APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1991, 0099-2240/91/102946-05$02.00/0 Copyright © 1991, American Society for Microbiology
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Transposon Tn916 Mutagenesis in Clostridium botulinum WEI-JEN LIN AND ERIC A. JOHNSON* Department of Food Microbiology and Toxicology, University of Wisconsin, 1925 Willow Drive, Madison, Wisconsin 53706 Received 23 April 1991/Accepted 24 July 1991
The study of toxinogenesis and other properties in Clostridium botulinum is limited by the absence of genetic methods that enable construction of defined mutants. In this study, tetracycline-resistant transposon Tn916 in Enterococcus faecalis was conjugatively transferred in filter matings to group I Clostridium botulinum strains Hall A and 113B. The Tn916 transfer frequencies to C. botulinum ranged from 10-8 to 10-5 Tcr transconjugant per recipient depending on the donor strain. Southern blot analyses of EcoRI or HindlIl chromosomal digests extracted from randomly selected Tcr transconjugants showed that the transposon inserted at different sites in the recipient chromosome, and the copy number of Tn916 varied from one to three. Tn916 insertion gave several different auxotrophic mutants. This approach should be useful for the study of genes important in growth, survival, and toxinogenesis in C. botulinum.
several other organisms (9, 13-15). Tn916 is a 16.4-kbp tetracycline-encoded conjugative transposon that was originally detected in Enterococcus (Streptococcus) faecalis DS16 (9, 10). Tn916 belongs to a group of tetracyclineresistant determinants (tetM) that have fertility properties and may be involved in multidrug transfers in pathogenic bacteria (10). It has been suggested that Tn916 moves by an excision-insertion mechanism that involves a circular intermediate (5, 6, 11, 19). Tn9J6 can insert into recipient chromosomes at multiple sites and cause insertional inactivation of various genes (10, 11, 13, 15). In this paper, we describe the introduction of Tn916 from Enterococcus faecalis into C. botulinum strains by a filter mating procedure. We found that Tn916 stably integrated and caused mutations in several genes which resulted in auxotrophic nutritional requirements. The system which we developed should be useful for the study of botulinum toxin and other properties of C. botulinum.
Clostridium botulinum produces seven known serotypes of neurotoxins (serotypes A, B, C1, D, E, F, and G), which have specific toxicities that range from 107 to 108 mouse 50% lethal doses per mg of protein (20). Botulinum neurotoxin types A, B, and E are mainly responsible for human botulism, and types A through E are important causes of botulism in domestic and wild animals (20). The various neurotoxins have been characterized biochemically and pharmacologically (7). Recently, by cloning small fragments of the toxin genes into Escherichia coli vectors, workers in several laboratories have determined the entire nucleotide sequence of the gene encoding botulinum neurotoxin type A and a partial sequence of the gene encoding botulinum neurotoxin type E (3, 4, 21). These studies also demonstrated that the genes are chromosomally located. However, little is known of the genetics and metabolic regulation of toxinogenesis in C. botulinum. The genetic analysis of C. botulinum toxinogenesis has been hampered because of a lack of effective genetic methodologies, including a mutation system and gene transfer systems (24). Genetic studies are further limited by the National Institutes of Health Recombinant DNA Advisory Committee restrictions on cloning of toxin gene fragments larger than 1 kb and other in vitro genetic manipulations with C. botulinum. Alternative methods for genetic analyses need to be developed for C. botulinum. Transposon mutagenesis potentially could be used as an effective in vivo method for obtaining mutations that affect toxinogenesis or other important characteristics of C. botulinum. Transposons have not been reported to occur naturally in C. botulinum, but they have been found in other pathogenic clostridia. Hachler et al. found a tetracycline-resistant determinant in Clostridium difficile that had substantial DNA homology to transposon Tn916 (12). Two Tn3-like transposons, Tn4451 and Tn4452, have been isolated from Clostridium perfringens (1); although these transposons are potentially useful for genetic studies, they have not been shown to transpose in C. perfringens. Among the several known transposons, Tn916 has been successfully transferred by conjugation to a variety of gram-positive bacteria, including Clostridium acetobutylicum (2), Clostridium tetani (22), and *
MATERIALS AND METHODS Bacterial strains and media. C. botulinum 113B and Hall A were provided by H. Sugiyama, University of Wisconsin, Madison. Enterococcus faecalis DS16C1, CG110, and CG180 and Escherichia coli CG120 and CG120LT were kindly provided by D. B. Clewell, University of Michigan, Ann Arbor. Natural isolate DS16 contains Tn916 on the chromosome (9), and strain DS16C1 is a derivative of strain DS16 and contains a plasmid (pAD2) that encodes resistance to erythromycin, kanamycin, and streptomycin. Plasmidless strain CG110 has two nontandem copies of Tn916 on the chromosome with linked chromosomal markers for resistance to rifampin and fusidic acid (10). Strain CG180 contains the same chromosomal markers as strain CG110 but has Tn916 on plasmid pAM180 (10). Escherichia coli CG120 and CG120LT harbor plasmids pAM120 and pAM12OLT, respectively. pAM120 was constructed (11) by ligating EcoRI fragment F' (an EcoRI fragment containing Tn916 prepared from pAD1) into an Escherichia coli vector, pGL101, and pAM12OLT has the same structure without Tn916. These plasmids were used in this study for preparing probes for Tn916. Enterococcus faecalis and C. botulinum were maintained and grown in brain heart infusion medium (Difco Laborato-
Corresponding author. 2946
