APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p. 1109-1115 0099-2240/78/0035-1109$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 35, No. 6
Printed in U.S.A.
Transduction in Bacillus thuringiensis CURTIS B. THORNE
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003 Received for publication 29 December 1977
Bacteriophage CP-51, originally reported as a generalized transducing phage for Bacillus cereus and B. anthracis, has been shown to carry out generalized transduction in several strains of B. thuringiensis. A newly isolated phage, CP54, which has a broader host range than CP-51, also mediates generalized transduction in B. thuringiensis. CP-51 and CP-54 are similar in size and morphology and are related serologically, but they are not identical. CP-54 is more cold labile than CP-51, and, as with CP-51, its stability both at 0 and 150C is enhanced by the presence of 0.02 M Mg2e. Some examples of cotransduction of linked markers in B. thuringiensis are presented, demonstrating the feasibility of chromosomal mapping in this organism. The rare occurrence of cross-transduction among strains of B. thuringiensis is probably a reflection of nonhomology rather than restriction, since phage itself did not appear to be restricted when grown on a particular host and assayed with other hosts as indicator. Strains ofBacillus thuringiensis produce proteinaceous parasporal crystals which are toxic for the larval stages of susceptible insects, and preparations of B. thuringiensis for use as insecticides are made commercially in the United States and several other countries (3). Although a number of investigators have studied the chemistry of the toxic protein crystals (2, 4, 5, 8, 11) and the physiological and morphological aspects of spore and crystal formation (6, 8-10, 19), the lack of a genetic exchange system in B. thuringiensis has hindered studies of genetic factors associated with crystal formation. This report deals with transduction in strains of B. thuringiensis mediated by two bacteriophages, CP-51 and CP-54. Phage CP-51 (13, 14, 16, 17) was isolated from soil in my laboratory and originally studied as a generalized transducing phage for B. cereus and B. anthracis. These studies have now been extended to include B. thuringiensis. The second phage, CP-54, also isolated from soil, has a broader host range with respect to B. thuringiensis strains than does CP51 and can mediate generalized transduction in strains which are not hosts for CP-51. MATERIALS AND METHODS Organisms. The strains of B. thuringiensis used
isolation of CP-51 (13), except that wild-type B. thuringiensis 4041 was the host organism and streptomycin was omitted from the medium. Media and cultural conditions. The minimal medium, Min 3, was composed of the following (in grams per liter): (NH4)2SO4, 2; KH2PO4, 6; K2HPO4, 14; sodium citrate, 1; glucose, 5; L-glutamic acid, 2; glycine, 0.2; thiamine hydrochloride, 0.01; MgSO4 .7H20, 0.2; FeCI3 6H20, 0.04; MnSO4 H20, 0.00025; pH adjusted to 7.0 with NaOH. The glucose and FeC!3 were sterilized separately. Min 3C contained, in addition to Min 3, 5 g of vitamin-free, acid-hydrolyzed casein (Nutritional Biochemicals Corp., Cleveland, Ohio) per liter. Enriched minimal medium, Min 3E, was Min 3 with 1% (vol/vol) L-broth added. NBY medium contained 8 g of nutrient broth (Difco Laboratories, Detroit, Mich.) and 3 g of Difco yeast extract per liter, pH 6.8. L-broth contained 10 g of Difco tryptone, 5 g of Difco yeast extract, and 10 g of NaCl per liter, pH 7.0. PA medium was composed of the following (in grams per liter): Difco nutrient broth, 8; NaCl, 5; MgSO4 7H20, 0.2; MnSO4 H20, 0.05; CaCl2 * 2H20, 0.15; pH adjusted to 6.0 with HCI. Peptone diluent contained 10 g of Difco peptone per liter. For solid media, 15 g of agar was added per liter. For soft agar overlays, 5 g of agar was added per liter of appropriate medium. Spores were grown on slants of potato agar (12) in screw-cap tubes (20 by 150 mm) inoculated with spores or vegetative cells and incubated at 37°C for 3 to 5 days. Growth from a single slant was suspended in 5 ml of water and held in a 65°C bath for 30 min. Recipient cells for transduction were grown in 250ml Erlenmeyer flasks containing 25 ml of L-broth and incubated at 37°C on a rotary shaker (250 rpm). Cultures were grown for 5 to 6 h from a 10% (vol/vol) transfer of a 15-h culture or for 6 to 7 h from an inoculum of about 2 x 108 spores per flask. Phage assays. Both phages were routinely assayed by the soft agar overlay technique, with spores of B.
and their sources are included in Table 1. Auxotrophic mutants of B. thuringiensis were isolated by the procedure of Iyer (7) after exposure of spores to ultraviolet (UV) light. B. cereus NRRL 569 was used as an indicator in phage assays. Bacteriophage. Phage CP-51 has been described previously (13, 14, 16, 17). Phage CP-54 was isolated from soil in a manner similar to that described for the 1109
APPL. ENVIRON. MICROBIOL.
1110 THORNE cereus 569 as the indicator, in PA agar as described for CP-51 (13) except that assay plates were incubated at 30°C. When Min 3 agar was used for phage assays, 0.1 ml of a 15- to 24-h culture of the appropriate strain of B. thuringiensis in L-broth was used as the indicator. Peptone diluent was used routinely for diluting phage lysates to be assayed. Propagation of phage. Both phages were propagated routinely in soft NBY or PA agar from plaques as described for CP-51 (13). In some instances, 107 plaque-forming units (PFU) from phage lysates rather than phage from individual plaques were used as the phage seed. Either spores (about 108) or cells (0.2 ml of a 16-h L-broth culture) served as the host inoculum. Propagation plates were incubated at 37°C for 18 to 20 h, and phage from each plate were harvested in 5 ml of NBY or PA broth and filtered through Millipore DA membranes (Millipore Corp., Bedford, Mass.). Phage lysates were stored at 15°C. Transductions. Routinely, 0.5 to 0.8 ml of culture containing about 109 cells per ml was mixed in a 20mm tube with 0.2 to 0.5 ml of phage preparation to give a multiplicity of infection of 1 to 4 and incubated on a shaker at 37°C for 30 min. Phage to be used in transductions was usually treated with UV light (13) to inactivate 95 to 99% of the PFU, and the multiplicity of infection was calculated on the basis of phage titer before UV treatment. Tests for spontaneous reversion were always run by substituting NBY or PA broth for the phage. After incubation, 0.1-ml samples were spread in duplicate on appropriate minimal agar plates. When phage antiserum was used to facilitate recovery of transductants, 0.1 ml of a 1:10 dilution was spread with the sample of transduction mixture. Plates were incubated at 37°C, and transductants were scored after 24 h when plated on Min 3C or after 36 to 48 h when plated on Min 3 or Min 3E. Phage antiserum. Antiserum to CP-51 was prepared as described for phage SP-10 (12).
