Prophage deletions selected by heat induction of bacteria lysogenic for λint6CI857

VIROLOGY

42,

225-228 (1970)

Prophage

Deletions

Selected

by Heat

Induction

of Bacteria

Lysogenic

for AinK The exposure to heat of lysogenic K12 cells character were due to a single deletion event. carrying a heat-inducible prophage XC,857 The cured cells which were still uvrf were in (1) results in virus induction and cell killing. minority. Out of four uvr+-cured survivors found three were bio+ (U639); the fourth Only 10e3 cells form colonies after overnight (U638) grew slowly in synthetic medium incubation on plates at 42” (,2,5). More than wit.hout biotin, suggesting that the bio 300 survivors were tested and were found operon was affected by the deletion. Since cured. When the int- mutation, which all the lysogens were gal-, it was more prevents prophage excision (4), was present, difficult to test for the presence of the bacthe surviving fraction was about 10vs. Two terial genes at the left of the prophage. types of survivors were found; (a) bacteria lysogenic for a defective prophage, (b) However, the presence of N gene markers in bacteria where the prophage is entirely or one of the bti-uwBlysogens (U635) suggests that prophage deletion with respect to partially deleted. Analysis of the defective lysogens (a) the left end of the prophage is also erratic. revealed t’hat two prophage mutations are Errat’ic excision of Xint&,857 and XbZC, necessary to prevent cell killing after induc857 prophages was already found by analyztion: one at the left of C, affecting the funcing the transducing particles released from tion of gene N, the second affect.ing at least the lysogens after heat induction (5, 7’) one of the genes contained in the early where particles transducing the bio-uvrB operon at the right of C, : zd, 0, or P (3). segment were detect’ed; they correspond to The subject of the present communication the deletions found by our selection methods. is a description of the second type, the However, the deletions found in our surdeletion survivors (b): Figure 1 shows the vivors are probably not caused by t’he aberdeletions found among the survivors of three rant heat induction: the induction of early strains: U600, U593, and U597. U600 is genes was found lethal (8, 9), and we would ret+ strain lysogenic for hint&+357 (5). not be able to find such cells among the U593 and U597 are derivatives of a ret- strain survivors. The overnight growth at the lysogenic for hint&$57 and Xintsred&1857 inducing temperatureusedinour experiments (5), respectively. A wide range of prophage probably selected only for deletions already deletions was found from &rain U600: they present prior to heat exposure. represent about, 95% of the heat survivors. A partially deleted prophage, unable to The deletion mutants are less frequent among confer immunity, was described as X cryptic the heat survivors from the lysogens U593 (11). Cryptic prophages can be found among and U597 (about 10 %>. Seventy-five percent nonimmune survivors after UV induction of t,hese survivors seemed completely deleted of X lysogens (12). They are also due to (cured), i.e., no standard X sus markers were aberrant’ phage-direct,ed excision induced by rescuable. The majority of these cured cells UV. The utt region at, both sides of cryptic from strain U600 became UV sensitive prophages is present (IS, 1.4). Although our (U637 in Fig. 1); this suggests that the loss selection of heat survivors yields bacteria of the prophage in these strains is due to a cant aining a wide range of different prophage deletion extending through the neighboring gene contents, the survivors containing A-J bacterial gene Curb (6). All the UV-sensitive and Q-J prophage with the right end of survivors test.ed were bio-. This may be deletion similar t,o that of cryptic phage expected if the prophage loss and UW- (Xcry on XcryQ+R+) are 5-10 times more 225

226

SHORT COMMUNICATIONS N Cl P 0 R A W B C 0 E F G H M L K I l3 PL PRx,y,C60 289 3 117 2 11 812 1 20 123 4 968 9 12 67 63 24 2

J att bio uvrg 6

numbers mutants

of sx used

Deletion mutants derived from U600

. . .. . . . . . . . . . . .

. * . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

-

GE:: O-J C-J

....

A-J t R-J

. . . .

....

. . . . -.

