VIROLOGY

84,547-550

(1978)

Isolation ADRIANA Section

de Radiobiologie

Cellulaire,

of Ultravirulent BAILONE Laboratoire Accepted

Mutants RAYMOND

AND

d’Enzymologie, October

of Phage A DEVORET

C.N.R.S.,

91190

Gif-sur-Yvette,

France

12,1977

Ultravirulent mutants of phage A, able to grow in the presence of high cellular levels of repressor, have been isolated from ~oZu3. These phages can be divided into three groups of increasing virulence reflecting the number of mutational steps required for their isolation. Multiple mutations in the operator regions leading to a decrease in the affinity of the A repressor for the operator sites seen to be responsible for the ultravirulent phenotype.

In a A lysogen the repressor coded by the c1 gene not only maintains the prophage in a dormant state but also prevents the development of a superinfecting A phage, this property being designated immunity of the lysogen (1). Recently new Escherchia coli strains have been constructed in which c1 gene expression is greatly amplified by the insertion of gene c1 into plasmids such as pKB252 (2, 3). Such plasmids produce elevated cellular levels of the A repressor as shown by the repressor-operator binding assay (3). A phage mutant, Al2 (l), able to overcome the immunity of a lysogen and therefore called virulent (Auir) (l), does not form plaques on a lawn of strain 294 (pKB252) (3). We wanted to correlate discrete elevated cellular levels of the A repressor with increasing degrees of immunity. We sought to isolate phage A mutants with stepwise increased virulence, that is, able to grow in cells containing more and more A repressor. Bacteria with higher levels of A repressor should therefore be immune to the less virulent phages. A determination of the degree of immunity with a phage test would also provide a simple means to estimate the level of the A repressor in the host cell. The bacterial strains used were all derivatives of E. coli K12. From GY 105, sensitive to A (4), were derived GY 105 (A)

and GY 105 (A)2, lysogenic for, respectively, one A prophage or two A prophages in tandem (41, and GY 105 carrying plasmid pKB202 (2) (Table 1). From 294 (pKB252) (3) the pKB252 plasmid was transferred to AB 1157 by F42&c+ mobilization. From NlOO thy (Adu021ulu3) (5, 6) we derived GY 4679 which had lost the Adu plasmid. The four strains, 294 (pKB2521, AB 1157 (pKB252)‘(F-lac+), GY 105 (pKB2021, and GY 105 (A)*, had, respectively, about 300, 100, 5, and 1.7 times higher cellular levels of A repressor than did GY 105 (A), the monolysogen used as a standard (see Fig. 1). The phage strains used, except xc190 cY17 (71, were all derivatives of phage All (1) and therefore carry the u2 and u3 mutations (81, respectively, located in the 0, and OR operator regions (91. Phage A639 was isolated as a conditional virulent phage. It forms plaques on a A lysogen at 40”, but not at 32”. Phage Al46 was isolated by infecting uv-irradiated GY 1211 bacteria (101 with All and plating the infected cells on a lysogenic host to select the mutant phage. Phage A12G was our Gif stock of the classical virulent phage Auir isolated by Jacob and Wollman (1); this phage is a ul mutant derivative of All. The supervirulent phage A326 (11) was given to us by D. Kaiser. We have found that a cellular level of A repressor equal to or above fivefold that of

547 0042-6822/78/0642-0547$02.00/O Copyright 0 19’78 by Academic Press, Inc. All rights of reproduction in any form reserved.

548

SHORT

COMMUNICATIONS TABLE

GENEALOGY

OF

1

ULTRAVIRULENT

Hosts

Phages

Sensitive GY 105 Immune GY 105 (A) Hyperimmune GY 105 (pKB202) AB 1157 (pKB252) (F&c+) 294 (pKB252)

MUTANT@

All

Al~[lo-lo 10-r

A916

Al46 1 5x10-1

A816

A169

A2169

110-a A2668

A,1,,1'"

B Reading from top to bottom in column 1, the bacterial strains have discrete increasing levels of A repressor; the control GY 105 has none. Each repressor level was selective for the phage mutants indicated on the same line. The derivation of each phage mutant is given by arrows. The phage mutants except for A12G and Al46 were isolated as plaque-former8 by plating concentrated stocks of their direct ancestor on host bacteria with a given selective repressor level. The putative frequency of appearance of each mutant is indicated near the arrows. Phages A2668 and A668 were selected as large plaque-formers among minute plaques formed by their respective ancestors A2169 and X169.

