A P22 bacteriophage mutant defective in antigen conversion

DISCUSSION

APiD

PRELIMINARY

Another attempt was made to recover virus from the same carrot tissue 50 days after the inoculation feeding. This time virus-free aseptically reared leafhoppers were allowed to feed 011 the inoculated carrot tissue for 10 days before being transferred to aseptically grown, healthy aster seedlings. These seedlings developed symptoms of aster yellows approximately 27 days after the leafhoppers were transferred to them, indicating that the carrot tissue culture had been infected by the virus. It seems likely that the longer acquisition feeding and the systemic virus infection of the carrot tissue accounted for the successful virus recovery. The reason for the comparatively long incubation period in the seedlings is not known. It is conceivable that the growing conditions and age of the seedlings, somewhat different from that of aseptic seedlings inoculated in earlier tests, were responsible for the protracted incubation. The positive diagnosis of aster yellows is more complicated in the case of an aseptic seedling, since the plant cannot be handled in the same manner as a potted plant, and wat’er vapor condensat,ion on the wall of a glass jar often obstructs direct observation of signs of disease. The virus-infected carrot tissue did not show any visible symptoms; it had the same color as normal tissue, and no clear difference in the rate of growth was observed between the infected and the normal tissue. At present’, no information is available about possible localization of aster yellows virus in the infected carrot tissue culture. Electron micros&pica1 investigations of the infected tissues are under way. The reason for the failure to recover the virus in the first transmission experiment is still obscure. illthough no virus was recovered from the tomato and the potato tissue cultures, this lack of recovery could be explained in part by the inability of leafhoppers to survive for long periods on these tissues, and in part by the failure of these cultures to grow. The potato and tomato tissue cultures may nevertheless have been infected with aster yellows virus in the same manr,er as the carrot tissue culture. Further tests are under way to check this point.

REPORTS

279

ACKNOWLEDGMENTS We wish to express our thanks to Dr. Waher Tulecke for supplying the plant tissue cultures used in this study, and to Miss Patricia Mehlhop for her excellent technical assistance. REFERENCES 1. BLACK, L. M., Growth Suppl. Sixth Growth Xymp , 79-84 (1947). 2. MARAMOROSCIX, K., P;ICXELL~ L. G., LITTAC, V. C., and GRACE, T. D. C.: Amt. Record E-L, 579 (1958). 3. MITsUHASHI, J., and M~~~~oRQs~T~, R., Pvx. Intern. Congr. Zool. Z&h, Washington, 63. G,, 1963 (published by the Congress in Washing-, ton, D. C.) Vol. I, p. 3 (1963). 4. MTTsUHASHI, J., and N~ARA~XOROSCH, K., Contrib. Boyce Thompson Inst. 22, 165-173 (1963). 5. CNEN, T., KILPATRICK? R. A., and RICH; A. E., Phytopathology 51, 799400 (1961). 6. MARAMOROSCH, R., Truns. N.Y. Acad. si~i. Ser. II 20, 383-393 (1958). 7. WALLIS, R. L.: U.S. Dept. Agr. ARS 33-55, l-15 (1960). 8. TCLECICE, W., niature (in press) (1964). JUN MaTsnHAsIII" KARL MARA~~~RoBcB~ Boyce Thompson Institute for Plant Resew& Yonkers, ATew York Accepted March 16, 1984 2 On leave from cultural Sciences, Japan.

A P22

the National Nishigahara,

Bacteriophage

Mutant

Antigen

Conversion”

Institute Kita-ku,

Defective

of AgriTok:~o,

in

P22 and related phages have the ability to elicit somatic antigen 1 production in Xalmonella typhinaurium. The antigen is produced both when the phage is in the prophage and when the phage is in the vegetative state (1-3). The properties of antigen 1 have been described (2, 4), and some attributes of the genet.ic structure of phage P22 have been analyzed (5, ?‘). The purpose of the present communication is to describe the isolation and properties of a mutant of bacteriophage P22 that, lacks the 1 This work, in part, was supported by Besearch Grant E-1650 of the National Institute of Allergy and Infectious Diseases, Public Health Service.