as temperate for any of the strains, it was much more lytic for some strains than for others. In PA agar the plaques on some strains were turbid or colony-centered, characteristic of plaques formed by temperate phages, but on minimal medium, plaques produced on the same strains were clear. In general, as will be discussed later, the lytic response was much more marked on minimal medium than on rich media, such as PA and NBY. Strain 4060 (B. thuringiensis subsp. thompsoni) was one of the strains which appeared to be least susceptible to lysis by CP51, and it was chosen for studies on transduction. Transduction with CP-51. Some results of transduction tests with strain 4060 are given in Table 2. Transductants were obtained at reasonably high frequencies with phage lysates that were treated with UV light to inactivate most of the PFU. However, as indicated in Table 2, there was considerable variation among mutants of the same strain with respect to survival of transductants under various conditions. Generally, the number of transductants was greater when phage was treated with UV light to inactivate 95 to 99% of the PFU. Plating transduction mixtures with phage antiserum was particularly beneficial when phage lysates were not treated with UV light, and, in some instances, antiserum improved both the number and the size of colonies even when phage had been treated with UV TABLE 1. B. thuringiensis strains used Strain
X
thuringien-
sis subsp.
Source
Host for growth CP-51 CP-54
RESULTS
Host range of CP-51. Previous tests (14) showed that the Sterne strain of B. anthracis and four of seven strains of B. cereus tested were hosts for CP-51. In the present studies with 21 strains of B. thuringiensis, all but 4 were able to serve as hosts (Table 1). Susceptibilities of the various strains to lysis by the phage were tested both by using each strain as an indicator in a phage assay and by using each strain as a potential host for phage propagation. The 4 strains listed as unsusceptible to CP-51 failed to show plaques when used as indicators and did not serve as hosts for phage propagation. Phage lysates prepared on the 17 susceptible strains in soft NBY or PA agar had titers ranging from 5 109 to 8 x 1010 PFU/ml. Lysates prepared by the same procedure with B. cereus 569 usually had titers of 2 x 1011 or more PFU/ml. The nature of the phage-host interaction varied greatly among the strains susceptible to CP51. Although the phage could not be classified
B.
4040 4041 4042 4043 4044 4045 4046 4047 4048 4049 4050 4055 4056 4057 4058 4059 4060 1715 ATCC 10792 ATCC 13367 ATCC 19266
NRRL^ NRRL aksti NRRL sotto dendrolimus NRRL NRRL kenyae NRRL galleriae NRRL entomocidus entomocidus-li- NRRL massol NRRL aizawai NRRL morrisoni
finitimus
tolworthi kurstaki
-
+
+
+ + + + + +
-
+
+ + + + + + + + + + + +
+ + + + + + + + + +
+ + + -
NRRL NRRL NRRL canadensis NRRL subtoxicus darmstadiensis NRRL NRRL toumanoffi NRRL thompsoni thuringiensis A. Yousten + R. Gordon + R. Gordon + + R. Gordon a + Strain served as no detectable activity. ^ NRRL, Agricultural Research Service, Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill.
host;-,
TRANSDUCTION IN B. THURINGIENSIS 1111
VOL. 35, 1978
TABLE 2. Transduction of mutants ofB. thuringiensis 4060 (subsp. thompsoni) with CP-51a CP-51 Phage
Recipient
lysate
1
2
PFU in-
Colonies/ml when plated on:
actbvated byUV
plicity of
99 99 99 99 0 0
10 2 1 0.5 10 2
4060 M9 Trp97
4060 M87 Nic97
antise-
Min 3E
rum
0
0
1,150 1,210 1,110 810
1,170
210b 310b
1,610 1,080
+
anti-
0
10
2
1,750
3,500
0
10
2
325b
20 1,300
130
40 2,235
97
2
0
30
35
35
97
2
270b
510b
700
4060 M25 Met-
Min 3C
serum
0
0
4060 M99 Trp-
Min 3
infection
0
4060 M9 Trp-
Min 3E
Min 3 +
Multi-
4,700
20 10 0 2 1,920 97 700 a Recipient cells were grown for 6 h in L-broth after a 10% transfer of a 16-h culture. Transduction mixtures 4060 M52 Leu-
contained an average of 8 x 108 celis and phage to give the indicated multiplicity of infection in a final volume of 1 ml. Both phage preparations were grown on wild-type 4060. b Colonies were poorly developed as a result of lysis by the phage.
light. Table 2 also demonstrates that the rich- thuringiensis subsp. alesti). As shown in Table ness of the plating medium influenced the num- 1, CP-54 was active on all strains of B. thurinber of transductants obtained. The best medium giensis tested. Like CP-51, it was also active on of those tested was Min 3C, which could be used B. cereus 569, and that organism was used roufor mutants requiring tryptophan, vitamins, pu- tinely as the indicator in CP-54 assays. When rines, or pyrimidines. With mutants requiring viewed in the electron microscope, CP-54 has a amino acids other than tryptophan, Min 3E gave structure very similar, if not indentical, to that improved results. In many instances, even when of CP-51 (15, 16). However, the two phages differ UV-treated phage was used, the colonies of in the following respects: (i) CP-54 has a broader transductants on minimal medium were poorly host range than that of CP-51; (ii) plaque mordeveloped and partially lysed, and this problem phologies are different; (iii) CP-54 is more cold could often be avoided by using enriched mini- labile than CP-51 (17); and (iv) although antimal. There was considerable variation in this serum prepared against CP-51 was also active respect among mutants of the same strain, and on CP-54, the two phages are not serologically sometimes it was necessary to plate transduction identical. Antiserum prepared against CP-51 had mixtures with phage antiserum even when en- K values (1) of 1,000 for CP-51 and 100 for CPriched minimal medium was used. Such an ex- 54. Previous studies with CP-51 (13, 15) showed ample is mutant M25 (Table 2). With appropriate combinations of UV treatment, plating me- that it is very unstable when stored at refrigerdium, and phage antiserum, it has been possible ator temperatures, and the optimum temperato obtain transductants of all mutants tested ture for stability was around 15°C. Table 3 comrepresenting a wide variety of auxotrophic pares the stabilities of CP-51 and CP-54 at 0 to markers. Transduction frequencies ranged from 15°C. CP-54 was more cold labile than CP-51, and, as with CP-51, its stability was enhanced 10-7 to 5 x 10-6/PFU. Host range and properties of CP-54. CP- by the addition of 0.02 M Mg2+ and/or 10% 54 was isolated from soil during a search for (vol/vol) dimethyl sulfoxide. Routinely, CP-54 transducing phages active on strain 4041 (B. lysates were stored at 15°C after the addition of
11 12 THORNE
APPL. ENVIRON. MICROBIOL. TABLE 3. Stabilities of CP-51 and CP-54a % of original PFU remaining after:
Diluent
Storage temp (0C)
7 days
24 h CP-51
CP-54
CP-51
15 0
100 43
100 2.4
100 8
NBY broth 0.02 M MgSO4
15 0
100 95
100 36
100 77
92 7.6
NBY broth + 10% (vol/vol) dimethyl sulfoxide
15 0
100 73
100 54
100 44
67 27
NBY broth
CP-54
2.6
56 10-4
x
97 15 100 100 100 NBY broth + 0.02 M MgSO4 and 10% di0 87 72 100 95 methyl sulfoxide a Phages were propagated on B. cereus 569 in NBY agar and harvested in NBY broth. Each lysate was then diluted 1:10 in the diluents listed and stored at 0 and 150C. The pH of each diluent was 6.8. The titers at the beginning of storage were 2.4 x 109 PFU/ml for CP-51 and 3.8 x 109 PFU/ml for CP-54.