a monolysogen prevents the growth of virulent phage ~12 and AC190 cY17. Such bacteria can therefore be designated as hyperimmune. Starting from All we have collected in successive steps phage A mutants, able to grow in hyperimmune bacteria, that we have defined as ultravirulent. These mutants can be divided into three groups of increasing virulence (see Table 2). For each host bacterium a given degree of immunity has been assigned on the basis of the efficiency of plating of the super-infecting phage mutants. Phages A12G and A39 served to distinguish the two main classes of host bacteria, the immune and the hyperimmune. Since phages AC190 cY17 and A639 displayed a reduced growth on a dilysogen, this property defined an immunity step between mono- and dilysogens. Likewise, the pattern of growth of the ultravirulent phage mutants also defined three degrees of hyperimmunity (Table 2). Ultravirulent mutants of group 3 (see Table 2), ~668 and A2668, form plaques on Adu carrying bacteria and thus are insensitive to the cro+ gene product, as is Avs326 (5, 12, 13). The last mutational change must affect a cro binding site. In contrast the super-virulent A326, which forms

‘O’I

104 104

10'

pp protein

102

3.102

FIG. 1. A Repressor levels in immune and hyperimmune A lysogens. The DNA-repressor binding as a function of the concentration of cell extract was performed as described in Refs. (4, 14, 15). Bacteria were grown in L broth, supplemented with 20 pi/ml of tetracycline for bacteria carrying pKB252. The reaction mixtures contained cell extracts prepared from GY 105 (A) (O), GY 105 (A)* (W, GY 105 (pKB202) (A), AB 1157 (pKB252) (F&c+) (m), and 294 (pKB252) (+). The concentration of the input 3H-labeled DNA was 4 pg per nitrocellulose filter. The amounts of A-specific DNA retained on the filters, corrected for the retention of Aimm434 DNA, are shown on the ordinate; the concentration of the cell crude extract is given on the abscissa as amount of protein per filter.

plaques on a Adu carrier (ll), does not grow on hyperimmune hosts. The us326 mutation to super-virulence located in O,, (9) strongly reduces the

549

SHORT COMMUNICATIONS TABLE EFFICIENCY

SuperEaf;ting

Virulent 0.9 0.8

VARIOUS HOSTP Hyperimmune pKB252/F&c+

pKB202

CAY

2.10-4 ----------

Acz9OcYl7 A639

2 ON

Immune (A)’

Non virulent All

OF PLATING

Adv Carrier pKB252

Adv02lu,v,

2.10-4

10-1

10-T

10-T

0.736 0.54*

10-7 10-1

lo-’ 10-1

10-1 lo-’

-

10-1 lo-’

lo-’ 10-7

10-m 10-a

5 x 10-1 10-6

10-7 10-7

10-d 10-N

8 lo-2b : I lo-‘b I --------we

10-4 10-4

I

I I :I L,,,,--,,,

A39

1

1

A12G

1

1

I I 10-s* ,I 4 x 10-a I-----------

1.5

0.336

A816

0.9 1

1

1

Al69 A2169

1 1

1.9 1.5

0.33

2.86

-

1

A668 A2668

1.3

1.9 1

0.33 0.5

3

2.4

0.6

1.1

1.4

1.5

0.6

1

1

10-a

lo-’

lo-’

10-l

Ultravirulent Al46

I :I II

Supervirulent A326

D On hosts with increasing repressor levels: Phage stocks at about lOi phages/ml were serially diluted from 10-l to lo-‘. Samples (0.1 ml) we incubated for 20 min with 0.3 ml of overnight bacterial cultures grown in LB + Thy + Ura and plate% with 3 ml of soft agar. Plaques were counted after overnight incubation at 37”. The efficiency of plating was calculated using GY 105 as standard strain. Calibration for optimal adsorption was done with phage imm434hA, which grew on all bacterial hosts. The vertical and horizontal dashed lines define, respectively, the degree of phage virulence and the degree of cellular immunity. On a Adu carrier host: Phage stocks were diluted from 10e3 to lo-‘. Plating was done as above. The efficiency of plating was calculated using as standard strain GY 4679, a Adu- segregant. b Small-sized plaques.

affinity of the 0, operator for the A repressor (14) without conferring an ultravirulent phenotype. This fact leads us to propose that one of the mutations producing the ultravirulent phenotype might lie in the 0, operator sites. Mutations conferring ultravirulence did not impair the ability of the mutated phage to make A repressor upon infection of a nonlysogenic host (Fig. 2). This means that ultravirulent mutants carry a functional c1 gene. Furthermore, ultravirulent mutants did not show any insertion or deletions detectable by electron microscopy of DNA-DNA hybrids (E. Boy de la Tour, personal communication).

