JOURNAL OF VIROLOGY, JUlY 1976, P. 220-227 Copyright ©D 1976 American Society for Microbiology
Vol. 19, No. 1 Printed in U.S.A.
Isolation and Characterization of tox Mutants of Corynebacteriophage Beta WALTER LAIRD AND NEAL GROMAN* Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195 Received for publication 6 August 1975
Seventeen nontoxinogenic (tox) mutants of corynebacteriophage beta have been isolated by using a tissue culture screening technique. The mutants fall into four major classes. Two of the classes, I and II, appear to contain missense and nonsense mutants, respectively. However, classes III and IV have not been previously described. Class III mutants produce two proteins (CRMs) seriologically related to diphtheria toxin, but efforts to demonstrate the presence of more than one tox gene have not been successful. Class IV mutants are phenotypically CRM-, failing to produce any detectable protein serologically related to diphtheria toxin. Genetic studies indicate that the mutations in class IV strains are not in a gene distinct from the structural gene for toxin, and that the CRMstrains retain at least a portion of that gene. A natural phage isolate, gamma, behaves in a completely parallel fashion to the class IV mutants. The production of tox+ recombinants through recombination of various pairs of tox phage mutants has been demonstrated. The implications of these findings for the natural history of diphtheria are discussed. In 1951, Freeman discovered (4) that nontoxinogenic strains of Corynebacterium diphtheriae became toxinogenic when lysogenized by certain corynebacteriophages. Since it was recently established that the structural gene for diphtheria toxin is present on the beta converting phage genome (14), the regulation of toxin production has now become a logical focus of attention. It has long been known that the synthesis of toxin is influenced by the amount of iron present in the culture medium (16). High yields of toxin are produced only under conditions of low iron concentration. It seems reasonable that there might be other genes or sites on the phage genome as well as on the host genome that function in the expression or regulation of the gene for toxin. In the last few years, various nontoxinogenic (tox) phage mutants have been isolated. These tox mutants appear to be affected in a structural gene for toxin (19), but regulatory mutants have not been described. However, if such mutants are to be found, it is likely that a large number of tox mutants will have to be examined. Until recently, the search for such mutants was cumbersome since it required skin testing on a rabbit. The development of a more convenient tissue culture method for toxigenicity testing (11) has made it possible to screen large numbers of mutagenized phage. This paper describes the isolation and partial characterization of seventeen tox mutants. The study
has concentrated on the characteristics of one class of mutants that appears to be new to the literature. MATERIALS AND METHODS
Strains of bacteria and phage. C. diphtheriae strains C7, C7(,3), and C7(y) were from our stock
culture collection. A. M. Pappenheimer, Jr., generously sent us strains of C7(,8f3) and C7Q(345), which have been renamed C7(p'0x-30) and C7(I3b0-5), respectively. The nomenclature proposed by Holmes (9) for tox mutants has been followed. Phage strain y'0o5 was produced by crossing 3tox45 with gamma phage vegetatively. All lysogenic strains to be described were derived by infecting stock strain C7 with the appropriate phage or phages. A series of double lysogens was prepared and genetically characterized as previously described (12). The original monolysogens, the heterologous superinfecting phages, and the derived double lysogens are listed in Table 1. Bacteriophage markers. All phages described in this paper plaque on strain C7. Beta phage plaques on C7(y) but not on C7(f3), whereas the reverse is true for gamma phage. Phage immunity will be designated as imm-/3 or imm-y. The mutations in nontoxinogenic strains isolated in our laboratory have been assigned arbitrary numbers beginning with 101. Seventeen tox mutants designated f3Z'°'0 through f3O0-"l7 will be described in Results. The tox gene in gamma phage has been designated tox-118. The phenotypic designation CRM+ will be used to identify the protein product of a tox gene that crossreacts with or is serologically identical with diphtheria toxin. A number following the symbol CRM
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TABLE 1. Heteroimmune double lysogensa Double lysogen (tox alleles only) Superinfecting phage 1 C7(imm-/3 tox-30) imm--y tox-45 C7(tox-45)(tox-30) 2 C7(imm--y tox-45) imm-/3 tox-30 C7(tox-30)(tox-45) 3 NA NA C7(tox-45)(tox+) 4 NA NA C7(tox+)(tox-45 ) 5 C7(imm--y tox-45) C7(tox-116)(tox-45) imm-,3 tox-116 6 imm--y tox-45 C7(tox-45)(tox-116) C7(imm-,8 tox-116) 7 imm-y tox-45 C7(tox-45)(tox- 117) C7(imm-,8 tox-117) 8 C7(imm-y tox-45) imm-f3 tox-117 C7(tox-117)(tox-45) 9 imm--y tox-118 C7(tox-118)(tox-45) C7(imm-,3 tox-45) 10 C7(imm-y tox-118) imm-/3 tox-45 C7(tox-45 )(tox-118) 11 C7(imm-y tox-118) C7(tox-116)(tox-118) imm-,8 tox-116 12 C7(imm-/3 tox-117) imm-y tox-118 C7(tox-118)(tox-117) 13 C7(imm-/3 tox-30) imm-y tox-45 C7(tox-45)(tox-30) 14 imm-y tox-45 C7(tox-45)(tox-30) C7(imm-,3 tox-30) a The order of the immunity markers is not given in the double lysogens since it is not crucial to the discussion and in many cases was not determined. However, in each case it was ascertained that phages carrying both immunities were present. NA indicates that information was not available. Mutations tox-116 and tox-l1 7, described in Table 2, and tox-118, present in gamma phage, are all phenotypically CRM-, while tox+ represents the wild-type toxin-producing allele. In strains 11 and 12, the origin and position of each tox allele cannot be specifically identified, but since phages with both immunities were detected, it is assumed that each of the parent phages contributed its tox allele to the double lysogen. Strain no.