MgSO4 to give a final concentration of 0.02 M. Stocks of B. cereus 569 spores infected with CP54 served as a primary source of the phage (13). Generally, strains that were hosts for both CP-51 and CP-54 were lysed to a greater extent with CP-54 than with CP-51. However, it is unlikely that CP-54 is a clear-plaque mutant of CP-51. Both phages formed colony-centered plaques on B. cereus 569, and both gave rise to mutants which were more lytic and formed clear plaques on 569. Such mutants derived from CP51 did not have the extended host range characteristic of CP-54. Efforts to isolate host range mutants of CP-51 which were active on strains 4040 and 4041 of B. thuringiensis were unsuccessful. In PA and NBY agar, CP-54 formed barely visible plaques on strain 4041 and no detectable plaques on strain 4040. In Min 3 agar, the phage produced very distinct and clear plaques on both strains. Although NBY and PA agar could not be used for CP-54 assays, both media were used successfully for phage propagation. Lysates prepared on 4040 and 4041 had titers greater than 1010 PFU/ml. Transduction with CP-54. The results of transduction with CP-54 (Table 4) were analogous in many respects to those with CP-51. The number of transductants was greater on Min 3E and 3C than on Min 3 as a result of less lytic activity on the richer media. Results of experiments not included here showed that CP-54 adsorbed very slowly to recipient cells, and plating transduction mixtures with phage antiserum would often reduce the number of transductants. For this reason antiserum was not used routinely. Serial passage of CP-54 resulted in rapid accumulation of more lytic mutants, and thus it was important for use in transduction to propa-
TABLE 4. Transduction of mutants of B. thuringiensis 4040 (subsp. finitimus) and 4041 (subsp. alesti) by phage CP-54' Recipient
Multiplicity of in-
fection
4040 M40Trp4040 M27 Trp4040 M7 Nic4041 M12Trp-
Colonies/ml when plated on:
Min 3 Min 3E Min 3C
0 2.5
15 900
0 4
300b
0 4
20"
20
0
1,050 10 1,300
15
1,740 0 220
0
0
0 2
860
4041 M13 Met-
0 2
25 420
4041 M26 His-
0 2
0 610
4041 M32 His-
0 2
20 1,010
0 690 a Transduction mixtures contained an average of 6 x 10" cells and phage to give the indicated multiplicity of infection in a final volume of 1 ml. Phage was grown on the homologous wild-type strain and was treated with UV light to inactivate 97% of the PFU. b Colonies were poorly developed as a result of lysis by the phage.
4041 M219 Arg-
0 2
gate phage from the most turbid plaques. Mutants of strains 4040, 4041, 4060, and 1715 were transduced with CP-54. CP-54, as well as
VOL. 35, 1978
TRANSDUCTION IN B. THURINGIENSIS
1113
CP-51, was too lytic for some strains to be useful with the two phages were the same, suggesting in transducing them. For example, even with that the failure to demonstrate cross-transducUV-treated phage no transductants were found tion was not related to the particular phage used. when mutants of strains 4044 and 4055 were The results with Trp- mutants of 4040 and 4041 tested with CP-51 and CP-54 grown on homol- were very much alike. Each strain was transduced equally well with phage grown on 4040, ogous wild-type cells. In those tests, lysis was so extensive, even on Min 3E, that the usual back- 4041, or 4045 (B. thuringiensis subsp. galleriae). ground of growth attributable to the limited The two were also transduced with lower but supply of nutrients was extremely sparse or non- similar efficiencies when 4042 (B. thuringiensis existent. subsp. sotto) and 4047 (subsp. entomocidus-liTests for cotransduction of linked massol) were the donors. All of the other 13 markers. The data in Table 5 are from experi- strains of B. thuringiensis and B. cereus 569 ments designed to test whether cotransduction were ineffective as donors. Failure to demonstrate cross-transduction in B. thuringiensis could be demonstrated with CP-54. The results suggest that CP-54 will be could possibly be a result of restriction in some useful for chromosomal mapping studies in this instances. However, no detectable restriction of organism. Two linkage groups were demon- phage itself has been observed among the limstrated (Table 5). One group included linkage of ited number of strains thus far tested. For extrp-1 to cys-1 and cys-2 but not to met-i. The ample, significant variation in efficiency of platsecond group included linkage of met-1 to arg-1 ing has not been observed with CP-54 grown on and arg-2 but not to arg-3. The cys-1 and cys-2 B. cereus 569, B. thuringiensis 4040, 4041, or mutants were able to grow on cysteine, methio- 4060 and assayed on all four strains. nine, homocysteine, or cystathionine, but not on sulfide. Mutations conferring this phenotype are DISCUSSION not represented on the current B. subtilis chroThe results presented here demonstrate that mosomal map (18). The met-I mutant has a strict requirement for methionine and may be phages CP-51 and CP-54 can carry out generalanalogous to metC or metD of B. subtilis (18). ized transduction in strains of B. thuringiensis. The arg-1 and arg-2 mutants were able to grow Because of the virulent nature of the two phages, difficulties were encountered in selecting and on arginine, ornithine, or citrulline and may be analogous to argO mutants of B. subtilis (18), scoring transductants of certain mutants. Howwhereas the arg-3 mutant which grew only on ever, with the use of phage that was treated with UV light to inactivate most of the PFU, along arginine may be analogous to argA. Tests for cross-transduction among with appropriately enriched plating medium, strains. Table 6 shows the results of testing the reasonable yields of transductants were obtained abilities of B. cereus 569 and 18 strains of B. with most mutants. The variation among muthuringiensis to serve as donors for transduction tants of the same strain with respect to suscepof mutants of 4040 (B. thuringiensis subsp. fin- tibility to lysis by the phage and difficulty in itimus), 4041 (subsp. alesti), and 4060 (subsp. recovering transductants is not understood. For thompsoni). With the three mutants of 4060 that strains that were hosts for both CP-51 and CPwere tested transductants were obtained only 54, CP-51 was generally less lytic than CP-54 with the parent strain as donor. In five of the and gave better yields of transductants. The data presented here demonstrate that tests with 4060, including the homologous cross, CP-51 and CP-54 were each used, and the results CP-54 can be used for linkage studies in B. TABLE 5. Tests for cotransduction of linked markers in B. thuringiensisa Cotranaduction (%) Recipient 23.2 (52/224)b 4041 M13 cys-1 4041 M12 trp-i 23.9 (65/272) 4041 M12 M12 trp-1 cys-2 4041 prototrophic 0 (0/224) 4041 M12 M266 trp-i met-I 4041 prototrophic 37.2 (146/393) 4041 M266 met-i 4041 FM119 arg-1 37.7 (46/122) 4041 M266 met-i 4041 M213 arg-2 0 (0/112) 4041 M266 met-i 4041 M226 arg-3 a When the recipients were Trp- Cys- double mutants, Trp+ transductants were selected on Min 3C and tested for Cys+ by picking colonies to Min 3 and Min 3 plus methionine. When the recipients carried single markers, transductants were selected on Min 3E supplemented with the donor requirement, and colonies were tested for the presence of the donor marker by picking to Min 3 and Min 3 supplemented with the amino acid Donor
in question. b Number of doubles/number of transductants tested.