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ries, Detroit, Mich.) containing 0.5% yeast extract. Appropriate antibiotics were added to selective media at the following concentrations: tetracycline, 10 ,ug/ml; erythromycin, 10 ,ug/ml; streptomycin, 1,000 ,ug/ml; kanamycin, 10 ,ug/ml; fusidic acid, 25 ,ug/ml; and rifampin, 25 ,ug/ml. Escherichia coli strains were maintained and grown in Luria-Bertani broth (17). C. botulinum isolation agar, which contains 5% egg yolk and three antibiotics (cycloserine, trimethoprim, and sulfamethaxazole), was used as a selective medium for C. botulinum (8). A defined synthetic medium, MI medium (23), was used for detecting auxotrophs of C. botulinum. C. botulinum cultures were grown anaerobically at 37°C in Hungate tubes or on plates in anaerobic jars (BBL). Filter mating procedures. The filter mating procedure which we used was basically the same as that described for C. tetani (22). Overnight cultures of donors and recipients were diluted 1:100 in fresh medium and allowed to incubate until they reached optical densities at 660 nm of 0.8 (for Enterococcus faecalis) and 0.6 (for C. botulinum). Donors and recipients were mixed at a ratio of 1:10 (vol/vol) in a final volume of 1.1 ml. The cells were washed once with medium and pipetted onto a 25-mm cellulose nitrate membrane filter (pore size, 0.45 ,um; Whatman) as described previously (22); the membrane filters were incubated anaerobically for 18 h at 37°C unless otherwise indicated. The cells were washed from a filter with fresh medium and plated onto agar containing appropriate selective antibiotics. Control preparations which contained only the recipient were used to determine the frequency of spontaneous mutation to tetracycline resistance. The transfer frequencies were calculated by dividing the number of Tcr transconjugants by the number of recipient cells in the mating mixture determined at the end of the mating. Chromosomal and plasmid DNA isolations. The method used to isolate C. botulinum genomic DNAs was based on previously described methods (2, 3, 16). An overnight culture of C. botulinum was inoculated (3%, vol/vol) into 200 ml of brain heart infusion broth containing 0.5% yeast extract and the appropriate antibiotics, and this preparation was incubated anaerobically at 37°C for 5 to 6 h to obtain exponentially growing cells, which were easier to lyse. The cells were harvested by centrifugation at 10,000 rpm for 15 min, and the pellets were resuspended in sucrose TE buffer (6.7% sucrose, 50 mM Tris, 10 mM EDTA; pH 8.0) and subjected to a temperature of 50°C for 10 min to inactivate DNase activity. After centrifugation at 10,000 rpm for 15 min, the cells were resuspended in sucrose TE buffer. Lysozyme was added to a final concentration of 1 mg/ml, and the mixture was incubated at 37°C for 10 min. Proteinase K and sodium dodecyl sulfate were added to final concentrations of 50 ,ug/ml and 1%, respectively, and this was followed by incubation at 50°C for 20 min, at 37°C for 3 h, or until lysis was complete. Sodium perchlorate was added to a final concentration of 1 M to the viscous lysate, and the mixture was extracted three times with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). The DNA was then precipitated by adding 2 to 2.5 volumes of ethanol (-20°C). The DNA was collected by spooling it onto a glass rod and was rinsed twice with 70% ethanol before it was dried and dissolved in 3 to 5 ml of TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0). The DNA solution was treated with RNase (final concentration, 50 ,ug/ml) at 37°C for 30 min and then extracted twice with phenol-chloroform. The suspension was precipitated again with ethanol before it was dissolved in 1 to 2 ml of TE buffer. Plasmids pAM120 and
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TABLE 1. Filter mating transfer of tetracycline resistance Enterococcus faecalis
C. botulinum
donor strain
recipient strain
DS16C1 DS16C1 CG110 CG110 CG180 CG180
113B Hall A 113B Hall A 113B Hall A
Transconjugant per
recipienta (6.2 (2.8 (3.2 (3.5 (1.4 (1.7
± 1.6) x ± 2.1) x ± 0.7) x ± 1.4) x ± 0.5) x ± 1.1) x
1o-7 10-7 10-5 10-5 10-6 10-5
a Averages + standard deviations from at least three experiments.
pAM12OLT from Escherichia coli CG120 and CG120LT, respectively, were isolated by using a rapid plasmid DNA purification kit (Bio 101, Inc., La Jolla, Calif.). Restriction analysis and Southern hybridization. Restriction enzyme digestion of DNA was performed as recommended by BRL, Gaithersburg, Md. DNA fragments were fractionated by horizontal electrophoresis on a 0.5% agarose gel that was run in 0.5x Tris-borate-EDTA buffer (17) at 2 V/cm overnight. A Southern analysis was carried out as described previously (17). DNA fragments were transferred from the agarose gel to a nitrocellulose membrane filter (Schleicher & Schuell, Inc., Keene, N.H.) or a nylon membrane filter (Gelman Sciences, Ann Arbor, Mich.) by using a vacuum blotting system (Pharmacia, Piscataway, N.J.). Plasmids pAM120 and pAM12OLT were labeled with [a-32P]dCTP by using a nick translation kit obtained from BRL. Prehybridization and hybridization with 32P-labeled probe were done at 42°C in the presence of 50% formamide. The membrane filter was then washed and subjected to autoradiography as described previously (17). Screening for auxotrophic mutants. After filter mating each cell mixture was plated onto C. botulinum isolation agar containing tetracycline to select for transconjugants. Randomly selected Tcr transconjugants of C. botulinum 113B were streaked onto plates containing brain heart infusion agar supplemented with 0.5% yeast extract. After 24 h of incubation, the plates were replicated onto plates containing minimal MI agar and MI agar supplemented with 1 g of casein acid hydrolysate per liter. Clones which did not grow on MI agar plates but grew on MI agar plates containing casein acid hydrolysate were analyzed further for specific amino acid requirements. RESULTS
Frequencies of Tcr transconjugants obtained from mating Enterococcus faecalis with C. botulinum. Initially, we determined favorable conditions for filter mating between Enterococcus faecalis and C. botulinum 113B. Varying the initial ratio of donor to recipient from 1:1 to 1:20 and the incubation
time from 18 to 30 h did not affect the transfer frequency. However, the growth stage of C. botulinum used for mating influenced the frequency of transfer; intergeneric transfer of Tcr from Enterococcusfaecalis CG110 was three to six times higher when mid-exponential-phase C. botulinum cells (A66, 0.6) were used than when late-exponential-phase earlyexponential-phase, or stationary-phase cells were used. On the other hand, transfer frequency was not changed when mid- or late-exponential-phase Enterococcus faecalis cells were used. Tetracycline resistance was readily transferred to C. botulinum 113B or Hall A from Tn916-bearing Enterococcus faecalis strains (Table 1). The transfer frequency depended
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FIG. 1. Restriction enzyme and Southern analyses of Tn916 insertions in C. botulinum 113B and five Tcr transconjugants. (A) Ethidium bromide-stained 0.5% agarose gel of EcoRI-digested DNAs (Lanes 1 through 6) and HindlIl-digested DNAs (lanes 7 through 12). (B) Autoradiogram of the DNAs shown in panel A after they were transferred to a nitrocellulose filter and probed with 32P-labeled pAM120::Tn916. Lanes 1 and 7, C. botulinum 113B; lanes 2 and 8, Tcr transconjugants from a mating between strains DS16C1 and 113B; lanes 3 through 6 and corresponding lanes 9 through 12, Tcr transconjugants from a mating between strains CG110 and 113B.
