Delayed Lysis with a Mutant of Salmonella Bacteriophage P22

Transcription

1 JOURNAL OF VIROLOGY, Sept. 1969, p Copyright 1969 American Society for Microbiology Vol. 4, No. 3 Printed in U.S.A. Delayed Lysis with a Mutant of Salmonella Bacteriophage P22 LARRY W. COHEN Seaver Laboratory of Biology, Pomona College, Claremoiut, Ccaliforlnia Received for publication 21 May 1969 A mutant of bacteriophage P22 (Lys-) was isolated which shows a plaque morphology on mixed plates comparable to the r+ plaques of the T-even phages. When Lys- and normal Lys+ plaques are juxtaposed on a petri dish, the Lys+ plaque exhibits a flat side adjacent to the Lys- plaque. The mutant is identical to P22 under an electron microscope, is inactivated at the same rate by antiserum and heat, and has the same kinetics of attachment. It does not plate on Salmonella lysogenic for phage P22 nor on strain St/22. In liquid culture, the lysis of mutant infections in M9CAA medium is delayed between and 40 min. Cells mixedly infected in M9CAA with Lys- and Lys+ phage lyse later than Lys+-infected cells and even later than Lys--infected cells. In unsupplemented M9 medium, however, mixedly infected cells again lyse later than Lys+-infected cells, but Lys--infected cells require more than 3 hr to lyse. In supplemented and unsupplemented M9 media, intracellular phage development and endolysin synthesis proceed in Lys- infections at least as rapidly as in Lys+-infected cells. In diluted infections, the latent and eclipse periods of Lysand Lys+ infections are indistinguishable. The possible mechanisms involved in the control and timing of lysis are discussed. T-even wild-type phage plated on E;scherichia coli B produce plaques characterized by their small size and diffuse borders. Mutant T4 phage (r-) produce larger plaques with more sharply defined halos (9, ). The wild type, designated r+, produces the smaller plaques because of the delayed lysis resulting from infection followed by superinfection of the bacterial host cells by phage released from the first cells to lyse (4). 2'When T-phage r+ and r- plaques lie in sufficiently close proximity on a petri dish, the r- plaque seems to be inhibited in its development, exhibiting a flat side adjacent to the r+ plaque or even an inverted crescent border. In temperate phage X a mutant showing delayed lysis has been observed (6). This paper describes a mutant of Salmonella phage P22, infections of which show many characteristics of lysis inhibition but, in contrast to the T-even phages, do not require superinfection. MATERIALS AND METHODS Bacteriophage strains. The phage used in the following experiments were wild-type S. typhimurium phage P22, clear mutant C25, and Lys-c2, a mutant derivative of the C25 stock resulting from ultraviolet light mutagenesis. The C2 gene mutation was described previously (12, 13). Note that, except where specifically designated, the phage used in the experiments were mutant at the c2 gene; the double mutant Lys-c2- will be referred to a Lysr and the single c2- mutant as Lys+. Bacterial strains. S. typhimurium LT-2 and derivative mutant strains were used in all experiments. A gal- mutant was used as background in platings on eosin methylene blue galactose-supplemented plates. Strain C 527 is a histidine C amber mutant; strain C is isogenic to strain C 527 but contains an amber suppressor (17). Strain St/22 is resistant to phage P22 but sensitive to variant P22 phages (see 19). Media. Buffered saline contained: NaCl, M; KH2PO4, M; Na2HPO4, M; ph 7.0. M9 medium supplemented with Casamino Acids (M9CAA and unsupplemented M9 medium (15) were used to culture cells. Nutrient agar, L broth, E M B galactose agar and soft agar for plating phage have been described (12). Ultraviolet light mutagenesis. The Lys- mutant was isolated after ultraviolet irradiation (Sylvania germicidal 15-w lamp) of host bacteria and P22 Lys+ phage. S. typhimurium LT-2 strain was irradiated for 15 sec (3 ml in a sterile 0-mm petri dish lid) at 90 cm, which yields %U survival of colonyformers on nutrient agar plates. The phage were given 1 sec of irradiation at 50-cm distance, yielding approximately 1% survival when plated on the irradiated bacteria (2, 16). The Lys- mutant was found among isolates selected for small plaque size. Determination of time of lysis of undiluted infected bacteria. S. typhimurium LT-2 in M9CAA medium or unsupplemented M9 was grown in log phase with aeration to a concentration of approximately 8 9