DISCUSSION

AND

PRELIMINARY

ability to elicit antigen 1 formation in X. typhimurium. When an X. typhimurium culture is infected with P22 phage,. diluted, and plated, most of the phage-producing colonies that grow up upon incubation contain antigen 1. Similarly, antigen 1 is detected in most colonies after plating a stable P224ysogenic culture. However, bacteria in some of the colonies lack detectable antigen 1. Bacteria descended from most of the antigen l-negative colonies are found to contain antigen 1 after growth in fresh medium. In addition, phage isolated from most of the antigen l-negative colonies convert sensitive cells to antigen 1 production with normal frequency. The failure of all P22infected cells to express the antigen 1 TABLE

1

FAILURE OF MUTANT al TO ELICIT ANTIGEN 1 PRODUCTION IN CLONES OF SURVIVING BACTERIA AFTER INFECTION WITH P22 PHAGE” Infecting

phage

p22+ P22 MgHs P22 al

Presence of antigen per total colonies tested

29/30 24/30 o/30

Immunity

to P22

phage per total colonies tested

30/30 27/30 29/30

a A logarithmic-phase culture of sensitive bacteria at a concentration of 2 X 108/ml was infected at a multiplicity of infection of 10 with either P22 wild type (P22+), a magnesium hypersensitive mutant of P22 phage (MgHs) kindly supplied by Dr. N. D. Zinder, or with the antigen-converting mutant (P22 al). After 10 minutes at 37”, the culture was suitably diluted and aliquots were spread on agar. Colonies appearing on the agar were tested after 18 hours’ incubation for the presence or absence of antigen 1 by the method described in the text, and for their sensitivity to a clear plaque mutant (~9 of ref. 7) of phage P22. The proportion of lysogenic and sensitive bacteria in clones, and the number of completely sensitive clones, is a function of the temperature of incubation (8). Under the conditions used in the above experiment immune clones are comprised of approximately 50% lysogenic bacteria. Repeated reisolations of immune clones after infection with P22+ phage gave rise to lysogenic bacteria that produced P22 phage and contained antigen 1 in the clone tested or upon subculture. In contrast, similar subclones after infection by P22 ai resulted in lysogenic, immune bacteria that always lacked antigen 1.

REPORTS TABLE

2

FAILURE OF MUTANT al TO ELICIT ANTIGEN PRODUCTION IN SENSITIVE BACTERIA AFTER INFECTION” Time after infection (minutes)

2 5 8 10 15 20 25 45

Infecting P22 wild type

0 0 + + + + -t f

1

phage

P22 mutant al 0 0 0 0 0 0 0 0

a Cells in logarithmic growth at a concentration of 8 X 108/ml were infected at a multiplicity of 5 and aeration was continued at 37”. At the times stated in the table, KCN was added at a final concentration of 0.001 M. After an additional 10 minutes’ incubation, one drop of the cell suspension was mixed with one drop of the adsorbed immune serum (thus monovalent for antigen 1). The presence or absence of agglutination was then determined as described in the text. b Plus sign indicates antigen 1 detected; 0 indicates no antigen 1 detected.

characteristic has been described before (1) and is common to other systems of antigen expression in bacteria (“form variation”) (0 Several mutants with abnormal converting properties were isolated from about 1% of the colonies which lacked antigen 1. Almost all these mutants still converted to antigen 1 production, but in the proportion of less than 50% of lysogenic colonies. Thus in these phage mutant-host complexes, form variation is enhanced. The mechanism underlying this variation has not been further investigated. In contrast one mutant (al) proved to be unable to elicit appreciable antigen 1 production in any lysogenie colonies. This type of mutant is rare, present in less than 2.5 X 10d4 of lysogenized colonies. All other properties of mutant al appear characteristic of P22 phage. Its plaque morphology, serological specificity, immunity and transducing abilities are the same as those of wild type P22; as shown below, it recombines normally with P22 genomes. For the detection of antigen 1, immune

DISCUSSION

AND

PRELIMIXARY TABLE

FMLPRE

ai

OF MUTANT

REPORTS

3

TO ELICIT

ANTIGEN

CONVEFSIOX Agglutination

S. typhinzurium

immune

prepared

serqm

Adsorbed

against:

with 5’. typhimuvium

strain:

of bacteriaa

Lysogenic for pz2+

Nonlysogenic

Lysagenir for P22 mutant al

____---_ Nonlysogenic Lysogenic Lysogenic

for for

P22+ P22+

Lysogenic Lysogenic

for for

mutant mutant

Q Plus

indicates

sign

Cross

Lysogenic Nonlysogenic Lysogenic Nonlysogenic Lysogenic

al al agglutination,

number

0 indicates

Parental

phage

for

P22+

for

P22

for

P22+

mutant

m3

a+ct

al

no agglutination

I).

mt

2).mt

II.

m3atc2

m3

III.

at

mtalc+

ct

mtatc2

Freqi,-

Analyzed

at

ci

17

0.40

al

ct

26

0.60

m3

al

ct

32

0.44

at

ct

41

0.56

20

I .OQ

I).

2). 1

mtatci

I

Cross

m3 t

0 0 0 0 0

2). m3

I).

(Control) Cross

Number

Classes mtalc2

0 + + 0 0

detected.

Recombinant

Genotypes I.