mainly on the donor strain. Enterococcus faecalis DS16C1 transferred Tcr at a frequency of ca. io7 Tcr transconjugant per recipient, while strains CG110 and CG180 transferred Tcr at a frequency of around i0-5 to 10-6 transconjugant per recipient. The intergeneric transfer of Tcr from Enterococcusfaecalis to C. botulinum (Table 1) occurred at the same frequency as intraspecies transfers between Enterococcus faecalis strains (data not shown). The frequencies of Tcr transfer to C. botulinum were similar to the frequencies reported previously for the transfer of Tn9J6 among streptococci (9, 10). No spontaneous Tcr mutants of C. botulinum were detected in independent experiments involving plating of controls in which C. botulinum was incubated on filters without Enterococcus faecalis and plated onto C. botulinum isolation agar containing tetracycline. The stability of Tc' was examined by serially transferring two transconjugants in growth media lacking tetracycline. After four such transfers, the individual colonies which were isolated (-45 colonies) and subcultured on antibiotic-containing media were all resistant to tetracycline. Detection of Tn916 integration sites in C. botulinum. To assess the insertion of Tn9O6, as well as the randomness of insertion sites, chromosomal DNAs were isolated from C. botulinum 113B and five randomly selected Tcr transconjugants from filter matings and analyzed for the presence of Tn9J6 by Southern hybridization (Fig. 1). The probe which we used was pAM120, which contains an entire copy of
Tn916. To demonstrate that the hybridization occurred only with Tn916, we also used a control probe, pAM12OLT, which is identical to pAM120 except that Tn916 is deleted (11). pAM120 but not pAM12OLT hybridized to the Tcr transconjugant DNA digests (data not shown). All five transconjugant digests tested hybridized to pAM120. Independent experiments in which we used eight transconjugants from other matings also resulted in hybridization. Unlike C. difficile DNA (12), chromosomal DNA from C. botulinum 113B did not exhibit any homology to Tn916 (Fig. 1B, lanes 1 and 7). Since Tn916 lacks an EcoRI restriction site (6), each copy of Tn916 results in the appearance of a single hybridized band on autoradiograms, except in the cases of tandem duplications or closely located nontandem insertions. The sizes of the hybridized DNA fragments depend on the locations of the bordering EcoRI sites on the chromosome. Since Tn916 contains a single HindIII site (6), Southern analyses should reveal two junction fragments for each copy of the transposon in the genome. Figure 1B shows that the five Tcr transconjugants tested produced different band patterns for HindIII digests (Fig. 1B, lanes 8 through 12), indicating that a variety of insertion sites were present. EcoRI digests (Fig. 1B, lanes 2 through 6) containing fragments that were longer than 16.4 kb were not resolved by electrophoresis, and the band patterns appeared to be similar. However, the EcoRI digests provided information on
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casein acid hydrolysate per liter. These clones were studied for their specific requirements for nine nonessential amino acids. We found that two clones required alanine, one clone required serine, and one clone required lysine. The other clones required two or more amino acids and were not studied in detail.
FIG. 2. Autoradiogram chromosomal DNAs from after hybridization with through 8 contained eight obtained after mating with
of
a
Southern blot of HindlIl-digested
C. botulinum 113B Tcr transconjugants
32P-labeled pAM120::Tn9J6. Lanes 1 different Tcr transconjugants that were the donor Enterococcusfaecalis CG180.
Tn9J6 copy number. The results suggested that four Tcr transconjugants harbored two copies of Tn9J6 (Fig. iB, lanes 2 and 4 through 6), which was confirmed by the presence of four hybridized bands in HindlIl digests (lanes 8 and 10 through 12). We believe that lanes 4 and 10 each contain two overlapping bands. One of the Tcr transconjugants harbored only one copy of Tn9J6 since a single band was
observed in the EcoRI-digested DNA (Fig.
1B,
lane 3)
and two bands were observed in the HindlIl-digested DNA (lane 9). The presence of multiple insertion sites into the C. botulinum genome was further confirmed by performing Southern analyses of eight additional randomly selected Tcr transconjugants from matings between strains CG180 and 113B. The eight Tcr transconjugants which we examined produced seven band patterns when they were subjected to Southern analyses. Five transconjugants had single Tn9J6 insertions, two had two copies of Tn9J6, and one had three copies of Tn9J6 in the chromosomal DNA (Fig. 2). The results of the Southern analyses confirmed that multiple copies of Tn9J6 were inserted into the C. botulinum chromosome with different Tn9J6 donors. Our results also demonstrated that Tn9J6 inserted into different sites on the chromosome and indicated that Tn9I6 can be used to develop a mutagenesis system in C. botulinum. Auxotrophic mutants. To demonstrate the effectiveness of Tn9J6 mutagenesis in C. botulinum, 200 randomly selected Tcr transconjugants were screened for auxotrophic mutations; 20 clones did not grow or grew poorly on MI medium, but 11 of these grew on minimal medium containing 1 g of
DISCUSSION Enterococcus (Streptococcus) faecalis transposon Tn916 was readily and stably introduced into C. botulinum 113B and Hall A cells by using a filter mating procedure. The frequencies of Tcr transfer from Enterococcus faecalis DS16C1, CG110, and CG180 to C. botulinum 113B and Hall A were 10-7, 10-5, and 10-6 transconjugant per recipient, respectively, values which were comparable to those obtained with other intergeneric matings (13, 22). The transfer frequency of Tn916 depended on a number of factors, including the conditions used in the filter mating procedure, the donor strain, and the stage of growth of C. botulinum. Changing the ratio of donor to recipient had little effect on transfer frequency. The donor strains varied considerably in their capacity to transfer Tn916, probably because of the position of Tn916 in the donor chromosome. Enterococcus faecalis CG110 transferred Tn916 at a frequency that was 2 orders of magnitude higher than the frequency obtained with Enterococcusfaecalis DS16C1 (Table 1). This result is comparable to the data obtained by Gawron-Burke and Clewell (10). Since both strains have Tn916 on the chromosome, Gawron-Burke and Clewell proposed that DNA sequences near Tn916 may affect the rate at which the transposon