Effects of nitrous acid on intracellular T2 bacteriophage

VIROLOGY

16, 444-451

Effects

(1962)

of Nitrous MARTHA

Acid

on intracellular

B. BAYLOR

AND

T2 Bacteriophage’

H. R. MAHLER

Marine Biological Laboratories, Woods Hole, Massachusetts, and Department of Chemistry,’ Indiana University, Bloomington, Indiana Accepted January

2, 1962

Nitrous acid has been found to be an effective mutagenic and inactivating agent for bacteriophage T2 in the vegetative phase. The mutagenic efficiency for infected 2- and &minute complexes of Escherichia coli B is at least as high as that observed with extracellular phage, both with respect to r and ht mutations. The kinetics of inactivation of infected complexes by nitrous acid has been shown to be sensitive to the stage of development and to the presence of chloramphenicol. INTRODUCTION

Nitrous acid has become a favorite agent in investigations of the ‘(genetic chemistry” of viruses and microorganisms because of its simplicity of application and relative specificity (review in Sinsheimer, 196Oa,b). The lethal and the mutagenic activity of this agent in the bacteriophages studied has been shown to follow “single-hit” kinetics. The mutants produced were exclusively homozygous in 9X174 (Tessman, 1959b), a virus containing a single-stranded DNA (Tessman, 1959a; Sinsheimer, 1959)) and heterozygous for T2 (Vielmetter and Wieder, 1959) and T4 (Tessman, 1959b), viruses containing double-stranded DNA. Schuster (1960a,b) and Vielmetter and Schuster (1960) studied the deamination rates of the constituent bases in various isolated DNA preparations as a function of time and pH and have compared these rates with those for the intracellular DNA of Escherichia coli and with the inactivation and mutagenesis rates of T2. Freese (1959; 1 These investigations have been supported by research grants from the National Institutes of Health: RG 5942C4 and E1854C2 to H. R. Mahler, and E2173C4 to Martha B. Baylor. The experimental work was performed at the Marine Biological Laboratories, Woods Hole, Massachusetts, during the summer of 1960. ’ Contribution No. 1031.

see also Bautz-Freese and Freese, 1961) and Vielmetter and Schuster (1960) have used experiments of this kind to establish the molecular basis of mutation and inactivation. The recent studies of Geiduschek (1961) on the cross-linking action of nitrous acid on isolated DNA are also pertinent to the mechanism of its action. We present here data bearing on mutagenesis by nitrous acid and on the kinetics of inactivation of infective centers of T2H in E. coli at different times after injection of the viral DNA. The inactivation kinetics appears to depend on the phase of development of the vegetative phage and in this respect shows both similarities and differences with the corresponding effects of ultraviolet irradiation and “suicide” by internal P”*-decay @tent, 1958, 1959; Stahl, 1959). MATERIALS

AND

METHODS

The bacteriophage T2H (Hershey) and its host E. coli B (strains S and H) and B/2 (strain 2bc) were used. Phages were assayed as described by Adams (1959). Free phage was determined after chloroform treatment of the adsorption mixture. The nitrous acid inactivation experiments were performed as described by Tessman (195913) in 0.05 M KNOZ in acetate-acetic acid buffer, pH 4.0, at room temperature (20-22”). 444