2 2 COHEN J. VIROL. bacteria/inl (Klett reading = 25, Xmax = 540 nm). The cells were centrifuged for min at 4,300 X g, and the pellet was washed with an 0.5 volume of phosphate-buffered saline and spun again for min at 4,300 X g. The cells were resuspended in an 0.1 volume of phosphate-buffered saline, transferred to a small aeration tube, and aerated at 37 C for 15 min. A 1-ml amount was then transferred into the icechilled side arm of each of three Nephelo culture flasks, infected with either mutant Lys-, Lys+, or both Lys- and Lys+ phage and retained in the cold saline an additional 15 min. The infected cells were warmed by addition to each flask of 9 ml of prewarmed growth medium and the flasks were transferred into a shaker water bath (37 C). Optical density readings were recorded at timed intervals; a sustained rapid decrease in optical density is indicative of cell lysis. During aeration in saline, the cells exhaust internal nutrients so that phage development begins synchronously at the time nutrient medium is restored. To exclude the possibility that observed results might be due to the phosphate saline treatment, synchronization of infection was accomplished by aerating the cells in 0.01 M NaCN or in unsupplemented M9 medium lacking glucose. Results comparable to those reported were obtained. One-step growth experiment. Bacteria were grown in M9CAA medium to a concentration of approximately 8 cells/ml, and 1-ml samples were transferred to each of two aeration tubes. The cultures were separately infected with Lys- or Lys+ phage at a multiplicity of infection of 5. After 5 min of incubation, two 50-fold serial dilutions were carried out with prewarmed M9CAA, and the last culture was aerated. At -min intervals, 0.1-ml samples were withdrawn, diluted in prechilled diluting broth, and plated for infective centers. Determination of the eclipse period. S. typhimurium LT-2, at a concentration of 8/ml, were infected with Lys- or Lys+ phage (multiplicity of infection = 5). At the times indicated, 0.1-ml samples were withdrawn into 4.9 ml of ice-chilled diluting broth, and then 0.1 ml from these was withdrawn into 4.7 ml of diluting broth containing four drops of chloroform. The tubes were shaken vigorously, allowed to stand for 30 min at room temperature, and plated for infective centers. Endolysin assay. E. coli B cells, at a concentration of 4 X 8/ml, were sensitized by suspension in an 0.33 volume of 0.1 M ethylenediaminetetraacetate- 1.0 M tris(hydroxymethyl) aminomethane-hydrochloride buffer (ph 8.0) for 5 min at 0 C, followed by centrifugation and resuspension to the original volume in ice-chilled distilled water (11). Samples (1.5 ml) were withdrawn from Lys-- and from Lys+-infected cultures (8/ml) at various times during the infection and transferred into ice-chilled nitrocellulose tubes containing 0.15 ml of 0.11 M NaCN. The samples were sonically treated to disrupt the cells, and 0.15 ml of the extract was added to 4.9 ml of sensitized E. coli B in a Klett-Summerson colorimeter tube. The tube was incubated at 28 C, and optical density readings were taken at 2-min intervals. RESULTS Inhibition of lysis. The results of plating mixtures of phage Lys- with Lys+ phage (both are clear mutants) are shown in Fig. 1. Proximity of the smaller mutant plaques to the zone of lysis of the larger phage plaques results in an apparent impairment of the lysis of the latter. The effect is observed when the two phage plaques in question are close together. The larger plaque is produced by the Lys+ phage and the smaller by the mutant Lys- phage. The white zones surrounding the smaller plaques are not halos but are caused by bacterial growth. Delayed lysis in liquid culture. The reduced size of the mutant plaque suggests that the latent period of the mutant is extended in time. The time of lysis of Lys--infected cells in liquid culture is observed to be delayed by 25 to 30 min. This delay period may extend in M9CAA to 40 min, and to more than 3 hr in unsupplemented M9 medium. Once lysis begins, the rate is the same for the Lys--infected cells as for Lys+infected cells. The time of lysis is determined by the genotype of the phage without apparent alteration of the timing by superinfection. This is consistent with the observation that phage P22 normally shows exclusion of a secondarily infecting phage (14). To determine whether Lys--infected cells, at the higher cell concentration (8/ml), require more oxygen than wild-type infected cells, Nephelo culture flasks were adapted to bubble pure watersaturated oxygen into the swirling liquid in the FIG. 1. Plaque morphology of Lys+ and Lys- phages on mixedly infected plates.