excised (10). In our study it was interesting that Enterococcus faecalis CG110 mated intragenerically with Enterococcus faecalis recipient strain JH2SS under anaerobic conditions had the same low transfer frequency (-10-8 transconjugant per recipient) as Enterococcus faecalis DS16C1, whereas when it was mated aerobically, an increase in frequency (ca. 10-6 transconjugant per recipient) was observed. It is possible that the anaerobic conditions used during mating may have cancelled some positive factors that are present under aerobic conditions and promote transfer of strain CG110. However, the frequency of intergeneric transfer of Tn916 from Enterococcus faecalis CG110 to C. botulinum strains was not decreased by the anaerobic incubation necessary for C. botulinum. Further developments in the mating system could increase the frequency of transfer of Tn916 from Enterococcusfaecalis to C. botulinum. Sasaki et al. (18) reported that the transfer frequency of a conjugative plasmid, pAM,1, between Enterococcus faecalis and Lactobacillus plantarum was enhanced when cells were mated on the front side of a membrane containing cellulose-mixed esters in which the front side had a larger pore size than the back side (0.45 rather than 0.2 ,um). Passage of water through the membrane also increased the frequency 10-fold, probably because of increased contact between the cells. Pretreatment of Bacillus anthracis cells with naficillin was found to increase the transfer frequency 10-fold in matings between Enterococcus faecalis and B. anthracis, possibly because it facilitated penetration of the donor DNA or interaction of the cells (13). Tn916 is a conjugative transposon which moves via an excision-insertion mechanism that is mediated by a circular intermediate (5, 19). Plasmids or bacteriophages are not involved in the transfer of Tn9J6 (6). The results of Southern analyses (Fig. 1 and 2) showed that all of the Tcr transconjugants which we examined had Tn916 inserted in the C.
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botulinum chromosome at different sites. The multiple insertions of Tn916 which we detected probably resulted from continous excision from the donor since Tn9O6 itself does not self-replicate (6). It has been shown in several previous studies that the target gene of Tn916 insertion is changed, resulting in a loss of gene function (10, 11, 13, 15). The isolation of variety of auxotrophs in our study also confirmed that the use of Tn916 can be an effective mutation method in C. botulinum. ACKNOWLEDGMENTS This work was supported by a Hatch grant and by contributions from the food industry. REFERENCES 1. Abraham, L. J., and J. I. Rood. 1987. Identification of Tn4451 and Tn4452, chloramphenicol resistance transposons from Clostridium perfringens. J. Bacteriol. 169:1579-1584. 2. Bertram, J., and P. Durre. 1989. Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum. Arch. Microbiol. 151:551-557. 3. Betley, M. J., E. Somers, and B. R. DasGupta. 1989. Characterization of botulinum type A neurotoxin gene: delineation of the N-terminal encoding region. Biochem. Biophys. Res. Commun. 162:1388-1395. 4. Binz, T., H. Kurazono, M. Wille, J. Frevert, K. Wernar, and H. Niemann. 1990. The complete sequence of botulinum neurotoxin type A and comparison with other neurotoxins. J. Biol. Chem. 265:9153-9158. 5. Caparon, M., and J. R. Scott. 1989. Excision and insertion of the conjugative transposon Tn916 involves a novel recombination mechanism. Cell 59:1027-1034. 6. Clewell, D. B., and C. Gawron-Burke. 1986. Conjugative transposons and the dissemination of antibiotic resistance in streptococci. Annu. Rev. Microbiol. 40:635-659. 7. DasGupta, B. R. 1990. Structure and biological activity of botulinum neurotoxin. J. Physiol. (Paris) 84:220-228. 8. Dezfulian, M., L. M. McCroskey, C. L. Hatheway, and V. R. Dowell, Jr. 1981. Selective medium for isolation of Clostridium botulinum from human feces. J. Clin. Microbiol. 13:526-531. 9. Franke, A. E., and D. B. Clewell. 1981. Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid. J. Bacteriol. 145:494-502. 10. Gawron-Burke, C., and D. B. Clewell. 1982. A transposon in Streptococcus faecalis with fertility properties. Nature (London) 300:281-284.
APPL. ENVIRON. MICROBIOL. 11. Gawron-Burke, C., and D. B. Clewell. 1984. Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria. J. Bacteriol. 159:214-221. 12. Hachler, H., F. H. Kayser, and B. Berger-Bachi. 1987. Homology of a transferable tetracycline resistance determinant of Clostridium difficile with Streptococcus (Enterococcus)faecalis transposon Tn916. Antimicrob. Agents Chemother. 31:10331038. 13. Ivins, B. E., S. L. Welkos, G. B. Knudson, and D. J. Leblanc. 1988. Transposon Tn916 mutagenesis in Bacillus anthracis. Infect. Immun. 56:176-181. 14. Jones, J. M., S. C. Yost, and P. A. Pattee. 1987. Transfer of the conjugal tetracycline resistance transposon Tn916 from Streptococcusfaecalis to Staphylococcus aureus and identification of some insertion sites in the Staphylococcus chromosome. J. Bacteriol. 169:2121-2131. 15. Kathariou, S., P. Metz, H. Hof, and W. Goebel. 1987. Tn9J6induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J. Bacteriol. 169:1291-1297. 16. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. 17. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning, a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 18. Sasaki, Y., N. Taketomo, and T. Sasaki. 1988. Factors affecting transfer frequency of pAM,1 from Streptococcus faecalis to Lactobacillus plantarum. J. Bacteriol. 170:5939-5942. 19. Scott, J. R., P. A. Kirchman, and M. G. Caparon. 1988. An intermediate in transposition of the conjugative transposon Tn9O6. Proc. Natl. Acad. Sci. USA 85:4809-4813. 20. Smith, L. D., and H. Sugiyama. 1988. Botulism, the organism, its toxins, the disease. Charles C Thomas Publisher, Springfield, Ill. 21. Thompson, D. E., J. K. Brehm, J. D. Oultram, T.-J. Swinfield, C. C. Shone, T. Atkinson, J. Melling, and N. P. Minton. 1990. The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene. Eur. J. Biochem. 189:73-81. 22. Volk, W. A., B. Bizzini, K. R. Jones, and F. L. Macrina. 1988. Inter- and intrageneric transfer of Tn916 between Streptococcus faecalis and Clostridium tetani. Plasmid 19:225-259. 23. Whitmer, M. E., and E. A. Johnson. 1988. Development of improved defined media for Clostridium botulinum serotypes A, B, and E. Appl. Environ. Microbiol. 54:753-759. 24. Young, M., N. P. Minton, and W. L. Staudenbauer. 1989. Recent advances in the genetics of the clostridia. FEMS Microbiol. Rev. 63:301-326.