Monolysogen
indicates the molecular weight of the protein. Strains producing no detectable cross-reacting material are phenotypically CRM-. A modification of the gel immunodiffusion test for toxigenicity of C. diphtheriae (12) was used to detect CRMs. It is possible to distinguish CRM30-, CRM45-, and CRM62-producing strains from one another by differences in the intensity of the lines of precipitation, the intensity decreasing significantly with the decrease in their molecular weight. These results are checked by a more specific test for these differences, the basis of which is illustrated in Fig. 1. In this test, unknown strains are spotted next to each of the known CRM-producing strains, and the presence or absence of spur formation is noted. The colony overlay test (COT) of Laird and Groman (11) was used for toxigenicity tests in the screen for nontoxinogenic strains of C7 lysogenized with mutagenized beta phage. Final confirmation of the nontoxinogenic mutants was obtained by the guinea pig intracutaneous test (1). Samples of stationaryphase cultures shaken at 37 C for 24 h in heart infusion broth (Difco) were used as inocula. Toxin and antitoxin. Diphtheria antitoxin and purified toxin were purchased from the Connaught Laboratories Ltd., Ontario, Canada. All media, the phage methods, and the method of genetic analysis of double lysogens were described previously (12). Phage methods include the plaque assay, tests for lysogeny, production of phage stocks, and standard conditions for UV irradiation. Mutagenesis of corynebacteriophage. C7(,B) was grown overnight at 37 C in 5 ml of heart infusion broth. The next morning the culture was diluted 1:5 in heart infusion broth and grown to a density of 108/ ml. The cells were pipetted into a petri dish and irradiated with UV for 5 min. The irradiated suspension was returned to a tube and shaken at 37 C for 15 min. N-methyl-N'-nitro-N-nitrosoguanidine
was then added to give a final concentration of 60 ,tg/ml. The cells were shaken for an additional 165 min. Finally, the cells were centrifuged and the supernate containing approximately 2 x 108 phage/ ml was filtered with a 0.4-,um membrane filter (Millipore Corp.). The lysate was tested for sterility and then refrigerated at 4 C. Isolation of tox phage mutants. The mutagenized phage lysate was diluted in heart infusion broth and plaqued on C7. The plates were incubated at 30 C rather than the usual 37 C in order to preserve any temperature-sensitive mutants that were present. The next day, sterile cylindrical applicator sticks (2 mm in diameter) were used to transfer material from the plaques to small tubes containing 1 ml of heart infusion broth. The tubes were incubated for 24 h at 30 C, during which time cells lysogenic for the phage became dominant. Each cell suspension was then tested for toxigenicity by the COT. Thirtyfour suspensions plus a positive and negative control were tested on each large tissue culture plate. Whenever a suspension gave a negative tissue culture test, a C7 lysogen containing the putative tox mutant of beta phage was isolated as follows. The suspension was filtered and spotted onto a fresh plate containing C7 cells in a soft-agar overlay prepared as the phage indicator plates. About 6 h later, when a large confluent plaque could be observed, an inoculum from the plaque was streaked onto a tryptose-yeast extract plate. Fifty isolated colonies were later picked and tested for lysogeny, and one presumptive lysogen was selected for further isolation. This isolation process was repeated until all 10 colonies picked from a plate gave a positive test for lysogeny. One colony from this last plate was streaked to heart infusion slants, incubated, and then placed in stock. Finally, the tox state of the lysogen was confirmed by repeating the COT and performing a guinea pig intracutaneous test.
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FIG. 1. Immunodiffusion tests of tox mutants. The plate on the left contains all 17 mutants described in Table 2 plus C7(,0, the tox+ control. The plate on the right demonstrates the ability to detect double-line producers (upper group) and to discriminate between strains producing CRM30, CRM45, and CRM62 (lower group). The diagrams were drawn from the actual plates and reveal more detail than the photographs. Gel electrophoresis. The various lysogenic strains were grown with rapid shaking for 24 h at 37 C in the CY medium of Pappenheimer et al. (17). The cultures were filtered through 0.45-,tm membrane filters, and the filtrates were dialyzed against distilled water for 48 h. The filtrates were then tested by the slab gel electrophoresis method of Maizel (13) or according to the disc gel method of Weber and Osborn (20)- to characterize the major proteins. Purified toxin was run as a control.
RESULTS Isolation and classification of tox mutants. Beta phage was mutagenized with nitrosoguanidine after induction of the lysogenic strain C7(,3) by UV. Of the 5,398 phages tested as lysogens of C7, 17 proved to be nontoxinogenic on tissue culture. All 17 mutants produced infectious phage in strain C7 and lysogenized it normally. Further characterization of the CRMs produced by these mutants including the serological tests illustrated in Fig. 1 has led to their arrangement into four classes (Table 2).
All tests were performed with the C7 lysogens of each mutant or with material derived therefrom. Class I consists of 10 mutants, each of which produces a protein that forms a line of identity with diphtheria toxin when tested by immunodiffusion against diphtheria antitoxin. On slab gel electrophoresis these proteins migrate identically with purifed toxin, indicating a molecular weight of approximately 62,000. These mutants probably represent missense mutations in the structural gene for toxin. The COT test for toxin was negative at both 30 and 37 C. Hence, the mutation to tox does not appear to involve a change in temperature sensitivity of the activity of the tox gene product. The mutant in subclass 1A gives a positive skin test but a negative tissue culture test. Two possible explanations are that the mutant protein has a low level of activity or is produced in smaller amounts, but further testing remains to be done.
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Class II mutants produce proteins that form lines of partial identity with toxin when tested against antitoxin by immunodiffusion. On slab gel electrophoresis only one of these proteins was detected, but based on the immunodiffusion test all appeared to be smaller than purified toxin. Either a deletion, a nonsense mutation in the structural gene for toxin, or preferential proteolysis could account for the shortened polypeptide. Class III mutants produce two proteins serologically related to toxin, two lines being detected in the immunodiffusion test. One line shows full identity with purified toxin, whereas the other shows only partial identity. An experimental test for the possibility of gene duplication will be described later, and some speculative explanations consonant with this phenotype will be taken up in the discussion. The class IV mutants do not produce a protein serologically related to toxin, nor are they capable of eliciting a positive guinea pig skin test. As a result, their CRM phenotype has been designated CRM-. No major extracellular polypeptide appeared on gel electrophoresis. These mutants were also tested by R. Holmes, using the sensitive reversed passive hemagglutination assay (10), and no evidence for crossreacting protein was obtained. Taken together, these results indicate that the intact toxin molecule is either not produced or is produced or
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excreted in very small amounts. The CRM phenotype could result from such mutational events as a deletion, a very early nonsense mutation in the tox structural gene leading to the production of small fragments of toxin, or a mutation in a regulatory site or gene. Complementation test of tox mutants. The first question investigated was whether the mutations in the CRM- class of mutants were in some gene other than the structural gene for toxin. Complementation tests were performed to test for this possibility by means of a series of double lysogens (Table 1). The order of the prophage markers in these strains, determined as previously described (12), is given in Table 3. To understand this table, it is important to recall that the proteins produced by the CRM30, CRM45, and CRM62 alleles of the tox gene can be distinguished in the immunodiffusion assay. The first four double lysogens in the table serve as controls. They demonstrate that two structural genes for toxin placed in trans in strain C7 can be expressed, and that the immunodiffusion test can detect both proteins. It is important to note that though tox+ recombinants may be present in these cultures (see next section), the amount of toxin they could produce would not be detected by the immunodiffusion assay. We have found that the test only becomes positive when approximately 10% or more of the cells are tox+.
TABLE 2. Tox mutants of converting corynebacteriophage beta tox testa CRM product' Mutant Class Mutant Class COT Guinea pig Immunodiffu- Electrophorein COT ssmlw Guinea pig Sion S1S mol wt C tox-101 62,000 C tox-102 62,000 C tox-103 62,000 tox-104 C 62,000
Reversiontested) to tox'
(no/no.
ND' ND ND 0/400 ND ND 0/1,000 ND ND ND ND 0/1,000 0/1,000 0/3,000 0/3,000 0/3,000 0/3,000
C tox-105 62,000 C tox-106 I 62,000 C tox-107 62,000 C tox-108 62,000 C tox-109 62,000 C tox-1lO + IA 62,000 P tox-ll 50,000 P tox-112 II P tox-113 P&C tox-114 III P&C tox-115 tox-116 IV tox-11 7 a -, No toxin detected. b C, Complete; P, partial antigenic homology with diphtheria toxin. In both strains showing P and C, two lines of identity were detected, one showing partial identity equivalent to that displayed by CRM30, and the other showing complete identity with diphtheria toxin, CRM62. -, No CRM product was detected. X ND, Not done.