3 ~~- VOL. 4, 1969 DELAYED LYSIS WITH PHAGE P flask. The increased oxygen had no effect on the are comparable, even though lysis of the Lysculture-as determined turbidimetrically-was time of lysis: the Lys+-infected cells lysed in the normal time period and the Lys--infected cells delayed by min and was less extensive. lysed some 40 min later. Lysozyme synthesis during Lys- and Lys+ Latent and eclipse periods. The results of a infections. The delay in lysis observed in the typical one-step growth experiment indicated that dense liquid cultures of Lys--infected cells could the latent period, after 2,500-fold dilution of the be due to delay in the synthesis of endolytic infected cells in M9CAA medium, was the same enzyme. Cells were grown in M9CAA medium; for Lys-- and Lys+-infected cells. Apparently the one culture was infected with Lys- phage and dilution allows lysis to occur which would be the other with Lys+ phage, each at a multiplicity delayed in Lys- infections at a cell concentration of. Klett readings were taken at intervals to of 8/ml. The burst size calculated per infective determine the time of lysis, and samples were center present after min of the infection was withdrawn and assayed for lysozyme. Lysis 266 for the Lys+ and 5 for the Lys- phage began in the Lys+-infected cells at 45 min and in infections, the yield of Lys- phage being approximately twofold greater than the Lys+. of the infection. The results of the lysozyme the Lys--infected cells at approximately 65 min The eclipse period, which represents the time assays (28 C) are presented in Fig. 3A and 3B. interval between infection and appearance of the Samples taken at 30 min of the infection showed first intracellular infective particles, is shown in higher enzyme activity in Lys--infected cells than Fig. 2. The eclipse periods oflys- and Lys+ phage in Lys+, but by 40 min the activities appeared to be about the same. Double infection of bacteria with Lys- and Lys+ phage. Simultaneous infection with Lys- and Lys+ mutant phage produced most interesting and unexpected results. The cells grown on M9CAA medium, washed, and aerated in phosphatebuffered saline were infected with Lys+ phage (multiplicity of infection = ), Lys- phage (multiplicity of infection = ), or both phage (multi- lol plicity of infection = for each). After allowing 5 min for attachment, prewarmed M9CAA medium was added to the cells in a side arm Nephelo culture flask (Fig. 4). The Lys- mutation apparently is not only dominant, but, in mixed infection with Lys+ phage, lysis is inhibited even more than hlj 0 r A I- a. 80 ; z 60-25t nf k Hi N y 40 - z 15 9 b, C K TIME AFTER INFECTION (min) INCUBATION TIME (minutes) 6 O FIG. 3. Lysozyme activities in samples taken at various times during Lys- and Lys+ infections and assayed on sensitized E. coli B at 28 C. (A) lysozyme assays of TIME AFTER INFECTION (Minutes) Lys+-infected cells; (B) assays of Lys--infected cells. FiG. 2. Intracellular development of phage particles 0, Sensitized cells with no cell extract; *, assay of in Lys- (-) and Lys+ (0) infections. Optical density cell extracts from samples taken at min; A, assay of readings and chloroform-lysed plating samples of the -min samples; A, assay of 30-min samples; A assay cultures were taken at the times indicated. of 40-min samples; 0), assay of 50-min samples.