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Transposon Tn916 Mutagenesis in Clostridium botulinum WEI-JEN LIN AND ERIC A. JOHNSON* Department of Food Microbiology and Toxicology, University of Wisconsin, 1925 Willow Drive, Madison, Wisconsin 53706 Received 23 April 1991/Accepted 24 July 1991
The study of toxinogenesis and other properties in Clostridium botulinum is limited by the absence of genetic methods that enable construction of defined mutants. In this study, tetracycline-resistant transposon Tn916 in Enterococcus faecalis was conjugatively transferred in filter matings to group I Clostridium botulinum strains Hall A and 113B. The Tn916 transfer frequencies to C. botulinum ranged from 10-8 to 10-5 Tcr transconjugant per recipient depending on the donor strain. Southern blot analyses of EcoRI or HindlIl chromosomal digests extracted from randomly selected Tcr transconjugants showed that the transposon inserted at different sites in the recipient chromosome, and the copy number of Tn916 varied from one to three. Tn916 insertion gave several different auxotrophic mutants. This approach should be useful for the study of genes important in growth, survival, and toxinogenesis in C. botulinum.
several other organisms (9, 13-15). Tn916 is a 16.4-kbp tetracycline-encoded conjugative transposon that was originally detected in Enterococcus (Streptococcus) faecalis DS16 (9, 10). Tn916 belongs to a group of tetracyclineresistant determinants (tetM) that have fertility properties and may be involved in multidrug transfers in pathogenic bacteria (10). It has been suggested that Tn916 moves by an excision-insertion mechanism that involves a circular intermediate (5, 6, 11, 19). Tn9J6 can insert into recipient chromosomes at multiple sites and cause insertional inactivation of various genes (10, 11, 13, 15). In this paper, we describe the introduction of Tn916 from Enterococcus faecalis into C. botulinum strains by a filter mating procedure. We found that Tn916 stably integrated and caused mutations in several genes which resulted in auxotrophic nutritional requirements. The system which we developed should be useful for the study of botulinum toxin and other properties of C. botulinum.
Clostridium botulinum produces seven known serotypes of neurotoxins (serotypes A, B, C1, D, E, F, and G), which have specific toxicities that range from 107 to 108 mouse 50% lethal doses per mg of protein (20). Botulinum neurotoxin types A, B, and E are mainly responsible for human botulism, and types A through E are important causes of botulism in domestic and wild animals (20). The various neurotoxins have been characterized biochemically and pharmacologically (7). Recently, by cloning small fragments of the toxin genes into Escherichia coli vectors, workers in several laboratories have determined the entire nucleotide sequence of the gene encoding botulinum neurotoxin type A and a partial sequence of the gene encoding botulinum neurotoxin type E (3, 4, 21). These studies also demonstrated that the genes are chromosomally located. However, little is known of the genetics and metabolic regulation of toxinogenesis in C. botulinum. The genetic analysis of C. botulinum toxinogenesis has been hampered because of a lack of effective genetic methodologies, including a mutation system and gene transfer systems (24). Genetic studies are further limited by the National Institutes of Health Recombinant DNA Advisory Committee restrictions on cloning of toxin gene fragments larger than 1 kb and other in vitro genetic manipulations with C. botulinum. Alternative methods for genetic analyses need to be developed for C. botulinum. Transposon mutagenesis potentially could be used as an effective in vivo method for obtaining mutations that affect toxinogenesis or other important characteristics of C. botulinum. Transposons have not been reported to occur naturally in C. botulinum, but they have been found in other pathogenic clostridia. Hachler et al. found a tetracycline-resistant determinant in Clostridium difficile that had substantial DNA homology to transposon Tn916 (12). Two Tn3-like transposons, Tn4451 and Tn4452, have been isolated from Clostridium perfringens (1); although these transposons are potentially useful for genetic studies, they have not been shown to transpose in C. perfringens. Among the several known transposons, Tn916 has been successfully transferred by conjugation to a variety of gram-positive bacteria, including Clostridium acetobutylicum (2), Clostridium tetani (22), and *
MATERIALS AND METHODS Bacterial strains and media. C. botulinum 113B and Hall A were provided by H. Sugiyama, University of Wisconsin, Madison. Enterococcus faecalis DS16C1, CG110, and CG180 and Escherichia coli CG120 and CG120LT were kindly provided by D. B. Clewell, University of Michigan, Ann Arbor. Natural isolate DS16 contains Tn916 on the chromosome (9), and strain DS16C1 is a derivative of strain DS16 and contains a plasmid (pAD2) that encodes resistance to erythromycin, kanamycin, and streptomycin. Plasmidless strain CG110 has two nontandem copies of Tn916 on the chromosome with linked chromosomal markers for resistance to rifampin and fusidic acid (10). Strain CG180 contains the same chromosomal markers as strain CG110 but has Tn916 on plasmid pAM180 (10). Escherichia coli CG120 and CG120LT harbor plasmids pAM120 and pAM12OLT, respectively. pAM120 was constructed (11) by ligating EcoRI fragment F' (an EcoRI fragment containing Tn916 prepared from pAD1) into an Escherichia coli vector, pGL101, and pAM12OLT has the same structure without Tn916. These plasmids were used in this study for preparing probes for Tn916. Enterococcus faecalis and C. botulinum were maintained and grown in brain heart infusion medium (Difco Laborato-
Corresponding author. 2946