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TABLE 3. Complementation studies of tox mutants in double lysogensa Double-
Order of tox alleles
CRMs pro-
lysogen
duced (mol wt
no.
Prophage 1
Prophage 2
1 2 3 4 5 6 7 8 9 10 11 12
tox-45 tox-30 tox-45 tox+ tox-11 6 tox-45 tox-45 tox-11 7 tox-118 tox-45 tox-116 tox-118
tox-30 tox-45 tox' tox-45 tox-45 tox-11 6 tox-11 7 tox-45 tox-45 tox-118 tox-118 tox-117
x
10-3)
30,45 30,45 45,62 45,62 45 45 45 45 45 45 None None
a In double lysogens 1 through 10, the phages are inserted in tandem as diagramed below. 1 2
T'
T'
The bacterial DNA is represented by the dotted line, and the phage DNAs are represented by the solid line. The arrows indicate the positions of the tox alleles in the prophages. The order of the two tox markers in double lysogens 11 and 12 is not known. Phages carrying mutations tox-30 and tox-45 produce CRM30 and CRM45, respectively; tox-116, tox117, and tox-118 are CRM- and tox+ produces diphtheria toxin, here designated CRM62.
The results with double lysogens 5 through 8 show that when a CRM -, tox mutation is placed in trans with the CRM+, tox-45 mutation, only the CRM45 protein is produced in detectable amounts. Thus the CRM-, tox mutation does not appear to be in a gene distinct from the toxin structural gene. More specifically, these results show that the CRM- mutant does not produce a negative regulator, nor does it lack a positive regulator. A negative regulator such as a repressor would be expected to turn off the production of CRM45 by tox-45, whereas a positive regulator produced by tox-45 would be expected to turn on the production of toxin by a structural gene in the CRM strain. The last four double lysogens in Table 3 will be discussed later in relation to phage gamma, which shares much in common with the CRM-
mutants.
Recombination tests with CRM-, tox mutants. Since complementation tests did not support the hypothesis that CRM- strains were mutant in a separate gene, the next question was whether the CRM- strains retained the structural gene for toxin in whole or in part. If the mutation represented either an operator, promoter, or early nonsense mutation, then the
entire gene would still be present. If this were true, then tox+ recombinants might be recovered if CRM-, tox mutants were crossed against CRM+, tox mutants. If the structural gene for toxin were totally deleted, then tox+ recombinants could not be produced. However, if the deletion were partial, then the outcome would depend on where the deletion had oc-
curred. Prophage recombination in doubly lysogenic cells was used to investigate this question. The procedure for collecting and characterizing monolysogenic segregants that carry phage recombinants was previously described (12). The CRM phenotypes of phage recombinants recovered from various double lysogens (described in Table 1) are shown in Table 4. Each double lysogen contains two different tox alleles. Strains 1, 13, and 14 serve as controls and show that intragenic recombination can be detected. An examination of recombinants from strains 6 and 7 show that CRM- mutants tox116 and tox-il 7 contain at least a portion of the structural gene for toxin as evidenced by the production of tox+ recombinants in crosses with tox-45-bearing strains. As a control for these matings, wild-type gamma phage was also crossed with a tox-45-bearing phage (strain 10) with the expectation that tox+ recombinants would not be found. Surprisingly, tox+ recombinants were recovered at a high rate. Thus it appears that gamma phage is a natural counterpart of the CRM- strains. Complementation tests were also performed with gamma phage. The data (Table 3) show that gamma phage, whiose tox gene is designated tox-118, gives the same results as the CRM- strains carrying tox-116 and tox-117 (see strains 9 and 10) and suggest (see strains 11 and 12) that the tox genes of all three strains may be identical. In summary, the results show that CRMTABLE 4. Intragenic recombination in the tox genea Order of tox al- No. of phage recombinants with leles indicated phenotypes Double Prolysogen Prono.
phage
phage CRM30 CRM45 CRM62 CRM-
1 2 tox-45 tox-30
11 2 1 9 14 2 18 1200 6 tox-116 2 6 638 7 tox-11 7 9 1054 tox-118 3 10 a Phages carrying mutations tox-30 and tox-45 produce CRM30 and CRM45, respectively; tox-116, tox-117, and tox-118 are CRM-. All strains carrying CRM62-producing recombinant phages were positive when tested for toxigenicity by the COT. 1 13
tox-45 tox-45 tox-45 tox-45 tox-45
tox-30 tox-30
1849 173 890
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mutants tox-116, tox-117, and tox-118 contain at least the distal one-fourth of the structural gene for toxin. This result would obtain if the CRM-, tox mutation was due either to a deletion affecting all or part of the proximal threefourths of the tox gene or to a point mutation situated somewhere in the same region of the gene. If the latter, it could be an early nonsense mutation or a mutation in an operator or promoter site. Since there are no suppressor strains available for C. diphtheriae, it is not possible to make a direct test for the presence of a nonsense codon. The possibility that the recovery of the tox strains was due to reversion rather than recombination was checked. Over 1,000 colonies each of strains C7(pt"x30), C7(pt1oX45), C7(,8toxlI 16), C7(,f3x-"7), and C7(yx-118) were tested by immunodiffusion. No serologically positive colonies were detected, and it is safe to say that the reversion rate of each of these mutants was at least an order of magnitude less than the recombination rate. Additional data given in Table 2 show that reversion to the tox+ state is very rare. Test for multiple tox genes in class III mutants. The fact that class III mutants produce two proteins serologically related to toxin suggests that they might contain two tox alleles in their genome. However, when phages from these strains were replaqued on C7 and fresh lysogens were reisolated from single plaques, the lysogens still exhibited the double-line phenotype on the in vitro plates. The possibility of gene duplication was also investigated. One characteristic of gene duplications is that they are frequently unstable and commonly one of the two genes is lost (3). The following experiments were performed with class III mutants to detect whether segregants that had lost the ability to produce one of the two toxin-related proteins were produced. Stock cultures of C7(3x-11"4) and C7(,&1x0-") were passaged in Tween-broth for 200 generations, after which 3,000 colonies from each culture were tested by immunodiffusion for the production of toxinrelated proteins. Tween prevents adsorption of phage (6) and was added to protect any segregants from being superinfected and having their original phenotype restored. All but three of the colonies behaved as the parent cells, producing two lines in the immunodiffusion test. The three colonies that failed to produce any line proved to be clones that had been "cured" of prophage and were now nonlysogenic. In effect, these clones serve as internal controls for the effectiveness of Tween-broth. Since class III mutants appear to be very stable, the possibility of gene duplication seems less