The highest degree of ultravirulence displayed by A668 and ~2668 was obtained after three mutational steps. This suggests that the ultravirulent phenotype results from the accumulation of mutations in the operator regions leading to a decrease in the affinity of the operator sites for the A repressor and the cro proteins. The limited set of ultravirulent mutants we obtained can be greatly expanded. There is certainly not a unique mutational pathway to produce ultravirulence in phage A. It is expected that a specific ultravirulent phenotype will depend on a given combination of mutations in the operator sites.

550

SHORT COMMUNICATIONS provided the A repressor overproducing strains. G. Kellenberger-Gujer and E. Boy de la Tour suggested and performed the electron microscopic examination of ultravirulent phage DNAs. REFERENCES 1. JACOB, F., and WOLLMAN, E..Ann. Inst. Pasteur 87, 653-673 (1954). 2. BACKMAN, K., Ph. D. thesis. Harvard University, Cambridge, 1977. 3. BACKMAN, K., PTMHNE, M., and GILBERT, W., Proc. Nat. (1976). 4.

10-l I loo

10' pg protein

lo2

FIG. 2. Repressor level in cells infected with A ultravirulent phages. Exponentially growing bacteria at 2 x lo* cells/ml in BT broth supplemented with 0.2% maltose were collected by centrifugation at 4”. The pellets were rinsed, resuspended in 0.01 A4 MgS04 at 2 x lOi cells/ml, and incubated for 30 min at 37”. The cells were then chilled, infected with phage at an m.o.i. of 6, and left for 30 min at 0 for adsorption; the cells were diluted 50-fold in prewarmed BT broth and incubated for 30 min at 37” with aeration before being quickly chilled to 0” in a methanol dry-ice bath and pelleted. Cells extracts from C600 bacteria infected with All (+), Al46 (O), Al69 (A), and A668 (‘I) were prepared and tested for ADNA operator binding as in Fig. 1. ACKNOWLEDGMENTS The technical assistance of M. Pierre is highly appreciated. We are thankful to M. Gellert for his help in constructing GY 105 (pKB202) and to A. Levine for advice in the repressor assay. We are grateful to K. Backman and M. Ptashne, who

5. 6. 7. 8.

9.

10. 11. 12. 13.

Acad.

Sci.

USA

73,

4174-4178

LEVINE, A., BAILONE, A., and DEVORET, R., submitted for publication. BERG, D., Virology 62, 224-233 (1974). KELLENBERCER-GUJER, G., BOY DE LA TOUR, E., and BERG, D. E., Virology 58, 576-585 (1974). PEREIRA DA SILVA, L., and JACOB, F., Ann. Inst. Pasteur 115, 145-158 (1968). HOPKINS, N., and PTASHNE, M., In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 571-574. Cold Spring Harbor Laboratories, New York, 1971. MANIATIS, T., PTASHNE, M., BACKMAN, K., KLEID, D., FLASHMAN, S., JEFFREY, A., and MAURER, R., Cell 5, 109-113 (19751. DELVAUX, A. M., and DEVORET, R., Mutat. Res. 7, 273-285 (1969). ORDAL, G., In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 565-570. Cold Spring Harbor Laboratories, New York, 1971. MATSUBARA, K., J. Mol. Eiol. 102, 427-439 (1976). MATSUBARA, K., AND KAISER, A. D., Cold Spring

Harb.

Symp.

Quant.

Biol.

33, 755-764

(1968). 14. ORDAL, G., and KAISER, A. D., J. Mel: Biol. 79, 709-722 (1973). 15. SHINAGAWA, H., and ITOH, T., Mol. Gen. Genet. 126, 103-110 (1973).