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ries, Detroit, Mich.) containing 0.5% yeast extract. Appropriate antibiotics were added to selective media at the following concentrations: tetracycline, 10 ,ug/ml; erythromycin, 10 ,ug/ml; streptomycin, 1,000 ,ug/ml; kanamycin, 10 ,ug/ml; fusidic acid, 25 ,ug/ml; and rifampin, 25 ,ug/ml. Escherichia coli strains were maintained and grown in Luria-Bertani broth (17). C. botulinum isolation agar, which contains 5% egg yolk and three antibiotics (cycloserine, trimethoprim, and sulfamethaxazole), was used as a selective medium for C. botulinum (8). A defined synthetic medium, MI medium (23), was used for detecting auxotrophs of C. botulinum. C. botulinum cultures were grown anaerobically at 37°C in Hungate tubes or on plates in anaerobic jars (BBL). Filter mating procedures. The filter mating procedure which we used was basically the same as that described for C. tetani (22). Overnight cultures of donors and recipients were diluted 1:100 in fresh medium and allowed to incubate until they reached optical densities at 660 nm of 0.8 (for Enterococcus faecalis) and 0.6 (for C. botulinum). Donors and recipients were mixed at a ratio of 1:10 (vol/vol) in a final volume of 1.1 ml. The cells were washed once with medium and pipetted onto a 25-mm cellulose nitrate membrane filter (pore size, 0.45 ,um; Whatman) as described previously (22); the membrane filters were incubated anaerobically for 18 h at 37°C unless otherwise indicated. The cells were washed from a filter with fresh medium and plated onto agar containing appropriate selective antibiotics. Control preparations which contained only the recipient were used to determine the frequency of spontaneous mutation to tetracycline resistance. The transfer frequencies were calculated by dividing the number of Tcr transconjugants by the number of recipient cells in the mating mixture determined at the end of the mating. Chromosomal and plasmid DNA isolations. The method used to isolate C. botulinum genomic DNAs was based on previously described methods (2, 3, 16). An overnight culture of C. botulinum was inoculated (3%, vol/vol) into 200 ml of brain heart infusion broth containing 0.5% yeast extract and the appropriate antibiotics, and this preparation was incubated anaerobically at 37°C for 5 to 6 h to obtain exponentially growing cells, which were easier to lyse. The cells were harvested by centrifugation at 10,000 rpm for 15 min, and the pellets were resuspended in sucrose TE buffer (6.7% sucrose, 50 mM Tris, 10 mM EDTA; pH 8.0) and subjected to a temperature of 50°C for 10 min to inactivate DNase activity. After centrifugation at 10,000 rpm for 15 min, the cells were resuspended in sucrose TE buffer. Lysozyme was added to a final concentration of 1 mg/ml, and the mixture was incubated at 37°C for 10 min. Proteinase K and sodium dodecyl sulfate were added to final concentrations of 50 ,ug/ml and 1%, respectively, and this was followed by incubation at 50°C for 20 min, at 37°C for 3 h, or until lysis was complete. Sodium perchlorate was added to a final concentration of 1 M to the viscous lysate, and the mixture was extracted three times with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). The DNA was then precipitated by adding 2 to 2.5 volumes of ethanol (-20°C). The DNA was collected by spooling it onto a glass rod and was rinsed twice with 70% ethanol before it was dried and dissolved in 3 to 5 ml of TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0). The DNA solution was treated with RNase (final concentration, 50 ,ug/ml) at 37°C for 30 min and then extracted twice with phenol-chloroform. The suspension was precipitated again with ethanol before it was dissolved in 1 to 2 ml of TE buffer. Plasmids pAM120 and
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TABLE 1. Filter mating transfer of tetracycline resistance Enterococcus faecalis
C. botulinum
donor strain
recipient strain
DS16C1 DS16C1 CG110 CG110 CG180 CG180
113B Hall A 113B Hall A 113B Hall A
Transconjugant per
recipienta (6.2 (2.8 (3.2 (3.5 (1.4 (1.7
± 1.6) x ± 2.1) x ± 0.7) x ± 1.4) x ± 0.5) x ± 1.1) x
1o-7 10-7 10-5 10-5 10-6 10-5
a Averages + standard deviations from at least three experiments.
pAM12OLT from Escherichia coli CG120 and CG120LT, respectively, were isolated by using a rapid plasmid DNA purification kit (Bio 101, Inc., La Jolla, Calif.). Restriction analysis and Southern hybridization. Restriction enzyme digestion of DNA was performed as recommended by BRL, Gaithersburg, Md. DNA fragments were fractionated by horizontal electrophoresis on a 0.5% agarose gel that was run in 0.5x Tris-borate-EDTA buffer (17) at 2 V/cm overnight. A Southern analysis was carried out as described previously (17). DNA fragments were transferred from the agarose gel to a nitrocellulose membrane filter (Schleicher & Schuell, Inc., Keene, N.H.) or a nylon membrane filter (Gelman Sciences, Ann Arbor, Mich.) by using a vacuum blotting system (Pharmacia, Piscataway, N.J.). Plasmids pAM120 and pAM12OLT were labeled with [a-32P]dCTP by using a nick translation kit obtained from BRL. Prehybridization and hybridization with 32P-labeled probe were done at 42°C in the presence of 50% formamide. The membrane filter was then washed and subjected to autoradiography as described previously (17). Screening for auxotrophic mutants. After filter mating each cell mixture was plated onto C. botulinum isolation agar containing tetracycline to select for transconjugants. Randomly selected Tcr transconjugants of C. botulinum 113B were streaked onto plates containing brain heart infusion agar supplemented with 0.5% yeast extract. After 24 h of incubation, the plates were replicated onto plates containing minimal MI agar and MI agar supplemented with 1 g of casein acid hydrolysate per liter. Clones which did not grow on MI agar plates but grew on MI agar plates containing casein acid hydrolysate were analyzed further for specific amino acid requirements. RESULTS
Frequencies of Tcr transconjugants obtained from mating Enterococcus faecalis with C. botulinum. Initially, we determined favorable conditions for filter mating between Enterococcus faecalis and C. botulinum 113B. Varying the initial ratio of donor to recipient from 1:1 to 1:20 and the incubation
time from 18 to 30 h did not affect the transfer frequency. However, the growth stage of C. botulinum used for mating influenced the frequency of transfer; intergeneric transfer of Tcr from Enterococcusfaecalis CG110 was three to six times higher when mid-exponential-phase C. botulinum cells (A66, 0.6) were used than when late-exponential-phase earlyexponential-phase, or stationary-phase cells were used. On the other hand, transfer frequency was not changed when mid- or late-exponential-phase Enterococcus faecalis cells were used. Tetracycline resistance was readily transferred to C. botulinum 113B or Hall A from Tn916-bearing Enterococcus faecalis strains (Table 1). The transfer frequency depended
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FIG. 1. Restriction enzyme and Southern analyses of Tn916 insertions in C. botulinum 113B and five Tcr transconjugants. (A) Ethidium bromide-stained 0.5% agarose gel of EcoRI-digested DNAs (Lanes 1 through 6) and HindlIl-digested DNAs (lanes 7 through 12). (B) Autoradiogram of the DNAs shown in panel A after they were transferred to a nitrocellulose filter and probed with 32P-labeled pAM120::Tn916. Lanes 1 and 7, C. botulinum 113B; lanes 2 and 8, Tcr transconjugants from a mating between strains DS16C1 and 113B; lanes 3 through 6 and corresponding lanes 9 through 12, Tcr transconjugants from a mating between strains CG110 and 113B.