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attractive and alternative explanations need to be investigated. DISCUSSION The results of the present study demonstrate that the COT (11) is an effective and efficient method for toxigenicity testing. The relative convenience of the test makes it possible not only to screen for tox mutants, but to undertake genetic studies in which toxigenicity is being followed. When the CRM phenotype is being considered, the immunodiffusion test is the test of choice. Using the COT, we isolated 17 tox mutants and grouped them into four classes (Table 2). Two of these classes (I and II) were previously reported by Uchida et al. (19), but classes III and IV are new. Using the immunodiffusion test, Holmes (9) has independently isolated seven tox mutants, among which are representatives of classes II and IV. Class III mutants produce two lines that cross-react with diphtheria toxin in the immunodiffusion test. Our evidence indicates that this is probably not due to gene duplication. One possible explanation is that as a result of a missense mutation the altered toxin molecule is more sensitive to protease action, and thus some fraction of the toxin molecules are reduced to fragments and produce a second line of partial identity with toxin. Another possibility is that class III mutants contain nonsense mutations that are suppressed or read through with sufficient frequency to yield intact toxin as well as a toxin fragment. Class IV mutants do not produce any protein detectable with antitoxin. Complementation and recombination studies have ruled out the production of a super-repressor, the loss of production of a positive regulator, or total deletion of the structural gene for toxin as explanations for this phenotype. There are at least three remaining possibilities, including an early nonsense mutation in the structural gene, operator or promoter mutations, and deletions in the proximal portion of the structural gene for toxin. Although the CRM- strains are the most likely candidates for the regulatory mutants we set out to isolate, further work is needed to rule out the alternative interpretations for their phenotype. A number of observations have been made that are potentially significant in the natural history of diphtheria. It has been demonstrated that recombination can occur between two tox phage mutants to yield tox+ phage. Recombinants that are tox+ are found as prophage in monolysogenic segregants of strains that were originally doubly lyosogenic for two tox mutants, and, of course, they appear as free phage
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with the potential for converting nontoxino- long-term balance between toxinogenic and genic strains to toxinogenicity. It is obvious nontoxinogenic strains, their importance in the that prophage recombination and the resulting genesis of diphtheria outbreaks also needs to be segregation of monolysogens represent poten- considered. tially important mechanisms for the generation ACKNOWLEDGMENTS of new tox+ strains. We wish to acknowledge technical assistance of In addition to the laboratory-produced mu- Grace Suzuki in this study. Wethealso wish to thank R. K. tants that participate in the regeneration of Holmes for making his manuscript available before publicatox+ strains, we have found that the natural tion. This investigation was supported by Public Health Servnontoxinogenic isolate, gamma phage (7), also ice research grant A1-10492 from the National Institute of participates in this process. Of additional inter- Allergy and Infectious Disease. est is that fact that gamma phage appears to be LITERATURE CITED the natural counterpart of the CRM mutants. It carries at least a portion of the tox gene, al- 1. American Public Health Association. 1970. Diagnostic procedures for bacterial, mycotic and parasitic infecthough it is phenotypically CRM -. It is also of tions, 5th ed. American Public Health Association, interest that yIOx+ phage, the natural tox+ Inc., New York. counterpart of gamma, was isolated from a pre- 2. Barksdale, L. 1959. Symposium on the biology of cells dominant number of toxinogenic strains in a modified by viruses or antigens. I. Lysogenic conversions in bacteria. Bacteriol. Rev. 23:202-213. survey by Groman and Memmer (8). G., and L. E. Bertani. 1974. Constitutive These findings with tox phage mutants call to 3. Bertani, expression of bacteriophage P2 early genes resulting that of Parsons mind the observations (18) from a tandem duplication. Proc. Natl. Acad. Sci. phage recovered from a nontoxinogenic strain U.S.A. 71:315-319. of C. diphtheriae could convert other nontoxi- 4. Freeman, V. J. 1951. Studies on the virulence of bacteriophage-infected strains of Corynebacterium diphnogenic strains to toxigenicity. In an extension theriae. J. Bacteriol. 61:675-688. Groman showed that of these observations, (5) 5. Groman, N. B. 1956. Conversion in Corynebacterium phage from a strain carrying two known nondiphtheriae with phages originating from nontoxigenic strains. Virology 2:843-844. toxinogenic phages could also convert a nontoxN. B., and D. Bobb. 1955. The inhibition of inogenic strain to toxingenicity. It was postu- 6. Groman, adsorption of Corynebacterium diphtheriae phage by be due could that this lated then phenomenon Tween 80. Virology 1:313-323. to (i) reversion of nontoxinogenic phage to the 7. Groman, N. B., and M. Eaton. 1955. Genetic factors of Corynebacterium diphtheriae conversion. J. Bactetox+ state by mutation, (ii) recombination beriol. 70:637-640. tween two nonconverting phages to yield some 8. Groman, N. B., and R. Memmer. 1958. Lysogeny and converting phage progeny, and (iii) phenotypic conversion in mitis and mitis-like Corynebacterium suppression of the tox+ allele in the original diphtheriae. J. Gen. Microbiol. 19:634-644. bacterial host. Our current evidence indicates 9. Holmes, R. K. 1976. Characterization and genetic mapping of nontoxinogenic (tox) mutants of corynebacterthat reversion is a rare event, but that recombiiophage beta. J. Virol. 19:195-207. nation does occur at a high rate under some 10. Holmes, R. K., and R. B. Perlow. 1975. Quantitative conditions. Examples of the third mechanism, assay of diphtherial toxin and of immunologically cross-reacting proteins by reversed passive hemagin which the production of toxin was suppressed glutination. Infect. Immun. 12:1392-1400. in one strain but was normal when the same 11. Laird, W., and N. Groman. 1973. Rapid, direct tissue phage was present in another strain, have been culture test for toxigenicity of Corynebacterium diphreported by both Barksdale (2) and Pappenheitheriae. Appl. Microbiol. 25:709-712. 12. Laird, W., and N. Groman. 1976. Prophage map of mer (15). converting corynebacteriophage beta. J. Virol. 19: It is obvious from past and present studies 208-219. that the significance of nontoxinogenic strains 13. Maizel, J. 1971. Polyacrylamide gel electrophoresis of to the natural history of diphtheria requires viral proteins, p. 179-240. In K. Maramorosch and H. Koprowski (ed.), Methods in virology, vol. 5. Acareassessment. The important element is that a demic Press, New York. variety of nontoxinogenic strains may exist 14. Murphy, J., A. M. Pappenheimer, Jr., and S. T. de that harbor phage carrying all or part of the Borms. 1974. Synthesis of diphtheria tox-gene prodstructural gene for toxin. The influence of these ucts in Escherichia coli extracts. Proc. Natl. Acad. Sci. U.S.A. 71:11-15. strains on the balance between nontoxinogenic A. M., Jr. 1971. The evolution of an and toxinogenic strains of C. diphtheriae in 15. Pappenheimer, infectious disease of man, p. 174-188. In J. Monod nature needs to be reconsidered. This is particand E. Borek (ed.), Microbes and life. Columbia ularly so with the demonstration that tox+ University Press, New York. monolysogens are segregated at a high fre- 16. Pappenheimer, A. M., Jr., and S. J. Johnson. 1936. Studies in diphtheria toxin production. 1. The effect quency from double lyosgens carrying two of iron and copper. Br. J. Exp. Pathol. 17:335-341. phages with different tox mutations. Aside 17. Pappenheimer, A. M., Jr., T. Uchida, and A. A. Harper. from the effects of these interactions on the 1972. An immunological study of the diphtheria toxin
VOL. 19, 1976 molecule. Immunochemistry 9:891-906. 18. Parsons, E. I. 1955. Induction of toxigenicity in nontoxigenic strains of C. diphtheriae with bacteriophage derived from non-toxigenic strains. Proc. Soc. Exp. Biol. Med. 90:91-93. 19. Uchida, T., A. M. Pappenheimer, Jr., and R. Greany. 1973. Diphtheria toxin and related proteins. 1. Isola-
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tion and properties of mutant proteins serologically related to diphtheria toxin. J. Biol. Chem. 248:38383844. 20. Weber, K., and J. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.