HNO, ON INTRACELLULAR

A typical inactivation experiment was carried out as follows: A 2-hour culture of E. coli B in nutrient broth was centrifuged and suspended in adsorption buffer containing 10 pg chloramphcnicol (CAP) per milliliter to a final cell density of 2.0 x 10s per milliliter. Phage was added at a concentration of 2.0 X 1Oi infectious particles per milliliter. Adsorption was allowed to proceed for 5 minutes at 37” with aeration. The infected cells were centrifuged, resuspended in cold CAP-adsorption buffer, and kept in an ice bath. Assays were run at this point to determine total phage and free phage concentration; the latt’er never exceeded 5% of the total. To initiate phage development aliquots from the infected cell mixture were diluted 1:30 into prewarmed broth. After stated intervals in broth at 37” with aeration, samples were removed and diluted 1: 20 into nitrous acid and also into buffer to deterniino tlic count before treatment C&j. Thereafter, at intervals of 60 seconds, dilutions I :50 or higher were made out of the nitrous acid-phage mixture and samples were plated. The reaction was followed for 5-6 minutes. “0 time” controls were taken directly from the adsorption mixture prior to incubation in broth. Free-phage controls at equivalent concentrations were treated with nitrous acid by the same procedure. In the study of mutagenesis many wildtype plaques from treated and untreated samples were examined for the mutant. form. Rapid-lysis (TI mutants and mottled plaques were dct’ermined by their plaque morphologics, and extended host range (ht) mmants by the technique of Baylor et al. ( 1957). The mutant plaques were stabbed and assayed for homogeneity as described by Tcssman ( 1959b). In multiple infection, multiplicity reactivation, and rcscuc experiments, mixtures of diffcrcnt, recognizable genotypes (usually r13h and wild type) were used to permit identification of mottled infective centers and recombinants. The multiplicity of infection was measured by cell killing, and the infective centers were assayed after treat,tnent wit,h anti-T2 serum to remove fret phagc.

345

T2 RESULTS

Kinetics Figure 1 shows the effect of nitrous acid on T2-infected complexes of E. coli both as a function of time of intracellular phage development and as a function of exposure to nitrous acid. Distinct stages of phagc development (a through e) can be characterized by the inactivation kinetics of infective centers of different ages. Stage a: The inactivation of infective centers in which there has been no phage development (O-time controls) is exponential with a rate (k) indistinguishable from that of free phage. S/S0 = e-ki = e-“; where S/S, is the fraction of surviving infective centers, k is the apparent first-order inactivation rate constant, t is the time, and h is tht number of “hits”. dtagc b: Within 1 minute after the initiation of development by dilution into INtrient medium, the inactivation curve shows a lag or shoulder which persists for 1-2 minutes after exposure to nitrous acid. The final slope of the curve, which is approximately equal to that of free phage, cxtrapolates above the origin to about two times the input. This pattern is maintained throughout the next 3 minutes of devclopmerit. Stage c: After 4 minutes and continuing through 6 minutes of development the kinetics reverts to that exhibited by O-time complexes-the curve is strictly exponential and not sensibly different from that of free phage. Stage d: Starting at 7 minutes the curve becomes progressively “multihit”; sensitivity to the agent reaches a minimum value at 9 minutes. St’agc c: Under the conditions of this cxperimcnt the first, intracellular phage becomes detectable at 11 minutes. At t,hat time (not shown on the graph) inactivation appears to be again strictly exponential. but with a new and lessened inactivation rate. WC have not studied this effect cxtenxively in this investigation. The charact’eristic kinetics of difYercnt developmental times are highly reproduci-

446

BAYLOR

-4ND MAHLER

1.0

0.8 0.6

$ F

0.1

2 0.08 k 0.06

I

I

I

I

III/

123456

I

I

I

I

I

I

123456 TIME OF EXPOSURE

TO HN02

(MINS)

FIG. 1. The inactivation of infected complexes by HNO? The experiments were performed as indicated in the text. Samples of infect)ed complexes were withdrawn from broth at 37” with aeration at the times indicated near each curve. S/S,, the surviving fraction, was determined in duplicate on the experiments in Fig. la and in triplicate on those in Fig. lb. The points are averages. In Fig. la, where several experiments with different periods of development are illustrated on the same curve (e.g., 1, 2, and 3 minutes; 4 and 6 minutes; 9 and 10 minutes), the size of the symbol indicates the standard deviation for each point; “+” is free phage. Temperature for la was 20” and for lb, 22”.

ble, as can be seen by comparing and lb. The Effects of Chloramphenicol

Figs. la (CAP)

The effects of CAP, an inhibitor of protein synthesis (Brock, 1960), on the system under consideration here are shown in Fig. 2. When CAP is added to nutrient broth at the same time as the infective centers (0 time) and incubation is allowed to proceed for 7.5 minutes in the presence of the antibiotic (curve l), the multihit pattern characteristic for this stage of development fails to develop and the kinetics of HNOz inactivation after incubation is essentially indistinguishable from that observed with free phage or O-time controls. Incubation

in the presence of the drug for intermediate periods of time (curve .2) leads to inactivation kinetics reminiscent, of, but not as pronounced as, those found in analogous experiments in the absence of CAP. Incubation for 4 minutes in the absence of CAP, followed by a period of 6 minutes in its presence, also prevents the change to the multihit pattern of stage d (curve 4). On the other hand, the characteristic twohit pattern observed during the first 3 minutes of development (stage b) changes in the direction of the single-hit pattern characteristic of 4- to 6-minute complexes (stage c) spontaneously, even in the presence of CAP and at 0” (Fig. 2b). It appears as if CAP slowed the change from stages a to h.

had relatively little effect on the change from b to c, and completely abolished the transition from c to d.