4 212 COHEN J. VIROL. (I) z J O TIME AFTER INFECTION (minutes) FIG. 4. Double infection of S. typhimurium in M9CAA with Lys- phage (0) and Lys+ phage (0) or with Lys- and Lys+ phage (A). Multiplicities of infection totaled in each infection. in Lys--infected bacteria. When the infections were synchronized by allowing attachment in the presence of 0.01 M NaCN followed by dilution into M9CAA medium, the results were the same. However, when unsupplemented M9 salts medium was used instead of M9CAA, the results indicated that the Lys- mutation in doubly infected cells is still dominant in that lysis is delayed. However, the lysis of the cells infected solely with Lys- phage is delayed for some 3 hr. When L broth was added to the unlysed Lys-- infected cells to a concentration (v/v) of 2.5%, lysis followed in 15 to min (3). Is the Lys a mutant of phage P22? Strains of S. typhimurium have been shown to yield a number of P22-related phages (18, 19) which can be distinguished from P22 on the basis of host range and morphological, physical, and immunological differences. The following observations lead to the conclusion that the Lys- isolate is a mutant of P22: we found it identical to P22 phage under an electron microscope (1). The bacterial strain which yielded the Lys- phage was treated with ultraviolet light followed by plating of the ultraviolet-treated bacteria; this treatment gave confluent bacterial growth over the surface of nutrient agar plates (39 plates having approximately 8 bacteria per plate) without yielding any phage. This mutant will form plaques on the bacterial background on which it was isolated. The mutants isolated will not plate on cells already lysogenic for phage P22 nor on strain St/22. Antiserum prepared against P22 Lys+c2- phage gave essentially identical K values with P22 Lys+c+ and Lys-c2- phage (Lys+c+, 7,7 min-; Lys-c2, 7,860 min-'), indicating that, on the basis of rates of inactivation in the presence of antiserum, the Lys-c2- phage is indistinguishable from wild type. These data were obtained from experiments in which Lys+c+ and Lys-c2- phage (distinguishable on the basis of plaque morphology) were inactivated together in the same reaction with antiserum. The Lys+c+ and Lys-c-2 phage heat inactivate (78 C) at identical rates and show identical kinetics of attachment to sensitive bacteria (98 % within 4 min). DISCUSSION A number of observations indicate that the Lys- phage is indeed a mutant derived from a Lys+ P22 and not a P22-related phage (18, 19): the phage are identical to P22 under an electron microscope and are serologically indistinguishable from P22. Heat inactivation data, kinetic studies of attachment, and the absence of plating ability on Salmonella lysogenic for P22 or on strain St/22 are also in agreement with the conclusion. It is unlikely that the Lys- phage was already present in the ultraviolet-treated bacteria as a prophage; ultraviolet light administered to the Salmonella LT-2 strain (on which the mutant was isolated and does plate) did not produce plaques. Furthermore, the Lys- mutant is a clear plaque former which would not normally be expected had it been a prophage in the host cells. The Lys- mutant of Salmonella phage P22 exhibits some of the growth characteristics of phages that are subject to lysis inhibition. It apparently inhibits normal P22 phage development when the two plaques are juxtaposed on a petri plate, shows delayed lysis in liquid cultures ofhigh bacterial titer especially in minimal growth medium, but shows normal timing of lysis when the culture is sufficiently diluted. Infected cells deliberately superinfected after and min of infection were not, however, delayed in their lysis if diluted. It appears, therefore, that dilution itself enables lysis to proceed at normal times. Perhaps delayed lysis in concentrated Lys-- infected cell suspensions is due to the accumulation of a lysozyme antagonist which, in the diluted cultures, is at too low a concentration to be effective. Attempts to show the presence in the culture fluid of a substance capable of inhibiting lysis have not yet been successful. Intracellular phage development proceeds normally in the Lys- mutant infections, and endolytic activity, as assayed on sensitized E. coli