Vol. 19, No. 1 Printed in U.S.A.
Isolation and Characterization of tox Mutants of Corynebacteriophage Beta WALTER LAIRD AND NEAL GROMAN* Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195 Received for publication 6 August 1975
Seventeen nontoxinogenic (tox) mutants of corynebacteriophage beta have been isolated by using a tissue culture screening technique. The mutants fall into four major classes. Two of the classes, I and II, appear to contain missense and nonsense mutants, respectively. However, classes III and IV have not been previously described. Class III mutants produce two proteins (CRMs) seriologically related to diphtheria toxin, but efforts to demonstrate the presence of more than one tox gene have not been successful. Class IV mutants are phenotypically CRM-, failing to produce any detectable protein serologically related to diphtheria toxin. Genetic studies indicate that the mutations in class IV strains are not in a gene distinct from the structural gene for toxin, and that the CRMstrains retain at least a portion of that gene. A natural phage isolate, gamma, behaves in a completely parallel fashion to the class IV mutants. The production of tox+ recombinants through recombination of various pairs of tox phage mutants has been demonstrated. The implications of these findings for the natural history of diphtheria are discussed. In 1951, Freeman discovered (4) that nontoxinogenic strains of Corynebacterium diphtheriae became toxinogenic when lysogenized by certain corynebacteriophages. Since it was recently established that the structural gene for diphtheria toxin is present on the beta converting phage genome (14), the regulation of toxin production has now become a logical focus of attention. It has long been known that the synthesis of toxin is influenced by the amount of iron present in the culture medium (16). High yields of toxin are produced only under conditions of low iron concentration. It seems reasonable that there might be other genes or sites on the phage genome as well as on the host genome that function in the expression or regulation of the gene for toxin. In the last few years, various nontoxinogenic (tox) phage mutants have been isolated. These tox mutants appear to be affected in a structural gene for toxin (19), but regulatory mutants have not been described. However, if such mutants are to be found, it is likely that a large number of tox mutants will have to be examined. Until recently, the search for such mutants was cumbersome since it required skin testing on a rabbit. The development of a more convenient tissue culture method for toxigenicity testing (11) has made it possible to screen large numbers of mutagenized phage. This paper describes the isolation and partial characterization of seventeen tox mutants. The study
has concentrated on the characteristics of one class of mutants that appears to be new to the literature. MATERIALS AND METHODS
Strains of bacteria and phage. C. diphtheriae strains C7, C7(,3), and C7(y) were from our stock
culture collection. A. M. Pappenheimer, Jr., generously sent us strains of C7(,8f3) and C7Q(345), which have been renamed C7(p'0x-30) and C7(I3b0-5), respectively. The nomenclature proposed by Holmes (9) for tox mutants has been followed. Phage strain y'0o5 was produced by crossing 3tox45 with gamma phage vegetatively. All lysogenic strains to be described were derived by infecting stock strain C7 with the appropriate phage or phages. A series of double lysogens was prepared and genetically characterized as previously described (12). The original monolysogens, the heterologous superinfecting phages, and the derived double lysogens are listed in Table 1. Bacteriophage markers. All phages described in this paper plaque on strain C7. Beta phage plaques on C7(y) but not on C7(f3), whereas the reverse is true for gamma phage. Phage immunity will be designated as imm-/3 or imm-y. The mutations in nontoxinogenic strains isolated in our laboratory have been assigned arbitrary numbers beginning with 101. Seventeen tox mutants designated f3Z'°'0 through f3O0-"l7 will be described in Results. The tox gene in gamma phage has been designated tox-118. The phenotypic designation CRM+ will be used to identify the protein product of a tox gene that crossreacts with or is serologically identical with diphtheria toxin. A number following the symbol CRM
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TABLE 1. Heteroimmune double lysogensa Double lysogen (tox alleles only) Superinfecting phage 1 C7(imm-/3 tox-30) imm--y tox-45 C7(tox-45)(tox-30) 2 C7(imm--y tox-45) imm-/3 tox-30 C7(tox-30)(tox-45) 3 NA NA C7(tox-45)(tox+) 4 NA NA C7(tox+)(tox-45 ) 5 C7(imm--y tox-45) C7(tox-116)(tox-45) imm-,3 tox-116 6 imm--y tox-45 C7(tox-45)(tox-116) C7(imm-,8 tox-116) 7 imm-y tox-45 C7(tox-45)(tox- 117) C7(imm-,8 tox-117) 8 C7(imm-y tox-45) imm-f3 tox-117 C7(tox-117)(tox-45) 9 imm--y tox-118 C7(tox-118)(tox-45) C7(imm-,3 tox-45) 10 C7(imm-y tox-118) imm-/3 tox-45 C7(tox-45 )(tox-118) 11 C7(imm-y tox-118) C7(tox-116)(tox-118) imm-,8 tox-116 12 C7(imm-/3 tox-117) imm-y tox-118 C7(tox-118)(tox-117) 13 C7(imm-/3 tox-30) imm-y tox-45 C7(tox-45)(tox-30) 14 imm-y tox-45 C7(tox-45)(tox-30) C7(imm-,3 tox-30) a The order of the immunity markers is not given in the double lysogens since it is not crucial to the discussion and in many cases was not determined. However, in each case it was ascertained that phages carrying both immunities were present. NA indicates that information was not available. Mutations tox-116 and tox-l1 7, described in Table 2, and tox-118, present in gamma phage, are all phenotypically CRM-, while tox+ represents the wild-type toxin-producing allele. In strains 11 and 12, the origin and position of each tox allele cannot be specifically identified, but since phages with both immunities were detected, it is assumed that each of the parent phages contributed its tox allele to the double lysogen. Strain no.