11 14
THORNE
APPL. ENVIRON. MICROBIOL.
TABLE 6. Tests for cross-transduction among strains of B. thuringiensis and B. cereus Transduction of B. thuringiensis recipients' Donor
Strain no.
subsp. thompsoni
.
. .
40 M87 NicL4u0. M40 Trp 4060 Mgg TW-
b
4041 M12 Trp-
B. thuringiensis subsp.
finitimus
4040
alesti sotto
4041 4042 4043 4044
dendrolimus kenyae gaUeriae entomocidus entomocidus-limassol aizawai morrisoni tolworthi kurstaki canadensis subtoxicus darmstadiensis toumanoffi thompsoni thuringiensis B. cereus
4045 4046 4047 4048
4049 4050 4055 4056 4057 4058 4049 4060 1715
+ + (100%) + (10-15%)
+ (100%) + + (10-15%)
-
+ (100%)
+ (100%)
-
+
-
-
-
b
-
-
-
-
_b
_
(10%)
+
(10-15%)
-
_
_b +b
569 _b a All tranaductions were carried out under conditions which produced transductants with phage grown on the wild-type parent strain. Symbols: +, transduction observed; -, no transductants detected. The numbers in parentheses compare the efficiencies of transduction and represent the number of transductants obtained in the heterologous cross as a percentage of the number obtained with the same recipient cells exposed to the same multiplicity of infection of phage grown on the homologous wild-type strain. bIn these tests, results with CP-51 and CP-54 were indistinguishable. Only CP-54 was used in all the other tests.
thuringiensis. Cotransduction of linked markers in B. cereus by CP-51 has already been demonstrated, and, presumably, that phage could be used for mapping in B. thuringiensis as welL Although the problems in recovering transductants of some mutants as a result of the virulent nature of the phages may preclude the assignment of precise cotransduction frequencies for certain markers, demonstration of linkage of such markers in a qualitative manner will be useful in map construction. Attempts to cross-transduce among various strains of B. thuringiensis were largely unsuccessful. The rare occurrence of cross-transduction is probably a reflection ofa lack of homology among the strains rather than restriction, since the phage itself did not appear to be restricted when grown on a particular host and assayed with other hosts as indicators. However, this test for restriction of phage was applied to only a few strains, and the possibility of restriction occurring in some of the crosses cannot be ruled out. A very limited number of markers were tested in the cross-transduction experiments, and thus little can be inferred concerning the extent of
homology among the various strains. Previous studies on CP-51 transduction with strains of B. cereus (16) showed that the strains rarely crossed with one another and that certain strains would cross for some markers and not for others. In those tests, mutants of B. thuringiensis were not tested for transduction, but CP-51 grown on B. thuringiensis strain NRS 1328 did transduce certain markers in B. cereus 569 and ATCC 6464 (16). ACKNOWLEDGMENT This work was supported by grant BMS75-13956 from the National Science Foundation.
LITERATURE CITED 1. Adams, NL H. 1959. Bacteriophages. Interscience Publishers, Inc., New York. 2. Bulla, L A., Jr., K. J. Kramer, and L. I. Davidson. 1977. Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis. J. Bacteriol.
130:375-383. 3. Falcon, L. A. 1971. Use of bacteria for microbial control, p. 67-95. In H. D. Burges and N. W. Hussey (ed), Microbial control of insects and mites. Academic Press Inc., New York. 4. Glatron, ML, K Lecadet, and R. Dedonder. 1972.
VOL. 35, 1978
5. 6.
7. 8. 9.
10. 11.
Structure of the parasporal inclusion of Bacillus thuringiensis Berliner: characterization of a repetitive subunit. Eur. J. Biochem. 30:330-338. Herbert, B. N., H. J. Gould, and E. B. Chain. 1971. Crystal protein of Bacillus thuringiensis var. tolworthi. Eur. J. Biochem. 24:366-375. Holmes, K. C., and R. E. Monro. 1965. Studies on the structure of parasporal inclusions from Bacillus thuringiensis. J. Mol. Biol. 14:572-581. Iyer, V. 1960. Concentration and isolation of auxotrophic mutants of sporeforming bacteria. J. Bacteriol. 79:309-310. Rogoff, M. H., and A. A. Yousten. 1969. Bacillus thuringiensis: microbiological considerations. Annu. Rev. Microbiol. 23:357-386. Short, J. A., P. D. Walker, R. 0. Thompson, and H. J. Somerville. 1974. The fine structure of Bacillus finitimus and Bacillus thuringiensis spores with special reference to the location of crystal antigen. J. Gen. Microbiol. 84:261-279. Somerville, H. J. 1971. Formation of the parasporal inclusion of Bacillus thuringiensis. Eur. J. Biochem. 18:226-237. Somerville, H. J., and H. V. Pockett. 1975. An insect
TRANSDUCTION IN B. THURINGIENSIS 1115
12. 13. 14.
15. 16.
17. 18.
19.
toxin from spores of BaciUus thuringiensis and Bacillus cereus. J. Gen. Microbiol. 87:359-369. Thorne, C. B. 1962. Transduction in BaciUus subtilis. J. Bacteriol. 83:106-111. Thorne, C. B. 1968. Transducing bacteriophage for BaciUus cereus. J. Virol. 2:657-4662. Thorne, C. B. 1968. Transduction in Bacillus cereus and BaciUus anthracis. Bacteriol. Rev. 32:358-361. Thorne, C. B., and S. C. Holt. 1974. Cold lability of BaciUus cereus bacteriophage CP-51. J. Virol. 14:1008-1012. Yelton, D. B., and C. B. Thorne. 1970. Transduction in Bacillus cereus by each of two bacteriophages. J. Bacteriol. 102:573-579. Yelton, D. B., and C. B. Thorne. 1971. Comparison of BaciUus cereus bacteriophages CP-51 and CP-53. J. Virol. 8:242-253. Young, F. E., and G. A. Wilson. 1975. Chromosomal map of BaciUus subtiiis, p. 596-614. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. Yousten, A. A., and M. H. Rogoff. 1969. Metabolism of Bacillus thuringiensis in relation to spore and crystal formation. J. Bacteriol. 100:1229-1236.