0 0 0 0 0

II

c2

23

---------I

-3

1II

2j

-------A

I + FIG. 1. Recombination

21I

I ‘I

c2 -

+:

t

tests that position the al mutation. Logarithmic phase broth cultures of 8. lyphimurium at a concentration of 2 X IO8 per milliliter were infected with the appropriate phage suspensions at a multiplicity of approximately 5 for each parental phage type. The suspension was kept at 37” for 5 minutes to allow adsorption. The mixture was then diluted into fresh broth to give approximately lo5 organisms per milliliter and was incubat’ed for 90 minutes. Chloroform was added to kill surviving bacteria, and the phage were assayed by the agar layer method. Bottom-layer agar (Bacto-tryptone, 10 g; Bacto-yeast extract, 5 g; KaCl, 9 g; glucose, 8 g; distilled water, 1 liter; adjusted to pH 7.0 with NaOH) was overlaid with 2.0 ml. of soft agar (Difco Bacto-nutrient broth, 8 g; NaCl, 5 g; Baltimore Biological Laborat,ory agar, 7 g). On this agar, an :n8 plaque shows a distinct white peripheral ring and the cIear pIaques are sharply differentiated from plaques with turbid centers. Lysogenic survivors in plaques recombinant for m3 and c2 were picked with a needle into 1.0 ml of broth, and the cultures were incubated for 3 hours at 37”. The bacteria were t,hen diluted and plaued. After 10-15 hours’ incubation, aggIutination tests with monovalent antigen 1 immune serum and ronfirmation of lysogeny using the virulent P22 vd clear plaque mutant (7) were performed. Usually5 colonies from each recombinant plaque were tested. When no positive agglutination per 5 colonies was noted, the phage clone was considered to contain the al marker. Two or more clearly positive reactions per 5 colonies signified the presence of a+. No cases were found where there was only one positive agglutination per 5 coIonies tested. The percentage of recombination between the m3 and cb markers was 13.9 and between cg and A21 was 5.7 (cf. 5).

282

DISCUSSION

AND

PRELIMINARY

sera were prepared in rabbits against X. typhimurium lysogenic for wild-type P22. These immune sera, presumedly containing antibodies toward antigens 1, 4, 5, and 12 were exhaustively adsorbed with nonlysogenie, P22sensitive bacteria to remove antibodies for antigens 4, 5, and 12. The presence of antigen 1 in an infected culture or in colonies removed from agar plates was demonstrated by mixing in a depression plate one drop of an appropriate dilution of the antiserum with one drop of the infected culture or of the suspended colony which had been adjusted to contain 5 X lo8 or more bacteria per milliliter. The presence or absence of cell agglutination was determined 20 minutes later at a magnification of 30 times. Reconstruction experiments showed that antigen l-containing bacteria could be readily detected although they constituted only 10% of the population of bacteria. The results presented in Table 1 are representative of several similar experiments. The data demonstrate the absence of antigen 1 in bacteria lysogenized with mutant al but its presence in most colonies derived from bacteria exposed to wild-type P22 phage or to a magnesium-hypersensitive mutant. The ability of the magnesiumhypersensitive mutant (MgHs) to elicit antigen 1 production on agar containing magnesium (0.1 M Mg++), in which all superinfection is eliminated (Zinder, personal communication, and Young, unpublished), demonstrates clearly that antigen 1 production takes place in lysogenic bacteria and is not merely due to superinfection of sensitive segregants. The results reported in Table 2 show that the mutant phage is unable to elicit detectable antigen 1 production in vegetative growth or in early stages preceding lysogenization. Antigen 1 can be detected 7-8 minutes after infection of sensitive bacteria with wild-type P22 phage. In confirmation of Zinder’s results (3), clear plaque mutants of P22, including US phage, while unable to lysogenize (?‘), do elicit antigen 1 production during vegetative growth (not shown in Table 2).

REPORTS

Cross adsorption tests were performed to determine whether mutant al actually fails to elicit antigen 1 or, rather, elicits an altered form of antigen 1 or some different antigen. The results, summarized in Table 3, show that the strain lysogenic for mutant a1 contained neither detectable antigen 1 nor any other detectable antigen not contained on nonlysogenic bacteria. The location on the phage linkage map of the al marker was determined by performing phage crosses, using mutants kindly supplied by Dr. M. Levine. Three markers are located on the P22 chromosome in the order m3 & h21 (5). The results of crosses involving the m3 and cd markers are summarized in the top portion of Fig. 1. An interpretation of the results is schematized in the lower portion of Fig. 1, and the experimental details are described in the legend to that figure. In cross II, hZ?l was also contained in the m3 a+ c.9 parent (not shown in Fig. 1). Thirteen of 15 ma+ cf hW1 recombinants also contained al, indicating close association of al and c+. These results, combined with the data shown in Fig. 1, demonstrate that the al marker is located between the m3 and c.9 markers and is nearer to the m3 than to the C,S? locus. We conclude that the property of conversion of X. typhimurium to antigen 1 production by the presence of P22 genomes is controlled, at least in part, by a gene on the phage chromosome located between the m3 and CS markers described by Levine and Curtiss (5). REFERENCES 1. STOCKER, B. A. D., J. Gen. Microbial. (1958). 2. STOCKER, B. A. D., Abstr. Intern. Congr. biol. 7th Coongr., 65 (1958). 3. ZINDER, N. D., Perspectives in Viral. New York, 1, 43-53. Wiley, New York 4. STOCKER, B. A. D., STAUB, A. M., TINELLI, and KOPACKA, B., Ann. Inst. Pasteur 505-523 (1960). 5. LEVINE, M., and CURTISS, R., ViroZogy 37(t371 (1960). 6. KAUFFMANN, F., In “Enterobacteriaceae”, pp. 49-51. Munksgaard, Copenhagen, 7. ZINDER, N. D., Virology 5, 291-326 (1958). 8. LURIA, S. E., FRASER, D. IX., ADAMS,