Monolysogen
indicates the molecular weight of the protein. Strains producing no detectable cross-reacting material are phenotypically CRM-. A modification of the gel immunodiffusion test for toxigenicity of C. diphtheriae (12) was used to detect CRMs. It is possible to distinguish CRM30-, CRM45-, and CRM62-producing strains from one another by differences in the intensity of the lines of precipitation, the intensity decreasing significantly with the decrease in their molecular weight. These results are checked by a more specific test for these differences, the basis of which is illustrated in Fig. 1. In this test, unknown strains are spotted next to each of the known CRM-producing strains, and the presence or absence of spur formation is noted. The colony overlay test (COT) of Laird and Groman (11) was used for toxigenicity tests in the screen for nontoxinogenic strains of C7 lysogenized with mutagenized beta phage. Final confirmation of the nontoxinogenic mutants was obtained by the guinea pig intracutaneous test (1). Samples of stationaryphase cultures shaken at 37 C for 24 h in heart infusion broth (Difco) were used as inocula. Toxin and antitoxin. Diphtheria antitoxin and purified toxin were purchased from the Connaught Laboratories Ltd., Ontario, Canada. All media, the phage methods, and the method of genetic analysis of double lysogens were described previously (12). Phage methods include the plaque assay, tests for lysogeny, production of phage stocks, and standard conditions for UV irradiation. Mutagenesis of corynebacteriophage. C7(,B) was grown overnight at 37 C in 5 ml of heart infusion broth. The next morning the culture was diluted 1:5 in heart infusion broth and grown to a density of 108/ ml. The cells were pipetted into a petri dish and irradiated with UV for 5 min. The irradiated suspension was returned to a tube and shaken at 37 C for 15 min. N-methyl-N'-nitro-N-nitrosoguanidine
was then added to give a final concentration of 60 ,tg/ml. The cells were shaken for an additional 165 min. Finally, the cells were centrifuged and the supernate containing approximately 2 x 108 phage/ ml was filtered with a 0.4-,um membrane filter (Millipore Corp.). The lysate was tested for sterility and then refrigerated at 4 C. Isolation of tox phage mutants. The mutagenized phage lysate was diluted in heart infusion broth and plaqued on C7. The plates were incubated at 30 C rather than the usual 37 C in order to preserve any temperature-sensitive mutants that were present. The next day, sterile cylindrical applicator sticks (2 mm in diameter) were used to transfer material from the plaques to small tubes containing 1 ml of heart infusion broth. The tubes were incubated for 24 h at 30 C, during which time cells lysogenic for the phage became dominant. Each cell suspension was then tested for toxigenicity by the COT. Thirtyfour suspensions plus a positive and negative control were tested on each large tissue culture plate. Whenever a suspension gave a negative tissue culture test, a C7 lysogen containing the putative tox mutant of beta phage was isolated as follows. The suspension was filtered and spotted onto a fresh plate containing C7 cells in a soft-agar overlay prepared as the phage indicator plates. About 6 h later, when a large confluent plaque could be observed, an inoculum from the plaque was streaked onto a tryptose-yeast extract plate. Fifty isolated colonies were later picked and tested for lysogeny, and one presumptive lysogen was selected for further isolation. This isolation process was repeated until all 10 colonies picked from a plate gave a positive test for lysogeny. One colony from this last plate was streaked to heart infusion slants, incubated, and then placed in stock. Finally, the tox state of the lysogen was confirmed by repeating the COT and performing a guinea pig intracutaneous test.
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FIG. 1. Immunodiffusion tests of tox mutants. The plate on the left contains all 17 mutants described in Table 2 plus C7(,0, the tox+ control. The plate on the right demonstrates the ability to detect double-line producers (upper group) and to discriminate between strains producing CRM30, CRM45, and CRM62 (lower group). The diagrams were drawn from the actual plates and reveal more detail than the photographs. Gel electrophoresis. The various lysogenic strains were grown with rapid shaking for 24 h at 37 C in the CY medium of Pappenheimer et al. (17). The cultures were filtered through 0.45-,tm membrane filters, and the filtrates were dialyzed against distilled water for 48 h. The filtrates were then tested by the slab gel electrophoresis method of Maizel (13) or according to the disc gel method of Weber and Osborn (20)- to characterize the major proteins. Purified toxin was run as a control.
RESULTS Isolation and classification of tox mutants. Beta phage was mutagenized with nitrosoguanidine after induction of the lysogenic strain C7(,3) by UV. Of the 5,398 phages tested as lysogens of C7, 17 proved to be nontoxinogenic on tissue culture. All 17 mutants produced infectious phage in strain C7 and lysogenized it normally. Further characterization of the CRMs produced by these mutants including the serological tests illustrated in Fig. 1 has led to their arrangement into four classes (Table 2).
All tests were performed with the C7 lysogens of each mutant or with material derived therefrom. Class I consists of 10 mutants, each of which produces a protein that forms a line of identity with diphtheria toxin when tested by immunodiffusion against diphtheria antitoxin. On slab gel electrophoresis these proteins migrate identically with purifed toxin, indicating a molecular weight of approximately 62,000. These mutants probably represent missense mutations in the structural gene for toxin. The COT test for toxin was negative at both 30 and 37 C. Hence, the mutation to tox does not appear to involve a change in temperature sensitivity of the activity of the tox gene product. The mutant in subclass 1A gives a positive skin test but a negative tissue culture test. Two possible explanations are that the mutant protein has a low level of activity or is produced in smaller amounts, but further testing remains to be done.
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Class II mutants produce proteins that form lines of partial identity with toxin when tested against antitoxin by immunodiffusion. On slab gel electrophoresis only one of these proteins was detected, but based on the immunodiffusion test all appeared to be smaller than purified toxin. Either a deletion, a nonsense mutation in the structural gene for toxin, or preferential proteolysis could account for the shortened polypeptide. Class III mutants produce two proteins serologically related to toxin, two lines being detected in the immunodiffusion test. One line shows full identity with purified toxin, whereas the other shows only partial identity. An experimental test for the possibility of gene duplication will be described later, and some speculative explanations consonant with this phenotype will be taken up in the discussion. The class IV mutants do not produce a protein serologically related to toxin, nor are they capable of eliciting a positive guinea pig skin test. As a result, their CRM phenotype has been designated CRM-. No major extracellular polypeptide appeared on gel electrophoresis. These mutants were also tested by R. Holmes, using the sensitive reversed passive hemagglutination assay (10), and no evidence for crossreacting protein was obtained. Taken together, these results indicate that the intact toxin molecule is either not produced or is produced or
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excreted in very small amounts. The CRM phenotype could result from such mutational events as a deletion, a very early nonsense mutation in the tox structural gene leading to the production of small fragments of toxin, or a mutation in a regulatory site or gene. Complementation test of tox mutants. The first question investigated was whether the mutations in the CRM- class of mutants were in some gene other than the structural gene for toxin. Complementation tests were performed to test for this possibility by means of a series of double lysogens (Table 1). The order of the prophage markers in these strains, determined as previously described (12), is given in Table 3. To understand this table, it is important to recall that the proteins produced by the CRM30, CRM45, and CRM62 alleles of the tox gene can be distinguished in the immunodiffusion assay. The first four double lysogens in the table serve as controls. They demonstrate that two structural genes for toxin placed in trans in strain C7 can be expressed, and that the immunodiffusion test can detect both proteins. It is important to note that though tox+ recombinants may be present in these cultures (see next section), the amount of toxin they could produce would not be detected by the immunodiffusion assay. We have found that the test only becomes positive when approximately 10% or more of the cells are tox+.
TABLE 2. Tox mutants of converting corynebacteriophage beta tox testa CRM product' Mutant Class Mutant Class COT Guinea pig Immunodiffu- Electrophorein COT ssmlw Guinea pig Sion S1S mol wt C tox-101 62,000 C tox-102 62,000 C tox-103 62,000 tox-104 C 62,000
Reversiontested) to tox'
(no/no.
ND' ND ND 0/400 ND ND 0/1,000 ND ND ND ND 0/1,000 0/1,000 0/3,000 0/3,000 0/3,000 0/3,000
C tox-105 62,000 C tox-106 I 62,000 C tox-107 62,000 C tox-108 62,000 C tox-109 62,000 C tox-1lO + IA 62,000 P tox-ll 50,000 P tox-112 II P tox-113 P&C tox-114 III P&C tox-115 tox-116 IV tox-11 7 a -, No toxin detected. b C, Complete; P, partial antigenic homology with diphtheria toxin. In both strains showing P and C, two lines of identity were detected, one showing partial identity equivalent to that displayed by CRM30, and the other showing complete identity with diphtheria toxin, CRM62. -, No CRM product was detected. X ND, Not done.
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TABLE 3. Complementation studies of tox mutants in double lysogensa Double-
Order of tox alleles
CRMs pro-
lysogen
duced (mol wt
no.