The mutagenic effect of nitrous acid is obtained on vegetative intracellular phage (DNA) with an efficiency at least as great as that observed with free phage. As seen in Table I, the 2- and j-minute complexes appear to be equally sensitive to the mutagenic action. The induced increase in the frequencies of the r and the ht phenotypes are also approximat’ely equal. As reported by Tessman (1959b) and by Viclmctter and Wieder (1959)) mutant plaques arising from nitrous acid-treated complexes produce either pure mutant or mixed wild-type and mutant progeny. There is an indication that the 2- and 5-minute complexes may differ in the proportion of pure and mixed rlones (Table 2). Mdtiplicity

Reactivation

and Rescue

A characteristic feature of radiation-inactivated phage particles is their ability to participate in genetic recombination with whole phage (rescue) and in multiplicity reactivation (Stahl, 1959). Phage inactivated by nitrous acid treatment can also undergo multiplicity reactivation, and genetic markers can be rescued during replication of whole phage. Since extensive investigations along these lines have already been reported by others (Harm, 1960; BautzFreese and Frecse, 1961) we shall make only a brief statement of our findings. In a typical experiment free phage were inactivated by nitrous acid to 1.7% survival corresponding to four ‘Lhits” per phage. The inactivated particles were Glen used to infect cells at different multiplicities of infection. The actual phage dosages per cell on the basis of ~11 killing mere 2.9, 1.0, and 0.01 (calculated from higher dosages), respectively. If the Poisson distribution is obeyed, 78% of tile bartcria in the 2.9 dosage and 27% of the bacteria in the 1.0 dosage should contain two or more inactive particles and thus be susceptible to mult8iplicity reactivation. The data showed that 1.0/1.9 X 0.78 = 0.67 of

\,, .\

TIME

T

OF F.XPOSIJRE

TO HNG,IMINS)

FIG. 2. The effect of chloramphenicol on inartivation kinetics. The experiments were performed as indicated in the text. Fig. 2a: Two free-phage controls, run on two successive days (symbols 0 and 0) simultaneously with various phases of the experiments, are shown. Curve 3, st,andard experiment, no CLIP added, HNO, inactivation started after 7.5 minutes of incubation in broth at 37”. Curve 1. similar to curve 3, but with CAP present throughout the time in broth. Curve 2, incubation in broth for 2 minutes (Ml or 5 minutes (A) in the presence of CAP. Curve ,$, incubation in broth for 4 minutes in the absence of C.W, followed by a second period of incubation in the lnesence of CAP in broth for 6 minutes. Fig. 21~: Currt~ 2, standard experiment, no CAP added, incubat,ion in broth allowed to proceed for 2.0 minut,es. CAP was then added and immediately thcreaftcr the HNO? keatrnrnt, was performed as usual. Ciirvc 1. incubation in hrotb for 2 minutes; then CAP was added, the sample was chilled in cracked ice, and 2 minutes later an ali( was withdrawn for HSO, treatment.

all multicomplexes in the higher dosage and 0.27/1.1 X 0.27 = 0.88 in t,he second dosage actually particiljated in multiplicity rt’actiration. In another experiment, free phage were inactivated to different levels of survival and used to infect bacteria at a constant

448

BAYLOR

AND

MAHLER

TABLE MUTAGENESIS

-

I

BY NITROUS ACIDS

-

Intracellular

Total no. examined I -

10 18 19 20

Total

Induced

No. and type of mutant r

ht

36,000” 16,000 3,100

19 10

I1 13 7

6,000

9

17

-^_-__ Total

P-Minute

-

Expt. no.