* * U635 * * U637 U636 U639 U610 -J I-J K-J L-J M-J

. . .

P-J O-J

Oeletion mutants derived from US93

.... . . . .

U613A-J * Q-J *

Deletion mutants derived from U597

. *..

.... ,...

U61LA-J

t

&j *

FIG. 1. Gene deletions found in the heat-inducible lysogens selected by heat treatment. Selection of the deletion mutants: The bacteria were grown in tryptone broth with aeration at 32” to stationary phase, centrifuged and resuspended in 1:50 of the original volume, then plated on tryptone plates with soft agar containing anti-X serum (K = 5 min-1 per plate) and incubated overnight at 42”. The surviving colonies were purified and tested. Test for gene presence: the presence of phage markers was tested by the marker rescue test (12) with the sus mutants of X indicated. The absence of immX region was tested by analysis of the progeny of hi434 vegetatively grown on the mutants (less than 10m9of iX in each case). In the case of U642 the rescue of the C&857 gene was performed with XC160: no turbid recombinant was found among more than lo4 plaques at 32” of the progeny of XC160 on U642 (three strains tested). The bio marker was tested by the capacity to grow on minimal medium supplemented with vitamin-free casamino acids (29&F), glucose (O.l%), and tryptophane (10-S) with andwithout biotin (1O--7). The UffrB function was tested by UV sensitivity. The sequence of the tested genes results from the work of several authors (10, 19, 20). Full line = the segment present. Dotted line = genes not tested. * = 5-10 times more frequent than the other classes.

frequent than the other types, all of which appear with comparable frequency. This suggests that there are weak spots between prophage genes P-Q and R-A. Most of the deletions found in our heat survivors comprise at least the region N-C,-0 of the prophage. This is consistent with the notion that N function and the X

DNA replication must be absent in order to allow nonimmune lysogens to survive. In some rare cases, however, the sus+ alleles of the gene N and/or 0 are still rescuable (U610 O-J, U635, U642 in Fig. 1). In spite of this, the results of the complementation tests summarized in Table 1 show that none of these strains is able to supply N or 0 func-

SHORT

COMMUNICATIONS TABLE

Original strain u593 U597

moo

Deletion derivative UG13-3 U613-2, 4 UG14-4a b U614-3 UG14-2 U635 U642 U(ilO-Sa-c e, g f d U610-13 UBlO-3 UGlO-14

Phage genes present

1” Plating efficiency (%) of

Number oft;;;$s susN7

P-J

Q-J P-J

Q-J

3 1 1 3 4 2 1 1 1 3 1

P-J R-J

susos

1 2 1 1

12-J (att-)N (att-)N, O-J O-J

Q-J

227

SUSPS 100 0.1 0.5

0.5
0.3
100 1 2
susR.S-54 100 100 100 100 100 0.5 80

100 3

Q The ability of the prophage genes to complement the superinfecting phage was measured as the efficiency of plat.ing on the st,rain to be t,ested of phages carrying a sus mutation of the gene tested, expressed as 7cof plating efficiency on a pm+ control (C600). Each number is an average of several experiments with ix and i434 sus mutants with the number of strains stated for each group. According to suitable control tests (S), plating efficiency of less than 0.1% accounts for the defect, in the particular gene, O&3% accounts for extragenetic recombination, over 30yc of plating efficiency reflects true t,ranscomplementation of the function tested.

tion to the superinfecting phage. Thus, in all deletion mutants obtained by heat select’ion both N and 0 genes are eit,her absent or silent. In this respect, members of the U642 set are of particular interest. Despite the presence of phage markers both at the left and at the right of the C, gene? neither the whole immunity region nor C&S57 were rescuable. Thus the C1 gene in these strains is probably deleted including at least the promoters of the t,wo early operons adjacent to it : pi of t,he gene N and pR of the x-0 operon (15). The functionof thegene P is transinducible by heteroimmune phage in a normal lysogen (16). P function can be transinduced even from an x- prophage where the x mutation is polar bot,h over Cl, and 0 gene (5). In Table 1 we see that some of the partially deleted prophages P-J complement the P function: U613-3, U610-13, whereas some others do not: U614-4. The inability of this last group of deletion mutants to complement the P function might suggest that the deletion extends within the P gene. But even in st.rains of the U610-2 set where sus8 and

