Mutation in gal U gene of E. coli blocks phage P1 infection

SHORT

Mutation

COMh~lUNICATIONS

in gal U Gene of E. coli

Blocks Phage

Pl Infection’

A new observation shows that phage Pl plates poorly on bacteria which are unable to ferment galactose (Gal-) by virtue of a mutation in gal U, the structural gene for uridine diphosphoglucose pyrophosphorylase (1, 2). By selecting on gal U bacteria, mutants of PI were found which plate effectively on gal U as well as adsorbing with increased efficiency to normal bacteria. Conversely, the selection of bacteria resistant to Pl yielded strains with mutations or deletions of gal U and adjacent genes. Pl cl? forms plaques on Shigella dysenteriae, on Escherichia coli 1112 and on E. coli C. It does not plate on K12 gal U (W4597) (3) nor on a transductant of E. coli C which has incorporated the gal U mutation of W4597. On another K12 derivative which also has gal U from W4597, Pl produces very faint plaques with an efficiency approaching 1. (These bacteria are all nonpermissive for a variety of amber and ochre phage mutants.) Plaques of Pl clr are found on W4597 and on C gal U at a frequency of 10m5 to lo-‘. When propagated, these prove to be genetically stable and to have properties similar to each other. One, called Pl clr g, was taken for further study. The plating properties of Pl clr and Pl clr g with respect to a variety of hosts show that genetic mutation rather than host modification is the basis of the g character (Table 1). Furthermore, Pl cZr g apparently is not dependent upon glucosylation by means of the gal U function, as are the T-even phages (4, 5). Table 1 also shows that restriction by host K12 of PI clr or Pl clr g grown on E. coli C is slight compared to that of phage X (0 The ability of Pl clr and Pl clr g to adsorb to bacteria was best measured by 1 This research was supported by a grant from National Science Foundation. 2 The Plkc stock used came originally from the collection of S. E. Luria. Pl clr was isolated from Plkc as a clear mutant, which gives partial lysis of Pl lysogens and is itself unable t,o lysogenize. It was identified as a mutation of gene cl by June R. Scott (Virology 36, 564-574. 1968). the

189

survival of bacteria (Table 2). Parental Pl clr shows no ability to kill in the absence of Ca*+, and little killing effect on a gal U bacterium (with 0.005 M Ca*+ present). Mutant Pl clr g displays a greater killing effect than Pl clr under all circumstances. The ability of PI clr g to kill in the absence of Ca?+ is reflected in the highly sectored appearance of bacterial colonies derived from bacteria surviving exposure to PI clr g and plated on a medium substantially free of Ca*+. Single-step growth measurements show burst sizes of lo-50 for Pl clr on gal U-t bacteria, 20-100 for Pl clr g on gal U+ or gal U-. Pl clr shows very little if any replication in gal U bacteria (even if Pl clr g infects simultaneously). PI ckr phages recovered from attempted passage through gal U bacteria are unable to form plaques on the same gal U strain. Probably they represent unadsorbed phage particles, but if they are descendants they have not been adapted during passage through gal U. The simplest interpretation would be that Pl clr cannot efficiently adsorb to, or inject its DIVA into, gal U mutants of E. coli. The ability of Pl clr and Pl clr g to transduce reflects their ability to interact with the recipient bacteria. Pl clr transduces a gal U- recipient at a frequency lo- to loo-fold lower than a gal Uf. In the absence of Ca2+ it transduces a gal U+ recipient with a frequency less than 5 % of the frequency in the presence of 0.005 M Ca*+. Pl clr g, on the other hand, transduces efficiently either with a host which is gal U- or in the absence of Ca*+. Under conditions of poor infection, transducing efficiency is expected to be greater than plaque-forming efficiency, since a single infection event suffices for transduction, whereas the difficulty is compounded for plaque formation. The role of the gal U function in this situation is indicated by the following: (1) Of the rare revertants of W4597 to Gal+, three regained full sensitivity to Pl clr, two regained partial sensitivity, and one remained resistant. (2) Among mutants of E. coli C (10-6-10-7) resistant to Pl clr, 4 out of 20 were Gal- and sensitive to Pl cZr g. The Gal+ PI-resistant types were nonreceptive