Vol. 35, No. 6
Printed in U.S.A.
Transduction in Bacillus thuringiensis CURTIS B. THORNE
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003 Received for publication 29 December 1977
Bacteriophage CP-51, originally reported as a generalized transducing phage for Bacillus cereus and B. anthracis, has been shown to carry out generalized transduction in several strains of B. thuringiensis. A newly isolated phage, CP54, which has a broader host range than CP-51, also mediates generalized transduction in B. thuringiensis. CP-51 and CP-54 are similar in size and morphology and are related serologically, but they are not identical. CP-54 is more cold labile than CP-51, and, as with CP-51, its stability both at 0 and 150C is enhanced by the presence of 0.02 M Mg2e. Some examples of cotransduction of linked markers in B. thuringiensis are presented, demonstrating the feasibility of chromosomal mapping in this organism. The rare occurrence of cross-transduction among strains of B. thuringiensis is probably a reflection of nonhomology rather than restriction, since phage itself did not appear to be restricted when grown on a particular host and assayed with other hosts as indicator. Strains ofBacillus thuringiensis produce proteinaceous parasporal crystals which are toxic for the larval stages of susceptible insects, and preparations of B. thuringiensis for use as insecticides are made commercially in the United States and several other countries (3). Although a number of investigators have studied the chemistry of the toxic protein crystals (2, 4, 5, 8, 11) and the physiological and morphological aspects of spore and crystal formation (6, 8-10, 19), the lack of a genetic exchange system in B. thuringiensis has hindered studies of genetic factors associated with crystal formation. This report deals with transduction in strains of B. thuringiensis mediated by two bacteriophages, CP-51 and CP-54. Phage CP-51 (13, 14, 16, 17) was isolated from soil in my laboratory and originally studied as a generalized transducing phage for B. cereus and B. anthracis. These studies have now been extended to include B. thuringiensis. The second phage, CP-54, also isolated from soil, has a broader host range with respect to B. thuringiensis strains than does CP51 and can mediate generalized transduction in strains which are not hosts for CP-51. MATERIALS AND METHODS Organisms. The strains of B. thuringiensis used
isolation of CP-51 (13), except that wild-type B. thuringiensis 4041 was the host organism and streptomycin was omitted from the medium. Media and cultural conditions. The minimal medium, Min 3, was composed of the following (in grams per liter): (NH4)2SO4, 2; KH2PO4, 6; K2HPO4, 14; sodium citrate, 1; glucose, 5; L-glutamic acid, 2; glycine, 0.2; thiamine hydrochloride, 0.01; MgSO4 .7H20, 0.2; FeCI3 6H20, 0.04; MnSO4 H20, 0.00025; pH adjusted to 7.0 with NaOH. The glucose and FeC!3 were sterilized separately. Min 3C contained, in addition to Min 3, 5 g of vitamin-free, acid-hydrolyzed casein (Nutritional Biochemicals Corp., Cleveland, Ohio) per liter. Enriched minimal medium, Min 3E, was Min 3 with 1% (vol/vol) L-broth added. NBY medium contained 8 g of nutrient broth (Difco Laboratories, Detroit, Mich.) and 3 g of Difco yeast extract per liter, pH 6.8. L-broth contained 10 g of Difco tryptone, 5 g of Difco yeast extract, and 10 g of NaCl per liter, pH 7.0. PA medium was composed of the following (in grams per liter): Difco nutrient broth, 8; NaCl, 5; MgSO4 7H20, 0.2; MnSO4 H20, 0.05; CaCl2 * 2H20, 0.15; pH adjusted to 6.0 with HCI. Peptone diluent contained 10 g of Difco peptone per liter. For solid media, 15 g of agar was added per liter. For soft agar overlays, 5 g of agar was added per liter of appropriate medium. Spores were grown on slants of potato agar (12) in screw-cap tubes (20 by 150 mm) inoculated with spores or vegetative cells and incubated at 37°C for 3 to 5 days. Growth from a single slant was suspended in 5 ml of water and held in a 65°C bath for 30 min. Recipient cells for transduction were grown in 250ml Erlenmeyer flasks containing 25 ml of L-broth and incubated at 37°C on a rotary shaker (250 rpm). Cultures were grown for 5 to 6 h from a 10% (vol/vol) transfer of a 15-h culture or for 6 to 7 h from an inoculum of about 2 x 108 spores per flask. Phage assays. Both phages were routinely assayed by the soft agar overlay technique, with spores of B.
and their sources are included in Table 1. Auxotrophic mutants of B. thuringiensis were isolated by the procedure of Iyer (7) after exposure of spores to ultraviolet (UV) light. B. cereus NRRL 569 was used as an indicator in phage assays. Bacteriophage. Phage CP-51 has been described previously (13, 14, 16, 17). Phage CP-54 was isolated from soil in a manner similar to that described for the 1109
APPL. ENVIRON. MICROBIOL.
1110 THORNE cereus 569 as the indicator, in PA agar as described for CP-51 (13) except that assay plates were incubated at 30°C. When Min 3 agar was used for phage assays, 0.1 ml of a 15- to 24-h culture of the appropriate strain of B. thuringiensis in L-broth was used as the indicator. Peptone diluent was used routinely for diluting phage lysates to be assayed. Propagation of phage. Both phages were propagated routinely in soft NBY or PA agar from plaques as described for CP-51 (13). In some instances, 107 plaque-forming units (PFU) from phage lysates rather than phage from individual plaques were used as the phage seed. Either spores (about 108) or cells (0.2 ml of a 16-h L-broth culture) served as the host inoculum. Propagation plates were incubated at 37°C for 18 to 20 h, and phage from each plate were harvested in 5 ml of NBY or PA broth and filtered through Millipore DA membranes (Millipore Corp., Bedford, Mass.). Phage lysates were stored at 15°C. Transductions. Routinely, 0.5 to 0.8 ml of culture containing about 109 cells per ml was mixed in a 20mm tube with 0.2 to 0.5 ml of phage preparation to give a multiplicity of infection of 1 to 4 and incubated on a shaker at 37°C for 30 min. Phage to be used in transductions was usually treated with UV light (13) to inactivate 95 to 99% of the PFU, and the multiplicity of infection was calculated on the basis of phage titer before UV treatment. Tests for spontaneous reversion were always run by substituting NBY or PA broth for the phage. After incubation, 0.1-ml samples were spread in duplicate on appropriate minimal agar plates. When phage antiserum was used to facilitate recovery of transductants, 0.1 ml of a 1:10 dilution was spread with the sample of transduction mixture. Plates were incubated at 37°C, and transductants were scored after 24 h when plated on Min 3C or after 36 to 48 h when plated on Min 3 or Min 3E. Phage antiserum. Antiserum to CP-51 was prepared as described for phage SP-10 (12).
as temperate for any of the strains, it was much more lytic for some strains than for others. In PA agar the plaques on some strains were turbid or colony-centered, characteristic of plaques formed by temperate phages, but on minimal medium, plaques produced on the same strains were clear. In general, as will be discussed later, the lytic response was much more marked on minimal medium than on rich media, such as PA and NBY. Strain 4060 (B. thuringiensis subsp. thompsoni) was one of the strains which appeared to be least susceptible to lysis by CP51, and it was chosen for studies on transduction. Transduction with CP-51. Some results of transduction tests with strain 4060 are given in Table 2. Transductants were obtained at reasonably high frequencies with phage lysates that were treated with UV light to inactivate most of the PFU. However, as indicated in Table 2, there was considerable variation among mutants of the same strain with respect to survival of transductants under various conditions. Generally, the number of transductants was greater when phage was treated with UV light to inactivate 95 to 99% of the PFU. Plating transduction mixtures with phage antiserum was particularly beneficial when phage lysates were not treated with UV light, and, in some instances, antiserum improved both the number and the size of colonies even when phage had been treated with UV TABLE 1. B. thuringiensis strains used Strain
X
thuringien-
sis subsp.