-______45,100 (9,100) 61,100 (25,100) ---

for r for ht

complex

7800

11

11

5,560 3,500 600 100 -_____

6 9 3

18 11

17,460

-

3

-.

29

Total no. examined

No. and type of mutant r

ht

8800 13,500 3,875 2,800 600 100

18 7 0 -

21 18 8 1

16,075

37

(::I 16,960

-- --__-

43

(:;I

_16.6

(2:::) __-____ Induced

-

S-Minute complex

No. and type of mutant Total no. examined ___ r ht

X 10e4

spontaneous

phage

-

Free phage

L

(1:::)

--__--

1.9 (3.7)

_--_

20,275 --_

25.3

.- ----

6.6

38

23.1 --__

6.4

---

23.7 ---_

9.2

5.9

-

Q All nitrous acid treatments were performed under the conditions described in the text for 2 minutes at 24l”C, which corresponds to 0.4 survivors ~1 hit on free-phage or 5-minute complexes. The spontaneous mutation rates were ~2.5 X 10m4for r and 4 X 10e4 for ht determined by the number of mutants per 29,000 plaques in the free-phage controls. b A dash (-) means not examined. c Very crowded plates ; if this experiment (no. 10) is omitted for this reason, the statistics shown in parentheses are obtained.

total (active plus inactive) multiplicity of infection. Although the free phage decreased to about 1% after 8 minutes in nitrous acid, the number of infective centers remained constant at 70% of their initial value. We also compared (Fig. 3) the kinetics of inactivation of singly infected and multiply infected (4 phage per cell) &minute complexes. Under these conditions 72% of the cells should receive both r and T+ phages and should yield mottled plaques; 72% of the plaques were found to be mottled. The rate of inactivation of the singly infected complexes was first order. While the actual shape of the inactivation curve for multiply infected complexes could not be determined without ambiguity the terminal slope is clearly very different from the one obtained

for singly infected complexes. The results of this experiment are consistent with the hypothesis that nitrous acid acts directly on vegetative phage instead of indirectly on some cellular component necessary for phage replication. Marker rescue was also demonstrated. Cells were multiply infected with two untreated wild-type phage together with six rlSh phage inactivated to 0.02 survival with HN02. From the total number of infected centers and from those containing both genotypes, the rescue efficiency can be computed at 89%. The results of these experiments and those above demonstrate the ability of treated phage to adsorb and inject and indicate that the primary action of nitrous acid is not on the external coat

HNO, TABLE

ON INTRACELLT-LAIR

2

S:AMIWNG OF I'I.ACJITX~FOR PRESENCE OF HETEROZYGOTES~ I

Two-minute complexes

/ I

Five-minute complexes

119

T2

tainable is similar in principle and supplementary to that derived from ultraviolet, S-radiation, and P-suicide (Sent, 1958; Stahl, 1959; Sent and Fuerst, 1960). Among the characteristic features which set it apart from the more ‘Lclassical” techniques art’ the following: (1) The deamination rates of at least seine of the bases (e.g., guaninc 1 in DNA arc relatively invariant regardless of physical environment, state of aggregation, conformation or configuration, and possibly even association with other entities suc~li as R?;A protein (Schuster, 196Oa, b I ; I 2 I I)(~amination lrads to substitution of an “unnatural” base without extensive brcakagc of either thts st’rand affcctcd directly or tllc

11The experiments are those of Table 1. Isolated plaques were picked and a sample replated. Pure clones are those in which the mutant showed a preponderance of >9:1. Chi square analysis shows the differences in distribution of pure and mixed clones between the 2. and 5-minute complexes significant at the 99% level. Tessman (195913) found 52y0 heterozygotes in extracellular T4. The numher of plaques per plate were 100 to 300. ,411 mut,ant plaques except those touching parental plaques were replated to test for homogeneit)y. To avoid bias, in experiments 18 through 20 the mutants were selected before identifying t,hr source of t#he plating.

of the phage particle. Bacteria inactivated by HNO, (to 0.1% by colony count) are nevertheless fully susceptible to infection (infective center count after anti-T2 serum trc>atment 1. Nitrous acid-treated infective centers which plate on nutrient agar will not burst and release phage if held in broth. The infectivcl center count falls gradually to about 1076 of the initial count in 1 hour. During this time chloroform treatment will not release phage. Infective centers treated with hydroxylamine at high salt concentration (Frccsc ct al., 1961 ) act similarly to nitrous acid-treated infective centers. DISCUSSION