190

SHORT

Pl

LYSATES

GROWN

COMMUNICATIONS TABLE TITERED

AND

1 ON VARIOUS Titer,

Titer

on Shigella

Pl

c1r.Ca .K czr g.c .K .C gal U .K gal U

a Designates

host

9.2 1.2 1.1 2.1 3.0 3.3

used in preparation TABLE

SURVIVAL

W1485 W4597, Gal+ revertant W4597 gal U

108 10’0 10’0 10’0 10’0 10’0

1.6 0.73 0.86 0.94 1.0 0.93

of the lysate.

2

OF BACTERIA EXPOSED FOR 20 MINUTES’

Bacteria

x x x x x x

TO Pl

PHAGES

Ca2+ .005 M

Pl clr

Pl clr g

+ + +

0.22 1.0 0.094 0.54

0.017 0.72 0.0068 0.56 0.039

a Bacteria (about 5 X lO*/ml) were in tryptone broth at 37”. Phage were added at multiplicity 46. Very similar figures were obtained in a repeat experiment.

to Pl clr g, indicating alternate modes of resistance to Pl, as is also known for temperate phages h and 680. The Gal- Plresistant mutants were restored to Gal+ by a high-frequency transducing phage 480-d gal U, showing their defect in galactose utilization to lie in the gal U gene (7, 8). In cultures of E. coli 1112 W1485, resistance to Pl cZr was more frequent (10d5) than in C and often accompanied by mucoid colony appearance; no Gal- were found in a small sampling. (3) As in other instances where positive selection for spontaneous mutants is possible (9, lo), it might be anticipated that selection for Pl-resistance would serve to discover bacteria with deletions of gene segments including the Pl-resistance locus. The map of E. coli in the vicinity of gal U is as follows (7, 8, 10, 11): ...

purB

suII1

gal

U

tdk-

relative

TYPES to Shigella

16, on

16 C

Pl

BACTERIAL

c gal u

K12

K gal U

10-T 10-e 0.84 0.94 1.2 1.4

0.39 1.4 0.33 1.3 0.33 1.2

10-S 10-S 0.23 1.3 0.33 1.6

C is E. coli C, K is E. coli K12.

By selection of Pl-resistance in a strain of E. coli C lysogenic for a @O-related prophage, there was found among 10 Gal- bacteria one, which in addition to becoming Gal- is no longer able to produce plaqueforming particles from its prophage. This strain was still immune to phages of its prophage type and was able to supply the phage P and R functions upon superinfection with a X phage lacking those functions. Since it was not possible by Pl transduction to separate genetically the Gal- character from the defective prophage, we assume the existence of a deletion covering the gal U gene and extending into the prophage. Thus, it is possible to select for mutation of the gal U gene as well as for deletions which enter the nearby prophage. In contrast to previous prophage deletions (IO), those generated here enter the prophage at the end specifying early phage functions. Analogous deletions extending into the early end of x have been reported recently (12). Deletions of gal U can also be found by selecting chlorate- or colicin Ez-resistance (S. Adhya, personal communication). Other cases are known in E. coli (5, IS), Bacillus subtilis (14) and Salmonella (16) where altered metabolism of galactose causes changes in cell wall structure which prevent infection by a variety of phages. REFERENCES 1. FUKASAWA, T., JOKURA, K., Biochem. Biophys. 125 (1962).

480 prophageNiPRAh prophage genes early + +-- late

ton B

K., and KURAHASHI, Res. Commun. 7, 121-

tryp operon . . .