Prophage 1
Prophage 2
1 2 3 4 5 6 7 8 9 10 11 12
tox-45 tox-30 tox-45 tox+ tox-11 6 tox-45 tox-45 tox-11 7 tox-118 tox-45 tox-116 tox-118
tox-30 tox-45 tox' tox-45 tox-45 tox-11 6 tox-11 7 tox-45 tox-45 tox-118 tox-118 tox-117
x
10-3)
30,45 30,45 45,62 45,62 45 45 45 45 45 45 None None
a In double lysogens 1 through 10, the phages are inserted in tandem as diagramed below. 1 2
T'
T'
The bacterial DNA is represented by the dotted line, and the phage DNAs are represented by the solid line. The arrows indicate the positions of the tox alleles in the prophages. The order of the two tox markers in double lysogens 11 and 12 is not known. Phages carrying mutations tox-30 and tox-45 produce CRM30 and CRM45, respectively; tox-116, tox117, and tox-118 are CRM- and tox+ produces diphtheria toxin, here designated CRM62.
The results with double lysogens 5 through 8 show that when a CRM -, tox mutation is placed in trans with the CRM+, tox-45 mutation, only the CRM45 protein is produced in detectable amounts. Thus the CRM-, tox mutation does not appear to be in a gene distinct from the toxin structural gene. More specifically, these results show that the CRM- mutant does not produce a negative regulator, nor does it lack a positive regulator. A negative regulator such as a repressor would be expected to turn off the production of CRM45 by tox-45, whereas a positive regulator produced by tox-45 would be expected to turn on the production of toxin by a structural gene in the CRM strain. The last four double lysogens in Table 3 will be discussed later in relation to phage gamma, which shares much in common with the CRM-
mutants.
Recombination tests with CRM-, tox mutants. Since complementation tests did not support the hypothesis that CRM- strains were mutant in a separate gene, the next question was whether the CRM- strains retained the structural gene for toxin in whole or in part. If the mutation represented either an operator, promoter, or early nonsense mutation, then the
entire gene would still be present. If this were true, then tox+ recombinants might be recovered if CRM-, tox mutants were crossed against CRM+, tox mutants. If the structural gene for toxin were totally deleted, then tox+ recombinants could not be produced. However, if the deletion were partial, then the outcome would depend on where the deletion had oc-
curred. Prophage recombination in doubly lysogenic cells was used to investigate this question. The procedure for collecting and characterizing monolysogenic segregants that carry phage recombinants was previously described (12). The CRM phenotypes of phage recombinants recovered from various double lysogens (described in Table 1) are shown in Table 4. Each double lysogen contains two different tox alleles. Strains 1, 13, and 14 serve as controls and show that intragenic recombination can be detected. An examination of recombinants from strains 6 and 7 show that CRM- mutants tox116 and tox-il 7 contain at least a portion of the structural gene for toxin as evidenced by the production of tox+ recombinants in crosses with tox-45-bearing strains. As a control for these matings, wild-type gamma phage was also crossed with a tox-45-bearing phage (strain 10) with the expectation that tox+ recombinants would not be found. Surprisingly, tox+ recombinants were recovered at a high rate. Thus it appears that gamma phage is a natural counterpart of the CRM- strains. Complementation tests were also performed with gamma phage. The data (Table 3) show that gamma phage, whiose tox gene is designated tox-118, gives the same results as the CRM- strains carrying tox-116 and tox-117 (see strains 9 and 10) and suggest (see strains 11 and 12) that the tox genes of all three strains may be identical. In summary, the results show that CRMTABLE 4. Intragenic recombination in the tox genea Order of tox al- No. of phage recombinants with leles indicated phenotypes Double Prolysogen Prono.
phage
phage CRM30 CRM45 CRM62 CRM-
1 2 tox-45 tox-30
11 2 1 9 14 2 18 1200 6 tox-116 2 6 638 7 tox-11 7 9 1054 tox-118 3 10 a Phages carrying mutations tox-30 and tox-45 produce CRM30 and CRM45, respectively; tox-116, tox-117, and tox-118 are CRM-. All strains carrying CRM62-producing recombinant phages were positive when tested for toxigenicity by the COT. 1 13
tox-45 tox-45 tox-45 tox-45 tox-45
tox-30 tox-30
1849 173 890
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mutants tox-116, tox-117, and tox-118 contain at least the distal one-fourth of the structural gene for toxin. This result would obtain if the CRM-, tox mutation was due either to a deletion affecting all or part of the proximal threefourths of the tox gene or to a point mutation situated somewhere in the same region of the gene. If the latter, it could be an early nonsense mutation or a mutation in an operator or promoter site. Since there are no suppressor strains available for C. diphtheriae, it is not possible to make a direct test for the presence of a nonsense codon. The possibility that the recovery of the tox strains was due to reversion rather than recombination was checked. Over 1,000 colonies each of strains C7(pt"x30), C7(pt1oX45), C7(,8toxlI 16), C7(,f3x-"7), and C7(yx-118) were tested by immunodiffusion. No serologically positive colonies were detected, and it is safe to say that the reversion rate of each of these mutants was at least an order of magnitude less than the recombination rate. Additional data given in Table 2 show that reversion to the tox+ state is very rare. Test for multiple tox genes in class III mutants. The fact that class III mutants produce two proteins serologically related to toxin suggests that they might contain two tox alleles in their genome. However, when phages from these strains were replaqued on C7 and fresh lysogens were reisolated from single plaques, the lysogens still exhibited the double-line phenotype on the in vitro plates. The possibility of gene duplication was also investigated. One characteristic of gene duplications is that they are frequently unstable and commonly one of the two genes is lost (3). The following experiments were performed with class III mutants to detect whether segregants that had lost the ability to produce one of the two toxin-related proteins were produced. Stock cultures of C7(3x-11"4) and C7(,&1x0-") were passaged in Tween-broth for 200 generations, after which 3,000 colonies from each culture were tested by immunodiffusion for the production of toxinrelated proteins. Tween prevents adsorption of phage (6) and was added to protect any segregants from being superinfected and having their original phenotype restored. All but three of the colonies behaved as the parent cells, producing two lines in the immunodiffusion test. The three colonies that failed to produce any line proved to be clones that had been "cured" of prophage and were now nonlysogenic. In effect, these clones serve as internal controls for the effectiveness of Tween-broth. Since class III mutants appear to be very stable, the possibility of gene duplication seems less
