected with phage T6 at an input multiplicity of 5. Unadsorbed phage (at 10 min) were removed by centrifugation and resuspension in fresh medium. After lysis, the resulting progeny phage were purified, and the DNA was extracted with phosphate buffersaturated phenol (8). These DNAs were then used as substrates for the addition of 14Cglucose from UDP- "4C-glucose in a crude phage T6-DNA glucosylating system (a sonically treated extract of E. coli B harvested 12 min after T6 infection) (6; Fig. 1). Since each reaction mixture contained 10 qg of DNA, the relative extent of glucosylation can be estimated from the graph. T6-tet-1-DNA appears to have about as many sites available for glucose addition as does T6-B/40-DNA,
We have reported previously the isolation and properties of some T-even phage-tolerant (tet) mutants of Escherichia coli B (2, 3). These strains seemed to be members of a class of "host-defective" bacterial mutants, also isolated in other laboratories (5; H. Revel, L. Simon, and T. Takano, personal communications), which are defective for some stage in phage development. However, it was suggested that the tet mutants were phenotypically similar to UDPG (uridine diphosphoglucose) (EC 2.7.7.9)-negative pyrophosphorylase strains (H. Revel, personal communication). Such strains could have been picked up by our isolation technique (2), which involved selection of bacterial colonies able to grow in soft (0.7%) agar in the presence of a large excess of phage T6 and T4. If growth of a colony started in an agar "space" devoid of phage, both tet and UDPG-negative strains could continue growth upon encountering phage particles, since infection of a single cell in the colony would produce noninfectious progeny and since the rest of the cells would continue to grow. With wild type, such encounters would produce enough infectious phage to lyse the entire colony. That our strains might be UDPG negative was suggested in part by the absence of detectable viable progeny (after T6 infection of tet and plating on E. coli B), poor adsorption of T4 by these strains, and the failure of the defect to be overcome by phage mutations (3). Indeed, preliminary evidence showed that the tet strains were phenotypically gal(i.e., no growth on galactose), as expected for UDPG-negative strains (7). Evidence presented here supports the idea that the tet mutants are lacking the enzyme UDPG pyrophosphorylase and produce nonglucosy50
151
NOTES
VOL. 11, 1973
TABLE 1. Burst size of phage T6 after infection of various strains of Escherichia coli B Burst size on
Strain B . B ...... .. 100 2 B/40 4 tet-1 tet-2 3
Krgl
77 18
20 50
TABLE 2. UDPG pyrophosphorylase activity Cell extract
,uMoles NADPH per hr per mg of protein
Escherichia coli B B/40
0.540 0.052 tet-1 0.106 tet-2 0.097 a Molar extinction coefficient for the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm = 6.22 cm2/Mmole. Protein assayed by the biuret method with bovine serum albumin as a standard. At 34 C.
whereas T6-tet-2-DNA has about 25% more available sites. The B/40 strain was checked for some of the tet chracteristics. Survival in broth after T6 infection (Fig. 2) and plating in the presence of T4/T6 mixtures (data not shown) were similar to patterns reported earlier for the tet strains (3). Other aspects of the metabolism of phage-infected tet cells (e.g., DNA replication) are currently being reinvestigated with the B/4,, strain. The above data provide proof that the E. coli B tet strains isolated in our laboratory are lacking the enzyme UDPG pyrophosphorylase and are thus unable to provide the substrate (UDPG) necessary for glucosylation of progeny phage DNA. However, this does not rule out the possibility of a secondary mutation in the tet strains. The greater extent of glucosylation of T6 DNA passed through B/40 (Fig. 1) is not accounted for by the levels of enzyme activity found in the respective cells (Table 2). In addition, the failure of phage T3 and T5 to plate on the tet strains, along with their difference in ultraviolet light sensitivity from each other and from E. coli B(2), suggest that the UDPG pyrophosphorylase deficiency may not be the only mutational alteration. To partially answer this question, several gal+ revertants of tet-2 were selected, all of which showed normal plating efficiency for phage T6. However, similar experiments with tet-1 gave mixed results (i.e., gal+ that were either T6+ or T6-), and this strain is still under investigation.
Reoction Time (min.)
FIG. 1. " C-glucose incorporation into various phage DNAs. Data represent 5% trichloroacetic acidinsoluble counts/min in 0.1 ml of reaction mixture containing DNA isolated from phage T6 passed through the indicated bacterial strains.
MOI
FIG. 2. Survival curves for bacteria infected in nutrient broth. Bacterial cultures at about 3 x 101 per ml were infected with phage T6 at the indicated multiplicities. At 6 min, samples were removed, diluted and plated for determination of colony-forming ability, MOI, Multiplicity of infection. This work was supported by Public Health Service research grant AI-08230 from the National Institute of Allergy and Infectious Diseases and grant 70-1013 from the American Heart Association. M.J.H. was a National Defense Education Act Title IV fellow.
LITERATURE CITED 1. Fukasawa, T., K. Jokura, and K. Kurahashi. 1963. Muta-
152
2.
:3. 4.
5.
NOTES
tions in Escherichia coli that affect uridine diphosphate glucose pyrophosphorvlase activity and galactose f'ermentation. Biochim. Biophys. Acta 74:608-620. Mathews, C. K. 1970). T-even bacteriophage-tolerant mutants of' Escherichia coli B. I. Isolation and preliminary characterization. J. Virol. 6:163-168. Mathews, C. K., and M. J. Hewlett. 1971. T-even bacteriophage-tolerant mutants of Escherichia coli B. II. Nucleic acid metabolism. J. Virol. 8:276-285. Mills, G. T., and E. B. Smith. 1965. Uridine diphosphoglucose, uridine diphosphogalactose, uridine triphosphate and uridine diphosphoglucuronic acid, p. 581595. In H-U. Bergmeyer (ed.), Methods of enzvmatic analysis. Academic Press Inc., New York. Pultizer, J. F., and M. Yanagida. 1971. Inactive T4 prog-
J. VIROL.
eny virus formation in a temperature-sensitive mutant of' Escherichia coli K12. Virology 45:539-554. 6. Revel, H. R., and C. P. Georgopoulos. 1969. Restriction of' nonglucosylated T-even bacteriophages by prophage Pt. Virology 39:1-17. 7. Svmonds, N., K. A. Stacev, S. W. Glover. .I. Schell, anld S. Silver. 1963. The chemical basis for a case ot' host induced modification in phage T2. Biochem. Biophys. Res. Commun. 12:22(0-222. 8. Thomas, C. A., and J. Abelson. 1966. The isolation and characterization of' DNA from bacteriophage, p. 55:3 561. on G. L. Cantoni and D. R. Davies (ed.), Procedures in nucleic acid research. Harper and Row, New York.