SHORT

COMMUNICATIONS

2. SUNDARARAJAN, T. A., RAPIN, A. M. C., and KALCKAR, H. Mr., Proc. Natl. Acad. Sci. U.S. 48, 2187-2193 (1962). 3. Collection of Dr. E. Lederberg. 4. HATTMAN, S., and FUKASAWA, T., Proc. Natl. Acad. Sci. U.S. 50,297-300 (1963). 5. SHEDLOVSKY, A., and BRENNER, S., Proc. Natl. Acad. Sci. U.S. 50, 300-305 (1963). 6. ARBER, W., and DUSSOIX, E., J. Mol. Biol. 5, 18-36 (1962). 7. ANDOH, T., and OZEKI, H., Proc. Nat2. Acad. Sci. U.S. 59,792-799 (1968). 8. LANDY, A., ABELSON, J., GOODMAN, H. M., and SMITH, J. I>., J. Mol. Biol. 29,457-471 (1967). 9. CURTISS, R., J. Bacterial. 89, 2840 (1965). 10. FRANKLIN, N. C., DOVE, W. F., and YANOFSKY, C., Biochem. Biophys. Res. Commun. 18, 910-923 (1965). 11. IGARASHI, K., HIRAGA, S., and YURA, T., Genetics 57, 643-654 (1967). 12. ADHYA, S., CLEARY, P., and CAMPBELL, A., Proc. Natl. Acad. Sci. U.S. 61,956962 (1968). IS. SYMONDS, N., STACEY, K. A., GLOVER, S. W., SCHELL, J., and SILVER, S., Biochem. Biophys. Res. Commun. 12, 220-222 (1963). 14. YOUNG, F. E., Proc. Natl. Acad. Sci. U.S. 58, 2377-2384 (1967). 15. STOCKER, B. A. D., Colloq. Internat. Centre Natl. Recherche Scientisyue. (In press) (1969). NAOMI

C. FRANKLIN

Department of Biological Sciences Stanford University Stanford, California 94506 Accepted February 2Y, 1969

Loss of Host-Controlled Modification

Restriction

of Phage

X in Escheri-

chia co/i K12 Previously with UV-Irradiated phage

and

Infected Coli-

T3l, 2

Host range properties of bacteriophages are altered not only by genetic mutation, but also by the process of host-controlled 1 This investigation was supported NSF Grant GB-5975 and by U.S.P.H.S. Grant CA-5016. 2 Taken in part from a dissertation by R. J. Grass0 to the State University York at Buffalo in partial fulfillment quirements for the degree of Ph.D.

in part by Training submitted of New of the re-

191

modification (4, 12). For example, phage X propagated in Escherichia coli C (designated as X.C) plates with an efficiency of only ~10-~ on E. coli K12, whereas X propagated in E. coli 1~12 (designated as X.K) plates with an efficiency of 1.0 on this host. The ability of Et coli K12 to prevent the growth of most X.C particles is termed host-controlled restriction. X.K particles, unlike X.C, are insensitive to the restriction mechanism in E. coli Ii12 because the virus carries Kl2-host specificity which is a nongenetic and as yet unidentified change in the structure of its D1VA (2). The occasional plaques which arise when X.C infects E. coli K12, result from the presence in E. coli K12 cultures of a few “special” cells which have lost their abilitv to restrict X.C, but still maintain their ability to modify, producing X.K progeny (4, 15). The frequency of these special cells is normally very low, but can increase if restricting cultures are exposed to ultraviolet light (UV) (4), elevated temperatures (11, 17), or by growth into stationary phase (13, 16). Recently, we showed that, in restricting E. coli Ii12 cultures, the special cell frequency for X.C increases rapidly after methionine deprivation (7,8). This observation may be related to the fact that X-adenosylmethionine (SAM) is a necessary cofactor for the K12restriction enzyme to act on X DNA (14). In considering the possibility that the methionine effect might be mediated through a reduction of the SAM pool, we have allowed X.C to infect E. co& 1~12 cultures that had been previously infected with UV-irradiated T3 (UV-T3), a phage capable of inducing an enzyme which cleaves SAM (6). We find that prior infection of 1~12 by UV-T3 impairs the K12-restriction of X.C and that the progeny phage which arise have failed to acquire Kl2-host specificity. The ability of UVT3 to inhibit modification was first reported by Klein (10) for the I’1 lysogen induced modification of phage Tl. More recently, Hirsch-Kauffman and Sauerbier (9) have reported results which are in the main similar to those described here, namely, that both restriction and modification of X are inhibited in cells infected with UV-T3. E. coli C, E. coli K12 strain W3350, and