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attractive and alternative explanations need to be investigated. DISCUSSION The results of the present study demonstrate that the COT (11) is an effective and efficient method for toxigenicity testing. The relative convenience of the test makes it possible not only to screen for tox mutants, but to undertake genetic studies in which toxigenicity is being followed. When the CRM phenotype is being considered, the immunodiffusion test is the test of choice. Using the COT, we isolated 17 tox mutants and grouped them into four classes (Table 2). Two of these classes (I and II) were previously reported by Uchida et al. (19), but classes III and IV are new. Using the immunodiffusion test, Holmes (9) has independently isolated seven tox mutants, among which are representatives of classes II and IV. Class III mutants produce two lines that cross-react with diphtheria toxin in the immunodiffusion test. Our evidence indicates that this is probably not due to gene duplication. One possible explanation is that as a result of a missense mutation the altered toxin molecule is more sensitive to protease action, and thus some fraction of the toxin molecules are reduced to fragments and produce a second line of partial identity with toxin. Another possibility is that class III mutants contain nonsense mutations that are suppressed or read through with sufficient frequency to yield intact toxin as well as a toxin fragment. Class IV mutants do not produce any protein detectable with antitoxin. Complementation and recombination studies have ruled out the production of a super-repressor, the loss of production of a positive regulator, or total deletion of the structural gene for toxin as explanations for this phenotype. There are at least three remaining possibilities, including an early nonsense mutation in the structural gene, operator or promoter mutations, and deletions in the proximal portion of the structural gene for toxin. Although the CRM- strains are the most likely candidates for the regulatory mutants we set out to isolate, further work is needed to rule out the alternative interpretations for their phenotype. A number of observations have been made that are potentially significant in the natural history of diphtheria. It has been demonstrated that recombination can occur between two tox phage mutants to yield tox+ phage. Recombinants that are tox+ are found as prophage in monolysogenic segregants of strains that were originally doubly lyosogenic for two tox mutants, and, of course, they appear as free phage
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with the potential for converting nontoxino- long-term balance between toxinogenic and genic strains to toxinogenicity. It is obvious nontoxinogenic strains, their importance in the that prophage recombination and the resulting genesis of diphtheria outbreaks also needs to be segregation of monolysogens represent poten- considered. tially important mechanisms for the generation ACKNOWLEDGMENTS of new tox+ strains. We wish to acknowledge technical assistance of In addition to the laboratory-produced mu- Grace Suzuki in this study. Wethealso wish to thank R. K. tants that participate in the regeneration of Holmes for making his manuscript available before publicatox+ strains, we have found that the natural tion. This investigation was supported by Public Health Servnontoxinogenic isolate, gamma phage (7), also ice research grant A1-10492 from the National Institute of participates in this process. Of additional inter- Allergy and Infectious Disease. est is that fact that gamma phage appears to be LITERATURE CITED the natural counterpart of the CRM mutants. It carries at least a portion of the tox gene, al- 1. American Public Health Association. 1970. Diagnostic procedures for bacterial, mycotic and parasitic infecthough it is phenotypically CRM -. It is also of tions, 5th ed. American Public Health Association, interest that yIOx+ phage, the natural tox+ Inc., New York. counterpart of gamma, was isolated from a pre- 2. Barksdale, L. 1959. Symposium on the biology of cells dominant number of toxinogenic strains in a modified by viruses or antigens. I. Lysogenic conversions in bacteria. Bacteriol. Rev. 23:202-213. survey by Groman and Memmer (8). G., and L. E. Bertani. 1974. Constitutive These findings with tox phage mutants call to 3. Bertani, expression of bacteriophage P2 early genes resulting that of Parsons mind the observations (18) from a tandem duplication. Proc. Natl. Acad. Sci. phage recovered from a nontoxinogenic strain U.S.A. 71:315-319. of C. diphtheriae could convert other nontoxi- 4. Freeman, V. J. 1951. Studies on the virulence of bacteriophage-infected strains of Corynebacterium diphnogenic strains to toxigenicity. In an extension theriae. J. Bacteriol. 61:675-688. Groman showed that of these observations, (5) 5. Groman, N. B. 1956. Conversion in Corynebacterium phage from a strain carrying two known nondiphtheriae with phages originating from nontoxigenic strains. Virology 2:843-844. toxinogenic phages could also convert a nontoxN. B., and D. Bobb. 1955. The inhibition of inogenic strain to toxingenicity. It was postu- 6. Groman, adsorption of Corynebacterium diphtheriae phage by be due could that this lated then phenomenon Tween 80. Virology 1:313-323. to (i) reversion of nontoxinogenic phage to the 7. Groman, N. B., and M. Eaton. 1955. Genetic factors of Corynebacterium diphtheriae conversion. J. Bactetox+ state by mutation, (ii) recombination beriol. 70:637-640. tween two nonconverting phages to yield some 8. Groman, N. B., and R. Memmer. 1958. Lysogeny and converting phage progeny, and (iii) phenotypic conversion in mitis and mitis-like Corynebacterium suppression of the tox+ allele in the original diphtheriae. J. Gen. Microbiol. 19:634-644. bacterial host. Our current evidence indicates 9. Holmes, R. K. 1976. Characterization and genetic mapping of nontoxinogenic (tox) mutants of corynebacterthat reversion is a rare event, but that recombiiophage beta. J. Virol. 19:195-207. nation does occur at a high rate under some 10. Holmes, R. K., and R. B. Perlow. 1975. Quantitative conditions. Examples of the third mechanism, assay of diphtherial toxin and of immunologically cross-reacting proteins by reversed passive hemagin which the production of toxin was suppressed glutination. Infect. Immun. 12:1392-1400. in one strain but was normal when the same 11. Laird, W., and N. Groman. 1973. Rapid, direct tissue phage was present in another strain, have been culture test for toxigenicity of Corynebacterium diphreported by both Barksdale (2) and Pappenheitheriae. Appl. Microbiol. 25:709-712. 12. Laird, W., and N. Groman. 1976. Prophage map of mer (15). converting corynebacteriophage beta. J. Virol. 19: It is obvious from past and present studies 208-219. that the significance of nontoxinogenic strains 13. Maizel, J. 1971. Polyacrylamide gel electrophoresis of to the natural history of diphtheria requires viral proteins, p. 179-240. In K. Maramorosch and H. Koprowski (ed.), Methods in virology, vol. 5. Acareassessment. The important element is that a demic Press, New York. variety of nontoxinogenic strains may exist 14. Murphy, J., A. M. Pappenheimer, Jr., and S. T. de that harbor phage carrying all or part of the Borms. 1974. Synthesis of diphtheria tox-gene prodstructural gene for toxin. The influence of these ucts in Escherichia coli extracts. Proc. Natl. Acad. Sci. U.S.A. 71:11-15. strains on the balance between nontoxinogenic A. M., Jr. 1971. The evolution of an and toxinogenic strains of C. diphtheriae in 15. Pappenheimer, infectious disease of man, p. 174-188. In J. Monod nature needs to be reconsidered. This is particand E. Borek (ed.), Microbes and life. Columbia ularly so with the demonstration that tox+ University Press, New York. monolysogens are segregated at a high fre- 16. Pappenheimer, A. M., Jr., and S. J. Johnson. 1936. Studies in diphtheria toxin production. 1. The effect quency from double lyosgens carrying two of iron and copper. Br. J. Exp. Pathol. 17:335-341. phages with different tox mutations. Aside 17. Pappenheimer, A. M., Jr., T. Uchida, and A. A. Harper. from the effects of these interactions on the 1972. An immunological study of the diphtheria toxin
VOL. 19, 1976 molecule. Immunochemistry 9:891-906. 18. Parsons, E. I. 1955. Induction of toxigenicity in nontoxigenic strains of C. diphtheriae with bacteriophage derived from non-toxigenic strains. Proc. Soc. Exp. Biol. Med. 90:91-93. 19. Uchida, T., A. M. Pappenheimer, Jr., and R. Greany. 1973. Diphtheria toxin and related proteins. 1. Isola-
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tion and properties of mutant proteins serologically related to diphtheria toxin. J. Biol. Chem. 248:38383844. 20. Weber, K., and J. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.