The experiments reported here indicate that nitrous acid treatment may provide a tool for a study of certain events responsible for the initial and crucial stages of phago development. The information ob-

TIME OF EXPOSURE

TO kN0,

(MINS)

FIG. 3. Nitrous acid inactivation of multiply infected complexes. Curve 1, control rxperiment (multiplicity 0.1 phage per bacterium), inactivation by HPU’OZ after incubation in broth for 5 minutes. Curvrs 2 and Y, hact,eria were infected in CAP buffer with a phage mixture (equal prol~rt,ions of rl and h) to multiplicity of 4. Among tht, zero-time infected centers, 72% contained both I’ and r+, i.e., gave mottled plaques (expected 72% ). The bacteria were incubated for 5 minutes in broth, then diluted into HNO? Currp ,” shows S/S, for all infected centers in the HXO, trch:ltment. Curve 3 shows S/S, for infected cclnters containing both T and r’.

450

BAYLOR

AND MAHLER

complementary one; (3) There is no great likelihood of energy transfer or migration to “sensitive” centers, as in the case of radiation inactivation, because damage remains in place at the point of application. The mutation frequency determined foi both r and ht mutants was -1.5 x lo-” mutants per hit per survivor for vegetative phage, a value comparable to that observed for free phage. Tessman (1959b) reported the value of 7 x 10v5 for the h mutation in free +X174 induced by HNO:! under similar conditions. Vielmetter and Wieder (1959) and Vielmetter and Schuster (1960) reported a rate of 3.2 x 1O-3 for T mutations in T2 at a different pH and NOsconcentration. The demonstration of nitrous acid mutagenesis on vegetative phage in 2- and 5minute complexes; the efficiency of this process compared to that obtained with free phage; and the available information concerning the chemical reactions brought about by HNO, make it likely that the entity subject to inactivation and mutagenesis is a nucleic acid, most probably DNA. The production of heterozygotes by treatment of free phage with HNO:! (Vielmetter and Wieder, 1959; Tessman, 1959b) has been taken as evidence that genetic information of T2 and T4 is carried separately in each strand of their DNA. The fact that an equal or greater proportion of heterozygotes is found in our experiments (52% in free T4, Tessman, 195913; 50% in the 5minute complexes and 73% in the 2-minute complexes, see Table 2) justifies a similar inference for vegetative phage. Delayed penetration of the HN0z3 could produce the shoulder or lag found in the lto 3-minute complexes. Fluctuating changes in permeability and refractoriness of the infected cell to penetration by external agents are well known (Adams, 1959; Lesley et al., 1951; and with certain assumptions would lead to a kinetic picture indistinguishable from the one observed. Since changes in permeability might well require 3 We are indebted to Drs. A. D. Hershey and A. H. Doermann for bringing this possibility to our attention.

protein or polypeptide synthesis, the sensitivity of the pattern to CAP might also be understandable. However, although the lap; is of less than 2 minutes duration, the 3minute complex still responds as if it were a l-minute complex. Also from the limited data available, the frequency of induced mutation appears to be the same for the 2and 5-minute complexes. For this reason an explanation in terms of an alteration in the state of the genetic material might not be inappropriate. The cause of the lag of the inactivation curve of the 2-minute complexes could be studied by comparing the disappearance of recombinants and recombinational heterozygotes following HN02 treatment. If the ratio of heterozygotes to recombinants remained constant throughout the inactivation period, penetration is a likely factor. If the ratio changed, other mechanisms should be invoked. The multiple-hit inactivation curve characteristic of the later developmental stages is susceptible to a satisfactory analysis. The results are comparable to those obtained with inactivation of T2 complexes by Xrays and to inactivation of T7 complexes by ultraviolet light. Although in the infected cell, materials other than viral DNA are subject to reaction with HNO% , nevertheless these results find a reasonable interpretation in terms of the production of many replicas of parental DNA, each capable of producing viable progeny. ACKNOWLEDGMENT We wish to thank Dr. Anita Hessler and Mrs. Natalie Edwards for their assistance with many of the experiments reported here, and Drs. Cyrus Levinthal and Simon Silver for their comments and suggestions concerning this manuscript. REFERENCES ADAMS, M.

