Relationship between K-reactivation and UV-reactivation of bacteriophage λ

VIROLOOY

36,303-305 (1968)

Short

Communications

different reactivat’ion mechanism, each of which repairs more than 90% of the lesions and UV-Reactivation of lethal in the double mutant. This conclusion has been reached already in an earlier paper Bacteriophage X (6, see also 8). 2. Survival of UV-irradiated phage Tl This paper reports experiments which (and also of T3 and T7, unpublished) is show that the mechanisms of UV-reactivation (UVR) are different for phages X and independent of the host’s Ret allele (+ or - , Fig. 1B). Therefore, a mutation in this T3. UVR is the additional repair of phage lesions that occurs when host bacteria are yet gene does not affect the repair mechanism stimulated by slight ultraviolet (UV) doses responsible for host cell reactivation of Tl HCR”; 4; compare 6’ and 8) before phage infection (1, 2). UVR of T3 is (l’ordinary which is abolished by the Her- mutation blocked in one type of UV-sensitive mutant only. of Escherichia coli (Her- or Uvr-), which, 3. Survival of UV irradiated phage h is however, allows UVR of phage X (5, 4). It will be shown on the other hand, that in a slightly reduced in Ret- as compared to Ret+, both in the presence of Her+ and even second type of W-sensitive mutant (Ret-) Her- alleles (Fig. 1C). Therefore, a mutation UVR of X is blocked, and that the two types in this ret gene does affect the “residual of mutation affect different reactivation HCR” of phage X still intact in all Hcrmechanisms. (Ret- mutants are characterized mutants (3, 4). by t,heir deficiency in genetic recombination, 4. In strain Ret-Her+, where this “residHer- by the lack of host cell reactivation ual HCR” of X is affected, no UVR of X is (HCR), i.e., by the inability to repair UV found (Fig. 2A), although “ordinary HCR” lesions in phages Tl, T3, T7, and X). is intact. UVR occurs, however, in strain Strains and procedures have been described Ret+ Her- (Fig. 2B; see ref. S), which is defiearlier (4-S). In addition, two non-host-cellHCR.” Therefore, the reactivating mutants Ret-Hcrland cient in “ordinary mechanism of UVR of X is related to or idenRet-Hers- have been isolated independently from the recombination deficient strain 152- tical with that of t’his “residual HCR” but not “ordinary HCR” (unless there is some Ret- of Meselson (6) after 2aminopurine common step undisturbed in both mutants). mutagenesis by the method of Howard5. The survival curve of UV irradiated Flanders et al. (7) modified by R. Hill (personal communication). Both double mu- X adsorbed to heavily irradiated Rec-Hcrhosts is still steeper than with unirradiated tants behaved ident,ically in all experiments hosts (Fig. 1C). Therefore, even Ret-Hcrreported. The figures show the characteristics of the does still perform some “residual HCR,” double mutant Ret-Hcqas compared to which may be due to a third mechanism of representatives of the two single mutant relatively low efficiency, types and to wild type. The following results These conclusions may reconcile two seemare found : ingly conflicting (9) viewpoints. Harm (5) 1. Both highly UV sensitive single mu- postulat’ed that UVR both of T3 and X is t,ants, Her- and Ret-, are over ten times due to UV-enhanced HCR. But. Kneser et more resistant to UV than is the double la. (4) found that UVR of X differs in mechmutant Ret-Hcr(Fig. 1A). Therefore, anism from “ordinary HCR.” However, if each single mut,ant, performs a (partially) t.he term HCR is only used stri&ly opera303 Relationship

between

K-Reactivation

304

SHORT

COMJZUNICATIONS

tionally covering all mechanisms of reactivation by the host of phage lesions, then both statements are compatible: UVR of T3 and “ordinary HCR” share a common mech-

anism (dark reactivation), as do UVR and “residual HCR” of phage X. This latter mechanism may be the same one as I
3

ICY

FIG. 1. Reactivation

of UV lesions by the strains used. (A) Survival of the double mutant Ret-Hcrl(A) after UV irradiation. For comparison strain Bs-1 is included (0). Curves 1 and 2 represent survival of the single mutants K12S Her-, and 152 Ret-, respectively (exponential phase cultures in all cases, plated on LT plates). The curve for W 3102 Rec+Hcr+ would be indistinguishable from the dose axis [see Kneser (S)]. (B and C) Survival of UV-irradiated phage Tl (B) and Xvir (C) with t,he indicated plating bacteria. A Ret-Hcrl-; A K12S Her-; 0 152 Ret-; l W 3102 Ret+ Her+; V phage Xvir adsorbed to Ret-HcrlUV irradiated with 4000 erg/mm* and complexes plated with Ret-Hcrl(corrected for loss of capacity). Single representative experiments.

uv DOSE (ercr /mm’) 0

500

1000

150

0 I

566

1000

1500 2000

25(I

FIG. 2. Survival of UV-irradiated phage Xvir (one dose, (A) : 920 erg/mm2; (B) : 115 erg/mm$) adsorbed to bacteria irradiated with increasing UV doses. Symbols as in Fig. 1. Upper curves (Cap.): Survival of unirradiated Xvir , i.e., capacity of the cells to reproduce X,+ The lower curves are corrected for this loss of capacity. Single representative experiments.