mainly on the donor strain. Enterococcus faecalis DS16C1 transferred Tcr at a frequency of ca. io7 Tcr transconjugant per recipient, while strains CG110 and CG180 transferred Tcr at a frequency of around i0-5 to 10-6 transconjugant per recipient. The intergeneric transfer of Tcr from Enterococcusfaecalis to C. botulinum (Table 1) occurred at the same frequency as intraspecies transfers between Enterococcus faecalis strains (data not shown). The frequencies of Tcr transfer to C. botulinum were similar to the frequencies reported previously for the transfer of Tn9J6 among streptococci (9, 10). No spontaneous Tcr mutants of C. botulinum were detected in independent experiments involving plating of controls in which C. botulinum was incubated on filters without Enterococcus faecalis and plated onto C. botulinum isolation agar containing tetracycline. The stability of Tc' was examined by serially transferring two transconjugants in growth media lacking tetracycline. After four such transfers, the individual colonies which were isolated (-45 colonies) and subcultured on antibiotic-containing media were all resistant to tetracycline. Detection of Tn916 integration sites in C. botulinum. To assess the insertion of Tn9O6, as well as the randomness of insertion sites, chromosomal DNAs were isolated from C. botulinum 113B and five randomly selected Tcr transconjugants from filter matings and analyzed for the presence of Tn9J6 by Southern hybridization (Fig. 1). The probe which we used was pAM120, which contains an entire copy of
Tn916. To demonstrate that the hybridization occurred only with Tn916, we also used a control probe, pAM12OLT, which is identical to pAM120 except that Tn916 is deleted (11). pAM120 but not pAM12OLT hybridized to the Tcr transconjugant DNA digests (data not shown). All five transconjugant digests tested hybridized to pAM120. Independent experiments in which we used eight transconjugants from other matings also resulted in hybridization. Unlike C. difficile DNA (12), chromosomal DNA from C. botulinum 113B did not exhibit any homology to Tn916 (Fig. 1B, lanes 1 and 7). Since Tn916 lacks an EcoRI restriction site (6), each copy of Tn916 results in the appearance of a single hybridized band on autoradiograms, except in the cases of tandem duplications or closely located nontandem insertions. The sizes of the hybridized DNA fragments depend on the locations of the bordering EcoRI sites on the chromosome. Since Tn916 contains a single HindIII site (6), Southern analyses should reveal two junction fragments for each copy of the transposon in the genome. Figure 1B shows that the five Tcr transconjugants tested produced different band patterns for HindIII digests (Fig. 1B, lanes 8 through 12), indicating that a variety of insertion sites were present. EcoRI digests (Fig. 1B, lanes 2 through 6) containing fragments that were longer than 16.4 kb were not resolved by electrophoresis, and the band patterns appeared to be similar. However, the EcoRI digests provided information on
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casein acid hydrolysate per liter. These clones were studied for their specific requirements for nine nonessential amino acids. We found that two clones required alanine, one clone required serine, and one clone required lysine. The other clones required two or more amino acids and were not studied in detail.
FIG. 2. Autoradiogram chromosomal DNAs from after hybridization with through 8 contained eight obtained after mating with
of
a
Southern blot of HindlIl-digested
C. botulinum 113B Tcr transconjugants
32P-labeled pAM120::Tn9J6. Lanes 1 different Tcr transconjugants that were the donor Enterococcusfaecalis CG180.
Tn9J6 copy number. The results suggested that four Tcr transconjugants harbored two copies of Tn9J6 (Fig. iB, lanes 2 and 4 through 6), which was confirmed by the presence of four hybridized bands in HindlIl digests (lanes 8 and 10 through 12). We believe that lanes 4 and 10 each contain two overlapping bands. One of the Tcr transconjugants harbored only one copy of Tn9J6 since a single band was
observed in the EcoRI-digested DNA (Fig.