H. (1959). “Bacteriophages.” Interscience, New York. BAUTZ-FREESE, E., and FREESE, E. (1961). Induction of reverse mutation and cross reactivation of nitrous acid-treated phage T4. Virology 13, 19-30. BAYLOR, M. B., HURST, D. D., ALLEN, S. L., and BERTANI, E. T. (1957). The frequency and distribution of loci affecting host range in the coliphage T2H. Genetics 42, 104-120.

HNOS ON INTRACELLIJLAR BROCK, T. P. (1961). Chloramphenicol. Bacterial. Revs. 25, 32-48. FREESE, E. (1959). On the molecular explanation of spontaneous and induced mutations. Brookhnzlett Symposia in Biol. No. 12, 63-75. FREESE, E., BAUTZ-FREESE, E., and BAUTZ, E. (1961). Hydroxylamine as a mutagenic and inactivating agent,. J. Mol. Bio2. 3, 133-143. GEII)URCIIEK, E. P. (1961). Reversible “DNA”. Proc. 9atl. Ad. Sci. U. S. 47, 95C958. HARM, W;. (1960). Vergleirhende Untersuchungen an HNOI-inaktivierten und UV-inaktiviert,en Bacteriophagen T4. Z. Vererbungslehre 91, 5262. LESLEY, S. M., FRENCH, R. C., GRAHAM, A. F., and VAX ROOYEK, C. E. (1951). Studies on relationship between virus and host cell; II. The breakdown of T2r’ bacteriophage upon infection of its host E.schc)*ichifr co2i. Can. J. Med. Sci. 29, 12% 143. SCHUSTER. H. (1960a). Die Reaktionsweise der Desoxyribonucleindure mit salpetriger Same. %. ~Vaturfotxh. 15b, 298-304. SCWU~TER, H. (1960b). The reaction of nitrous acid with deoxyribonucleic acid. Biochem. Biophys. ICesearch Commons. 2, 320-323. SISSHEIMER. R. L. (1959). A single-stranded deoxyribonucleic acid from bacteriophage gX-174. J. .2101. Bid. 1, 43-53. SISSIIEIMER, R. L. (196Oa). The biochemistry of genetic factors. Ann. Rev. Biochem. 29, 503-524. SINSHEIMER, R. 1,. (1960b). Nucleic acids of the bnc?erial viruses. 1,~ “The Nucleic Acids” (E.

T2

451

Chargaff and J. N. Davidson, eds.), Vol. 3, pp. 187-244. Academic Press, New York. STAHL, F. W. (1959). Radiobiology of bacteriophage. In “The Viruses” (F. M. Burnet, and W. M. Stanley, eds.), Vol. 2, pp. 353-385. .Icademic Press, New York. STEST, G. S. (1958). Mating in the reproduction of bacterial viruses. Advances in Virus R~scwrrh 5, 95-149. STENT, G. S. (1959). Intracellular multiplication of bacterial viruses. In “The Viruses” (F. M. Burnct and W. M. Stanley, eds.), Vol. 2, pp. 237-280. Academic Press, New York. STEIYT, G. S., and FUERST, C. R. (1960). Genetic and physiological effects of the decay of incorporated radioactive phosphorus in bacterial viruses and bacteria. Adtlnnws in Bid. and Mc~rl. I’h!/s. 7, l-75. TESSMAN, I. (1959:~). Some unusual properties of the nucleic acid in bacteriophages S13 and +X174. Virology 7,263-275. TESS>~A~, I. (1959b). Mutagenesis in phages +X174 and T4 and properties of the genetic material. Virology 9,375-385. VIELMETTER, R., and SCHUSTER, H. (1960). Die Basenspezifitat bei der Induktion van Mutationen durch salpetrige Same im Phagen T2. %. Naturforsch. 15b, 304-311. VIELMETTER, R., and WIEDER, C. M. (1959). Mutagene und inaktivierende Wirkung salpetriger Siure auf frcie Partikel des Phagen T2. Z. h’utzcrforsch. 14b, 312-317.