Source
Host for growth CP-51 CP-54
RESULTS
Host range of CP-51. Previous tests (14) showed that the Sterne strain of B. anthracis and four of seven strains of B. cereus tested were hosts for CP-51. In the present studies with 21 strains of B. thuringiensis, all but 4 were able to serve as hosts (Table 1). Susceptibilities of the various strains to lysis by the phage were tested both by using each strain as an indicator in a phage assay and by using each strain as a potential host for phage propagation. The 4 strains listed as unsusceptible to CP-51 failed to show plaques when used as indicators and did not serve as hosts for phage propagation. Phage lysates prepared on the 17 susceptible strains in soft NBY or PA agar had titers ranging from 5 109 to 8 x 1010 PFU/ml. Lysates prepared by the same procedure with B. cereus 569 usually had titers of 2 x 1011 or more PFU/ml. The nature of the phage-host interaction varied greatly among the strains susceptible to CP51. Although the phage could not be classified
B.
4040 4041 4042 4043 4044 4045 4046 4047 4048 4049 4050 4055 4056 4057 4058 4059 4060 1715 ATCC 10792 ATCC 13367 ATCC 19266
NRRL^ NRRL aksti NRRL sotto dendrolimus NRRL NRRL kenyae NRRL galleriae NRRL entomocidus entomocidus-li- NRRL massol NRRL aizawai NRRL morrisoni
finitimus
tolworthi kurstaki
-
+
+
+ + + + + +
-
+
+ + + + + + + + + + + +
+ + + + + + + + + +
+ + + -
NRRL NRRL NRRL canadensis NRRL subtoxicus darmstadiensis NRRL NRRL toumanoffi NRRL thompsoni thuringiensis A. Yousten + R. Gordon + R. Gordon + + R. Gordon a + Strain served as no detectable activity. ^ NRRL, Agricultural Research Service, Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill.
host;-,
TRANSDUCTION IN B. THURINGIENSIS 1111
VOL. 35, 1978
TABLE 2. Transduction of mutants ofB. thuringiensis 4060 (subsp. thompsoni) with CP-51a CP-51 Phage
Recipient
lysate
1
2
PFU in-
Colonies/ml when plated on:
actbvated byUV
plicity of
99 99 99 99 0 0
10 2 1 0.5 10 2
4060 M9 Trp97
4060 M87 Nic97
antise-
Min 3E
rum
0
0
1,150 1,210 1,110 810
1,170
210b 310b
1,610 1,080
+
anti-
0
10
2
1,750
3,500
0
10
2
325b
20 1,300
130
40 2,235
97
2
0
30
35
35
97
2
270b
510b
700
4060 M25 Met-
Min 3C
serum
0
0
4060 M99 Trp-
Min 3
infection
0
4060 M9 Trp-
Min 3E
Min 3 +
Multi-
4,700
20 10 0 2 1,920 97 700 a Recipient cells were grown for 6 h in L-broth after a 10% transfer of a 16-h culture. Transduction mixtures 4060 M52 Leu-
contained an average of 8 x 108 celis and phage to give the indicated multiplicity of infection in a final volume of 1 ml. Both phage preparations were grown on wild-type 4060. b Colonies were poorly developed as a result of lysis by the phage.
light. Table 2 also demonstrates that the rich- thuringiensis subsp. alesti). As shown in Table ness of the plating medium influenced the num- 1, CP-54 was active on all strains of B. thurinber of transductants obtained. The best medium giensis tested. Like CP-51, it was also active on of those tested was Min 3C, which could be used B. cereus 569, and that organism was used roufor mutants requiring tryptophan, vitamins, pu- tinely as the indicator in CP-54 assays. When rines, or pyrimidines. With mutants requiring viewed in the electron microscope, CP-54 has a amino acids other than tryptophan, Min 3E gave structure very similar, if not indentical, to that improved results. In many instances, even when of CP-51 (15, 16). However, the two phages differ UV-treated phage was used, the colonies of in the following respects: (i) CP-54 has a broader transductants on minimal medium were poorly host range than that of CP-51; (ii) plaque mordeveloped and partially lysed, and this problem phologies are different; (iii) CP-54 is more cold could often be avoided by using enriched mini- labile than CP-51 (17); and (iv) although antimal. There was considerable variation in this serum prepared against CP-51 was also active respect among mutants of the same strain, and on CP-54, the two phages are not serologically sometimes it was necessary to plate transduction identical. Antiserum prepared against CP-51 had mixtures with phage antiserum even when en- K values (1) of 1,000 for CP-51 and 100 for CPriched minimal medium was used. Such an ex- 54. Previous studies with CP-51 (13, 15) showed ample is mutant M25 (Table 2). With appropriate combinations of UV treatment, plating me- that it is very unstable when stored at refrigerdium, and phage antiserum, it has been possible ator temperatures, and the optimum temperato obtain transductants of all mutants tested ture for stability was around 15°C. Table 3 comrepresenting a wide variety of auxotrophic pares the stabilities of CP-51 and CP-54 at 0 to markers. Transduction frequencies ranged from 15°C. CP-54 was more cold labile than CP-51, and, as with CP-51, its stability was enhanced 10-7 to 5 x 10-6/PFU. Host range and properties of CP-54. CP- by the addition of 0.02 M Mg2+ and/or 10% 54 was isolated from soil during a search for (vol/vol) dimethyl sulfoxide. Routinely, CP-54 transducing phages active on strain 4041 (B. lysates were stored at 15°C after the addition of
11 12 THORNE
APPL. ENVIRON. MICROBIOL. TABLE 3. Stabilities of CP-51 and CP-54a % of original PFU remaining after:
Diluent
Storage temp (0C)
7 days
24 h CP-51
CP-54
CP-51
15 0
100 43
100 2.4
100 8
NBY broth 0.02 M MgSO4
15 0
100 95
100 36
100 77
92 7.6
NBY broth + 10% (vol/vol) dimethyl sulfoxide
15 0
100 73
100 54
100 44
67 27
NBY broth
CP-54
2.6
56 10-4
x
97 15 100 100 100 NBY broth + 0.02 M MgSO4 and 10% di0 87 72 100 95 methyl sulfoxide a Phages were propagated on B. cereus 569 in NBY agar and harvested in NBY broth. Each lysate was then diluted 1:10 in the diluents listed and stored at 0 and 150C. The pH of each diluent was 6.8. The titers at the beginning of storage were 2.4 x 109 PFU/ml for CP-51 and 3.8 x 109 PFU/ml for CP-54.