sus29 markers of 0 gene are stil1 present and the deletion being thus shorter than in P-J prophages, the expression of P is prevented in most cases. This creates a paradox: the P gene can be turned on in strains where almost the entire x-0 segment is absent. On the other hand, in some strains where part of the x-0 segment is still detectable, the function of P is prevented. Thus, a partial deletion of x-0 can still exert a polar effect upon P function. This polar effect does not apparently depend on t’he distance of the right deletion end from P. R gene was transinducible in our deletion lysogens of the Q-J group in agreement with similar findings in immune and cryptic lysogens (16,17). This was found even in one strain of the Q-J group, U610-3, where t’he rescue by susQl17 was very inefficient, suggesting that the Q gene of the prophage was affected by the deletion. This is in agreement with the results (18) that even in a Qprophage the R gene can be turned on by the superinfecting Qf phage. On the other hand, the absence of R complementation in both R-J lysogens where t.here was no

228

SHORT

COMMUNICATIONS

susQ117 rescue (U610-14, U614-2) might reflect a partial deletion of the R gene itself. ACKNOWLEDGMENTS The author thanks Prof. Enrico Calef for helpful discussions and review of the manuscript. This work has partly been supported by EURATOMC.N.R.-C.N.E.N. contract, No. 012-61-12 BIAI. R.EFERENCES 1. SUSSMAX, R., and JACOB, F., C.R. Acad. Sci. Paris 254, 1517-1519 (1962). 2. CALEF, E., and NEUBAUER, Z., Cold Spring Harbor Symp. Quant. Biol. 33,765-767 (1968). 3. NEUBAUER, Z., and CALEF, E., J. Mol. Biol. in press (1970). 4. ZISSLER, J., Virology 31,189 (1967). 5. GOTTESMAN, M. E., and YARMOLINSKY, M. B., J. Mol. Biol. 31,487-505 (1968). 6. HOWARD-FLANDERS, P., BOYCE, R. P., and THERIOT, L., Genetics 53, 1119-1136 (1965). 7. GOTTESMAN, M. E., and YARMOLINSKY, M. B., Cold Spring Harbor Symp. Quant. Biol. 33, 735-747 (1968). 8. SLY, W. S., EISEN, H. A., and SIMINOVITCH, L., Trirology 34, 112-127 (1968).

9. CALEF, E., and NEUBAUER, Z., unpublished results. 10. ADHYA, S., CLEARY, P., and CAMPBELL, A., Proc. Nat. AcadSci. U.S. 61,956-962 (1969). 11. FISCHER-FANTUZZI, L., and CALEF, E., Virology 23, 209-216 (1964). 12. MARCHELLI, C., PICA, L., and SOLLER, A., Virology 34, 650-663 (1968). 13. FISCHER-FANTUZZI, L., Virology 32, 18-32 (1967). 14. SOLLER, A., and MARCHELLI, C., Atti Ass. Genet. Ital. 14, 54-64 (1969). 15. KUMAR, S., B$VRE, K., GUHA, A., HRADECNA, Z., MAHER, V. M., and SZYBALSKI, W., Nature (London) 221, 823-825 (1969). 16. THOMAS, R., J. Mol. Biol. 22, 79-95 (1966). 17. DAHL, D., SOLLER, A., and CALEF, E., J. Mol. Biol. 32, 639-658 (1968). 18. DAMBLY, C., COUTURIER, M., and THOMAS, R., J. Mol. Biol. 32, 67-81 (1968). 19. CAMPBELL, A., Virology 14, 22-32 (1961). 20. PARKINSON, J. S., Genetics 59, 311-325 (1968). ZDENEK NEUBAUER International Laboratory of Genetics and Biophysics Naples, Italy Accepted June b, 1970