SHORT

COMMUNICATIONS

ence in UV sensitivity between strains Ii12 and B of Ii:. coli (5). In fact the similarity of t,he UV survival curves of B and Bs-1 with those of Ret- and Ret-Her-, respectively (E’ig. 1A and ref. fi) has led to the supposition that B and Ret- lack the same reactivation mechanism, KR. I wish to thank ?\Irs. R. Ruppel for competent technical assistance, I>r. M. LIeselson for bacterial strains, and I>rs. I’. Starlinger and F. Stahl for criticisms. This work was supported by the Delltsche Forschlmgsgemeinschaft~. REFERENCES 1. W~GLIG, J. J., Proc. -l’atl. &ad. fki. c,‘.s. 39, 628-636 (1953). 2. WICIGLE, J. J., and l)u~rmcco, R., Ezperientia 9, 272 (1953). 3. HARM, W., Z. Verebungslehre 94, 67-79 (1963). 4. KNI:SICR, II., L~ETZGER, K., and SAUERRIER, W., Virology 2i, 213-221 (1965). 5. KNJCW:R, II., Z. Vexrbungslehre 9i, 102-110 (1965). 6. KSJZSEIL, H., Biochem. Biophys. Res. Commun. 22, 383-387 (1966). 7. HOWARD-FLANDERS, P., and THERIOT, L., Genetics 47,1219-1224 (1962). 8. HOAVARD-FLANDBRS, P., and BOYCE, R., Radiafion IZes., Suppl. 6, 156-184 (1966). 9. 11 \RM, w., Virology 29,494 (1966). HUBERT KNESER’ Institut fiir Genetib der Cniversitiit zu K&n Kiiln-lindenthal, Weyertal 121 U’est German77 Accepted July 4, 1968

-I_

1Present address: Institute Biology, n7403.

Inhibition

University

of Oregon,

of Tobacco Guanidine

of Molecular Eugene,

Oregon

Necrosis Virus by Carbonate

Finding plant virus control chemicals poses many problems. A chemical that inactivates or kills t,he virus may also injure or kill the host plant. Xot enough is known to explain how and what kind of a chemical is apt to inhibit a virus without harming the host, but extensive screening of diverse types of chemicals has been done (1, S-6). Recently, guanidine hydrochloride has been reported to inhibit, the cytopathic effect and

:3O.j

infectivit,y of some animal RSX viruses (7-10). Here, the effect of guanidinc cat‘bon&e (GO,) on tobacco necrosis virus (TNV) is reported. As far as t,he author is akvare, no guanidine salt has been reported before t,his to inhibit plant virus infection. To det#ermine the effect of GCO, on TSV infectivity, Phase&s wdyaris 1,. var. Saxa was used as a test plant.. Unless otherwise &ted, treatments were done on the intact1 primary leaves att,ached to the plantIs grown in J-inch clay pots. Virus inoculum \\ as applied by gently rubbing a glass spatula lvith a ground-glass face over the leaf su~f:tcc. Crude extract’s were prepared by crushing fresh TXV-infected leaves of P. ~zrlya~is in demineralized water. Cleared extracts were prepared by centrifuging the crude extract at) 10,000 rpm for 20 min at O-4”. One milliliter of these extracts or aqueous solution of purified TKV prepared by the m&hod of Ha\\-den and Erie (2) was mixed wivith 4 ml of various concent8rations of GCOs prepared in demineralized water to determine whether rxposure of TNV to GCOa irt vif~ decreased infectivity. For a cont,rol, w-ater \\-a~ used instead of GCOa solution. The mixtures were kept. for 0, 30, and 60 min at, room temperature (20-25”) before inoculat,ion. One half of each leaf was inoculated with t,he mixt,ure of chemical and virus, and the other half 1~:~sa control inoculated with virus mixed wit,h water. Results t’ypical of several experiments (Table 1) demonstrate that infection was prevented, and t,he leaves were not injured, by mixtures cont’aining GCO, (Fig. 1). There was no effect of time (up to 60 min) on infectdvity of in vitro exposure of virus to (K‘Oa. Different concentrations of GCO, lvere alkaline, and the inhibit,ory level (0.05 dl) was pH 10.5. To elucidate the role of this high pH, 0.05 X GCOy was first, adjusted to pH 7.0 wit,h dilute hydrochloric acid before mixing with TSV and inoculating. At pH 7.0, 0.05 M GCO, was highly toxic to the leaves. To further clarify the role of the high pH and also of the carbonat’e of GCOa , TSV was mixed with carbonate-bicarbonate buffer of pH 10.5. Thereafter, the mixtures of ‘IXV and 0.05 M GCOa at. either pH 10.5 or 7.0 (adjusted with HCI) and carbonate-bicarbonat.e buffer were first, inoculated to the leaves and then dialyzed to remove t’he salts