18,

ix

MicroSymp, (1959). R., 98, 10,

(1951). J. N.,

DISCUSSION

AND

PRELIMINARY

REPORTS

283

have made it at least plausible that the bottom component corresponds to the inBOBBY G. YOUNG~ fectious particle, the other components LYational Cancer Institute being no more infectious than is compatible National Institutes of Health with their contamination by bottom comBethesda 14, ;Maryland ponent. The RK\‘B content of the tot,al YOSHXMURA FIJKAZAWA~~~ preparation is 18-2291, (4, 6) and does not PHILIP E. HARTMAB seem to vary greatly between the different The Johns Hopkins University components. Department of Biology Baltimore 18, Maryland ,4MV shares this multiple charact’er Accepted March 80, 1964 (several components occurring together in the virus preparation) with several other z Present address: The Johns Hopkins Uniplant viruses and bacteriophages (?‘-I 1 j) versit,y, Department of Biology, Baltimore 18, and perhaps it might be a very general XIaryland. feature of the more simple viruses, observ3 Present address : Juntendo University, Department of Bacteriology, Tokyo, Japan. able only with finer procedures of analytical 4 Post-Doctoral Research Fellow on a National ultraeentrifugation and gradient cent& Institutes of Health, U. S. P. H. S. Post-Doctoral fugation. Considering the existence of these Training Grant No. ZG-504. “multiple” viruses as a more or less general phenomenon, it seemed worth while to study the mutual relationships of the memb’ers The Base Composition of Ribonucieic Acids of such a preparation. Unfortunately the simple method of comparing component, from Alfalfa Mosaic Virus Components proportions under different conditions in sedimentation diagrams was precluded by Alfalfa mosaic virus (A&IV) was first the very pronounced Johnston-OgsQon cfisolated by Ross (1) and physically characfeet exhibited by the AMV preparations. A terized by Lauff er and Ross (2). It appeared to be rather unstable. With the less ad- different approach had to be chosen, and was found in the determination of t#l-iebase vanced ultracentrifugation methods of that ratios of the RiYA’s of the separate compotime, nevertheless, sometimes a bimodal nents. The method used was that of Smith sedimentation pattern was observed. Investigations by Bancroft and Kaesberg in and Markham (12). Hydrolysis of the RNA in its protein coat 1958 and subsequently (3, 4) showed AMV was performed in 1 N IICl at 100°C for I preparations to be composed of several hour. Chromatograms were run on Rhatcomponents, separable by centrifugation man no. I paper with Wyatt’s solvent (i3js techniques, but inseparable by electroSpots were cut out, cut in strips, aid eiwted phoretical or serological methods. Prelimovernight in 0.1 N HCl. Ultraviolet (UY) inary investigations suggested the protein absorption was measured at the respective subunits of the components to be idenblanks in a tical, the difference between the virus and maxima against appropriate To its relat’ed viruslike particles residing in Unicam SP 500 spectrophotomet’er. test the met’hod in our hands, we controlled their particle weight and perhaps in their the reproducibility with yeast RNA (Britquaternary structure. ish Drug House) and found it to be satisThe different components were characfactory. In 32 determinations in 4 series terized by their sedimentation constants as follows: 99 S (bottom component), 89 S maximal (Twas 0.5 mole %. c tended to rise when determinations had to be made wit#h (middle component), 74 S (top component very small quantities. As another check on 6), and 68 S (top component a). In electron our technique, we determined the base micrographs the particles appeared as ratio of T’YXV of Markham’s grou.p I short rods of uniform diameter (240 nm) and variable length (ZOO-650 nm). Banand found values that compare favorably croft and Kaesberg (4) and Bancroft (5) with t,hose of Symons et al. (14) (Table l), and

Symp.

BCRROCS,

Quant.

J.

Biol.

W., Cold 23, 71-82

Spring

(1958).

Harbor