MgSO4 to give a final concentration of 0.02 M. Stocks of B. cereus 569 spores infected with CP54 served as a primary source of the phage (13). Generally, strains that were hosts for both CP-51 and CP-54 were lysed to a greater extent with CP-54 than with CP-51. However, it is unlikely that CP-54 is a clear-plaque mutant of CP-51. Both phages formed colony-centered plaques on B. cereus 569, and both gave rise to mutants which were more lytic and formed clear plaques on 569. Such mutants derived from CP51 did not have the extended host range characteristic of CP-54. Efforts to isolate host range mutants of CP-51 which were active on strains 4040 and 4041 of B. thuringiensis were unsuccessful. In PA and NBY agar, CP-54 formed barely visible plaques on strain 4041 and no detectable plaques on strain 4040. In Min 3 agar, the phage produced very distinct and clear plaques on both strains. Although NBY and PA agar could not be used for CP-54 assays, both media were used successfully for phage propagation. Lysates prepared on 4040 and 4041 had titers greater than 1010 PFU/ml. Transduction with CP-54. The results of transduction with CP-54 (Table 4) were analogous in many respects to those with CP-51. The number of transductants was greater on Min 3E and 3C than on Min 3 as a result of less lytic activity on the richer media. Results of experiments not included here showed that CP-54 adsorbed very slowly to recipient cells, and plating transduction mixtures with phage antiserum would often reduce the number of transductants. For this reason antiserum was not used routinely. Serial passage of CP-54 resulted in rapid accumulation of more lytic mutants, and thus it was important for use in transduction to propa-
TABLE 4. Transduction of mutants of B. thuringiensis 4040 (subsp. finitimus) and 4041 (subsp. alesti) by phage CP-54' Recipient
Multiplicity of in-
fection
4040 M40Trp4040 M27 Trp4040 M7 Nic4041 M12Trp-
Colonies/ml when plated on:
Min 3 Min 3E Min 3C
0 2.5
15 900
0 4
300b
0 4
20"
20
0
1,050 10 1,300
15
1,740 0 220
0
0
0 2
860
4041 M13 Met-
0 2
25 420
4041 M26 His-
0 2
0 610
4041 M32 His-
0 2
20 1,010
0 690 a Transduction mixtures contained an average of 6 x 10" cells and phage to give the indicated multiplicity of infection in a final volume of 1 ml. Phage was grown on the homologous wild-type strain and was treated with UV light to inactivate 97% of the PFU. b Colonies were poorly developed as a result of lysis by the phage.
4041 M219 Arg-
0 2
gate phage from the most turbid plaques. Mutants of strains 4040, 4041, 4060, and 1715 were transduced with CP-54. CP-54, as well as
VOL. 35, 1978
TRANSDUCTION IN B. THURINGIENSIS
1113
CP-51, was too lytic for some strains to be useful with the two phages were the same, suggesting in transducing them. For example, even with that the failure to demonstrate cross-transducUV-treated phage no transductants were found tion was not related to the particular phage used. when mutants of strains 4044 and 4055 were The results with Trp- mutants of 4040 and 4041 tested with CP-51 and CP-54 grown on homol- were very much alike. Each strain was transduced equally well with phage grown on 4040, ogous wild-type cells. In those tests, lysis was so extensive, even on Min 3E, that the usual back- 4041, or 4045 (B. thuringiensis subsp. galleriae). ground of growth attributable to the limited The two were also transduced with lower but supply of nutrients was extremely sparse or non- similar efficiencies when 4042 (B. thuringiensis existent. subsp. sotto) and 4047 (subsp. entomocidus-liTests for cotransduction of linked massol) were the donors. All of the other 13 markers. The data in Table 5 are from experi- strains of B. thuringiensis and B. cereus 569 ments designed to test whether cotransduction were ineffective as donors. Failure to demonstrate cross-transduction in B. thuringiensis could be demonstrated with CP-54. The results suggest that CP-54 will be could possibly be a result of restriction in some useful for chromosomal mapping studies in this instances. However, no detectable restriction of organism. Two linkage groups were demon- phage itself has been observed among the limstrated (Table 5). One group included linkage of ited number of strains thus far tested. For extrp-1 to cys-1 and cys-2 but not to met-i. The ample, significant variation in efficiency of platsecond group included linkage of met-1 to arg-1 ing has not been observed with CP-54 grown on and arg-2 but not to arg-3. The cys-1 and cys-2 B. cereus 569, B. thuringiensis 4040, 4041, or mutants were able to grow on cysteine, methio- 4060 and assayed on all four strains. nine, homocysteine, or cystathionine, but not on sulfide. Mutations conferring this phenotype are DISCUSSION not represented on the current B. subtilis chroThe results presented here demonstrate that mosomal map (18). The met-I mutant has a strict requirement for methionine and may be phages CP-51 and CP-54 can carry out generalanalogous to metC or metD of B. subtilis (18). ized transduction in strains of B. thuringiensis. The arg-1 and arg-2 mutants were able to grow Because of the virulent nature of the two phages, difficulties were encountered in selecting and on arginine, ornithine, or citrulline and may be analogous to argO mutants of B. subtilis (18), scoring transductants of certain mutants. Howwhereas the arg-3 mutant which grew only on ever, with the use of phage that was treated with UV light to inactivate most of the PFU, along arginine may be analogous to argA. Tests for cross-transduction among with appropriately enriched plating medium, strains. Table 6 shows the results of testing the reasonable yields of transductants were obtained abilities of B. cereus 569 and 18 strains of B. with most mutants. The variation among muthuringiensis to serve as donors for transduction tants of the same strain with respect to suscepof mutants of 4040 (B. thuringiensis subsp. fin- tibility to lysis by the phage and difficulty in itimus), 4041 (subsp. alesti), and 4060 (subsp. recovering transductants is not understood. For thompsoni). With the three mutants of 4060 that strains that were hosts for both CP-51 and CPwere tested transductants were obtained only 54, CP-51 was generally less lytic than CP-54 with the parent strain as donor. In five of the and gave better yields of transductants. The data presented here demonstrate that tests with 4060, including the homologous cross, CP-51 and CP-54 were each used, and the results CP-54 can be used for linkage studies in B. TABLE 5. Tests for cotransduction of linked markers in B. thuringiensisa Cotranaduction (%) Recipient 23.2 (52/224)b 4041 M13 cys-1 4041 M12 trp-i 23.9 (65/272) 4041 M12 M12 trp-1 cys-2 4041 prototrophic 0 (0/224) 4041 M12 M266 trp-i met-I 4041 prototrophic 37.2 (146/393) 4041 M266 met-i 4041 FM119 arg-1 37.7 (46/122) 4041 M266 met-i 4041 M213 arg-2 0 (0/112) 4041 M266 met-i 4041 M226 arg-3 a When the recipients were Trp- Cys- double mutants, Trp+ transductants were selected on Min 3C and tested for Cys+ by picking colonies to Min 3 and Min 3 plus methionine. When the recipients carried single markers, transductants were selected on Min 3E supplemented with the donor requirement, and colonies were tested for the presence of the donor marker by picking to Min 3 and Min 3 supplemented with the amino acid Donor
in question. b Number of doubles/number of transductants tested.