5 VOL. 4, 1969 DELAYED LYSIS WITH PHAGE P B, is the same or even higher than normal at all stages of the infection. Harris et al. (8) described mutants of phage X which produce lysozyme but do not lyse, and Emrich (5) described a "spackle" (S-) mutant of phage T4 which lyses even though no lysozyme is produced. It is clear that the presence of lysozyme is insufficient to induce lysis; lysis is strongly influenced by at least one other phage-induced product (6, 7). A normal function of a second gene product, perhaps the S+ gene product described by Emrich, may be considered as antagonistic to lysozyme, stabilizing the cell membrane or wall, or both, until late in the infection. Since the S+ in T4 is dominant, it is presumed to produce the active product. Emrich's mutant T4 S- is abnormally sensitive to lysis from without and, like r-, it is not subject to lysis inhibition. This suggests that lysis inhibition might be the result of stimulated production of the S+ gene product. In the T-even system, the r+ locus could be considered to control the rate of production of S+ substance. Speculating further, the P22 Lys- mutant and certain mutants of X (8) would be over-producers of that stabilizing factor. Indeed, double infections with Lys- and Lys+ mutant phage indicate that the Lys- mutation is dominant, and Harris reported that infection of induced wild-type X lysogens with mutant ts9b (the lysis-inhibited X mutant) results in delayed lysis as if lysis were impeded by the ts9b gene product. The phage type most prevalent, and hence classified as wild type among the virulent T-even phages, is subject to lysis inhibition, whereas it is the rare mutant form of temperate phages X and P22 which shows delayed lysis. In phage T4 infections, delayed lysis is associated with much higher yields of phage per infected bacterium. This higher yield is observed in infections with the Lys- mutant of P22, especially in infections in unsupplemented M9 medium. Preliminary observations in our laboratory, however, indicate that the Lys- mutation reduces the efficiency of phage integration and lysogeny. If selection in temperate phages operates in favor of the capacity to lysogenize, this might account for the rarity of lysis-inhibited types among temperate phages X and P22. ACKNOWLEDGMENTS I am indebted to Mary Showers for her able and dedicated assistance during the conduct of the experiments, to Gerhard Ott for his fine photography, to Fred Eiserling (UCLA) for electron microscopy of the mutant phage, and to N. Yamamoto and D. Berkowitz for their generous contributions of S. typhimnurium LT-2 strain St/22 and strains 527 and This work was supported by Public Health Services grant Al from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Anderson, T. F On the fine structure of the temperate bacteriophages P1, P2 and P22. Proc. European Regional Conf. Electron Microscopy, Delft. Almqvist and Wiksell, Uppsala. 2. Campbell, A Sensitive mutants of bacteriophage X. Virology 14: Cohen, L. W Delayed lysis, with Salmoniella bacteriophage P22: induction of lysis by addition of cysteine or histidine to the growth medium. J. Virol Doermann, A. H Lysis and lysis inhibition with Escherichia coli bacteriophage. J. Bacteriol. 55: Emrich, J Lysis of T4 infected bacteria in the absence of lysozyme. Virology 35: Groman, N. B., and G. Suzuki Relation of endolysin to lysis by lambda bacteriophages. J. Bacteriol. 84: Groman, N. B., and G. Suzuki Quantitative study of endolysin synthesis during reproduction of lambda phages. J. Bacteriol. 86: Harris, A. W., D. W. A. Mount, C. R. Fuerst, and L. Siminovitch Mutations in bacteriophage lambda affecting host cell lysis. Virology 32: Hershey, A. D. 1946a. Mutation of bacteriophage with respect to type of plaque. Genetics 31: Hershey, A. D. 1946b. Spontaneous mutations of bacterial viruses. Cold Spring Harbor Symp. Quant. Biol. 11: t. Jacob, F., C. R. Fuerst, and E. L. 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