11 14
THORNE
APPL. ENVIRON. MICROBIOL.
TABLE 6. Tests for cross-transduction among strains of B. thuringiensis and B. cereus Transduction of B. thuringiensis recipients' Donor
Strain no.
subsp. thompsoni
.
. .
40 M87 NicL4u0. M40 Trp 4060 Mgg TW-
b
4041 M12 Trp-
B. thuringiensis subsp.
finitimus
4040
alesti sotto
4041 4042 4043 4044
dendrolimus kenyae gaUeriae entomocidus entomocidus-limassol aizawai morrisoni tolworthi kurstaki canadensis subtoxicus darmstadiensis toumanoffi thompsoni thuringiensis B. cereus
4045 4046 4047 4048
4049 4050 4055 4056 4057 4058 4049 4060 1715
+ + (100%) + (10-15%)
+ (100%) + + (10-15%)
-
+ (100%)
+ (100%)
-
+
-
-
-
b
-
-
-
-
_b
_
(10%)
+
(10-15%)
-
_
_b +b
569 _b a All tranaductions were carried out under conditions which produced transductants with phage grown on the wild-type parent strain. Symbols: +, transduction observed; -, no transductants detected. The numbers in parentheses compare the efficiencies of transduction and represent the number of transductants obtained in the heterologous cross as a percentage of the number obtained with the same recipient cells exposed to the same multiplicity of infection of phage grown on the homologous wild-type strain. bIn these tests, results with CP-51 and CP-54 were indistinguishable. Only CP-54 was used in all the other tests.
thuringiensis. Cotransduction of linked markers in B. cereus by CP-51 has already been demonstrated, and, presumably, that phage could be used for mapping in B. thuringiensis as welL Although the problems in recovering transductants of some mutants as a result of the virulent nature of the phages may preclude the assignment of precise cotransduction frequencies for certain markers, demonstration of linkage of such markers in a qualitative manner will be useful in map construction. Attempts to cross-transduce among various strains of B. thuringiensis were largely unsuccessful. The rare occurrence of cross-transduction is probably a reflection ofa lack of homology among the strains rather than restriction, since the phage itself did not appear to be restricted when grown on a particular host and assayed with other hosts as indicators. However, this test for restriction of phage was applied to only a few strains, and the possibility of restriction occurring in some of the crosses cannot be ruled out. A very limited number of markers were tested in the cross-transduction experiments, and thus little can be inferred concerning the extent of
homology among the various strains. Previous studies on CP-51 transduction with strains of B. cereus (16) showed that the strains rarely crossed with one another and that certain strains would cross for some markers and not for others. In those tests, mutants of B. thuringiensis were not tested for transduction, but CP-51 grown on B. thuringiensis strain NRS 1328 did transduce certain markers in B. cereus 569 and ATCC 6464 (16). ACKNOWLEDGMENT This work was supported by grant BMS75-13956 from the National Science Foundation.
LITERATURE CITED 1. Adams, NL H. 1959. Bacteriophages. Interscience Publishers, Inc., New York. 2. Bulla, L A., Jr., K. J. Kramer, and L. I. Davidson. 1977. Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis. J. Bacteriol.
130:375-383. 3. Falcon, L. A. 1971. Use of bacteria for microbial control, p. 67-95. In H. D. Burges and N. W. Hussey (ed), Microbial control of insects and mites. Academic Press Inc., New York. 4. Glatron, ML, K Lecadet, and R. Dedonder. 1972.
VOL. 35, 1978
5. 6.
7. 8. 9.
10. 11.
Structure of the parasporal inclusion of Bacillus thuringiensis Berliner: characterization of a repetitive subunit. Eur. J. Biochem. 30:330-338. Herbert, B. N., H. J. Gould, and E. B. Chain. 1971. Crystal protein of Bacillus thuringiensis var. tolworthi. Eur. J. Biochem. 24:366-375. Holmes, K. C., and R. E. Monro. 1965. Studies on the structure of parasporal inclusions from Bacillus thuringiensis. J. Mol. Biol. 14:572-581. Iyer, V. 1960. Concentration and isolation of auxotrophic mutants of sporeforming bacteria. J. Bacteriol. 79:309-310. Rogoff, M. H., and A. A. Yousten. 1969. Bacillus thuringiensis: microbiological considerations. Annu. Rev. Microbiol. 23:357-386. Short, J. A., P. D. Walker, R. 0. Thompson, and H. J. Somerville. 1974. The fine structure of Bacillus finitimus and Bacillus thuringiensis spores with special reference to the location of crystal antigen. J. Gen. Microbiol. 84:261-279. Somerville, H. J. 1971. Formation of the parasporal inclusion of Bacillus thuringiensis. Eur. J. Biochem. 18:226-237. Somerville, H. J., and H. V. Pockett. 1975. An insect
TRANSDUCTION IN B. THURINGIENSIS 1115
12. 13. 14.
15. 16.
17. 18.
19.
toxin from spores of BaciUus thuringiensis and Bacillus cereus. J. Gen. Microbiol. 87:359-369. Thorne, C. B. 1962. Transduction in BaciUus subtilis. J. Bacteriol. 83:106-111. Thorne, C. B. 1968. Transducing bacteriophage for BaciUus cereus. J. Virol. 2:657-4662. Thorne, C. B. 1968. Transduction in Bacillus cereus and BaciUus anthracis. Bacteriol. Rev. 32:358-361. Thorne, C. B., and S. C. Holt. 1974. Cold lability of BaciUus cereus bacteriophage CP-51. J. Virol. 14:1008-1012. Yelton, D. B., and C. B. Thorne. 1970. Transduction in Bacillus cereus by each of two bacteriophages. J. Bacteriol. 102:573-579. Yelton, D. B., and C. B. Thorne. 1971. Comparison of BaciUus cereus bacteriophages CP-51 and CP-53. J. Virol. 8:242-253. Young, F. E., and G. A. Wilson. 1975. Chromosomal map of BaciUus subtiiis, p. 596-614. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. Yousten, A. A., and M. H. Rogoff. 1969. Metabolism of Bacillus thuringiensis in relation to spore and crystal formation. J. Bacteriol. 100:1229-1236.