1B,
lane 3)
and two bands were observed in the HindlIl-digested DNA (lane 9). The presence of multiple insertion sites into the C. botulinum genome was further confirmed by performing Southern analyses of eight additional randomly selected Tcr transconjugants from matings between strains CG180 and 113B. The eight Tcr transconjugants which we examined produced seven band patterns when they were subjected to Southern analyses. Five transconjugants had single Tn9J6 insertions, two had two copies of Tn9J6, and one had three copies of Tn9J6 in the chromosomal DNA (Fig. 2). The results of the Southern analyses confirmed that multiple copies of Tn9J6 were inserted into the C. botulinum chromosome with different Tn9J6 donors. Our results also demonstrated that Tn9J6 inserted into different sites on the chromosome and indicated that Tn9I6 can be used to develop a mutagenesis system in C. botulinum. Auxotrophic mutants. To demonstrate the effectiveness of Tn9J6 mutagenesis in C. botulinum, 200 randomly selected Tcr transconjugants were screened for auxotrophic mutations; 20 clones did not grow or grew poorly on MI medium, but 11 of these grew on minimal medium containing 1 g of
DISCUSSION Enterococcus (Streptococcus) faecalis transposon Tn916 was readily and stably introduced into C. botulinum 113B and Hall A cells by using a filter mating procedure. The frequencies of Tcr transfer from Enterococcus faecalis DS16C1, CG110, and CG180 to C. botulinum 113B and Hall A were 10-7, 10-5, and 10-6 transconjugant per recipient, respectively, values which were comparable to those obtained with other intergeneric matings (13, 22). The transfer frequency of Tn916 depended on a number of factors, including the conditions used in the filter mating procedure, the donor strain, and the stage of growth of C. botulinum. Changing the ratio of donor to recipient had little effect on transfer frequency. The donor strains varied considerably in their capacity to transfer Tn916, probably because of the position of Tn916 in the donor chromosome. Enterococcus faecalis CG110 transferred Tn916 at a frequency that was 2 orders of magnitude higher than the frequency obtained with Enterococcusfaecalis DS16C1 (Table 1). This result is comparable to the data obtained by Gawron-Burke and Clewell (10). Since both strains have Tn916 on the chromosome, Gawron-Burke and Clewell proposed that DNA sequences near Tn916 may affect the rate at which the transposon excised (10). In our study it was interesting that Enterococcus faecalis CG110 mated intragenerically with Enterococcus faecalis recipient strain JH2SS under anaerobic conditions had the same low transfer frequency (-10-8 transconjugant per recipient) as Enterococcus faecalis DS16C1, whereas when it was mated aerobically, an increase in frequency (ca. 10-6 transconjugant per recipient) was observed. It is possible that the anaerobic conditions used during mating may have cancelled some positive factors that are present under aerobic conditions and promote transfer of strain CG110. However, the frequency of intergeneric transfer of Tn916 from Enterococcus faecalis CG110 to C. botulinum strains was not decreased by the anaerobic incubation necessary for C. botulinum. Further developments in the mating system could increase the frequency of transfer of Tn916 from Enterococcusfaecalis to C. botulinum. Sasaki et al. (18) reported that the transfer frequency of a conjugative plasmid, pAM,1, between Enterococcus faecalis and Lactobacillus plantarum was enhanced when cells were mated on the front side of a membrane containing cellulose-mixed esters in which the front side had a larger pore size than the back side (0.45 rather than 0.2 ,um). Passage of water through the membrane also increased the frequency 10-fold, probably because of increased contact between the cells. Pretreatment of Bacillus anthracis cells with naficillin was found to increase the transfer frequency 10-fold in matings between Enterococcus faecalis and B. anthracis, possibly because it facilitated penetration of the donor DNA or interaction of the cells (13). Tn916 is a conjugative transposon which moves via an excision-insertion mechanism that is mediated by a circular intermediate (5, 19). Plasmids or bacteriophages are not involved in the transfer of Tn9J6 (6). The results of Southern analyses (Fig. 1 and 2) showed that all of the Tcr transconjugants which we examined had Tn916 inserted in the C.
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botulinum chromosome at different sites. The multiple insertions of Tn916 which we detected probably resulted from continous excision from the donor since Tn9O6 itself does not self-replicate (6). It has been shown in several previous studies that the target gene of Tn916 insertion is changed, resulting in a loss of gene function (10, 11, 13, 15). The isolation of variety of auxotrophs in our study also confirmed that the use of Tn916 can be an effective mutation method in C. botulinum. ACKNOWLEDGMENTS This work was supported by a Hatch grant and by contributions from the food industry. REFERENCES 1. Abraham, L. J., and J. I. Rood. 1987. Identification of Tn4451 and Tn4452, chloramphenicol resistance transposons from Clostridium perfringens. J. Bacteriol. 169:1579-1584. 2. Bertram, J., and P. Durre. 1989. Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum. Arch. Microbiol. 151:551-557. 3. Betley, M. J., E. Somers, and B. R. DasGupta. 1989. Characterization of botulinum type A neurotoxin gene: delineation of the N-terminal encoding region. Biochem. Biophys. Res. Commun. 162:1388-1395. 4. Binz, T., H. Kurazono, M. Wille, J. Frevert, K. Wernar, and H. Niemann. 1990. The complete sequence of botulinum neurotoxin type A and comparison with other neurotoxins. J. Biol. Chem. 265:9153-9158. 5. Caparon, M., and J. R. Scott. 1989. Excision and insertion of the conjugative transposon Tn916 involves a novel recombination mechanism. Cell 59:1027-1034. 6. Clewell, D. B., and C. Gawron-Burke. 1986. Conjugative transposons and the dissemination of antibiotic resistance in streptococci. Annu. Rev. Microbiol. 40:635-659. 7. DasGupta, B. R. 1990. Structure and biological activity of botulinum neurotoxin. J. Physiol. (Paris) 84:220-228. 8. Dezfulian, M., L. M. McCroskey, C. L. Hatheway, and V. R. Dowell, Jr. 1981. Selective medium for isolation of Clostridium botulinum from human feces. J. Clin. Microbiol. 13:526-531. 9. Franke, A. E., and D. B. Clewell. 1981. Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid. J. Bacteriol. 145:494-502. 10. Gawron-Burke, C., and D. B. Clewell. 1982. A transposon in Streptococcus faecalis with fertility properties. Nature (London) 300:281-284.
APPL. ENVIRON. MICROBIOL. 11. Gawron-Burke, C., and D. B. Clewell. 1984. Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria. J. Bacteriol. 159:214-221. 12. Hachler, H., F. H. Kayser, and B. Berger-Bachi. 1987. Homology of a transferable tetracycline resistance determinant of Clostridium difficile with Streptococcus (Enterococcus)faecalis transposon Tn916. Antimicrob. Agents Chemother. 31:10331038. 13. Ivins, B. E., S. L. Welkos, G. B. Knudson, and D. J. Leblanc. 1988. Transposon Tn916 mutagenesis in Bacillus anthracis. Infect. Immun. 56:176-181. 14. Jones, J. M., S. C. Yost, and P. A. Pattee. 1987. Transfer of the conjugal tetracycline resistance transposon Tn916 from Streptococcusfaecalis to Staphylococcus aureus and identification of some insertion sites in the Staphylococcus chromosome. J. Bacteriol. 169:2121-2131. 15. Kathariou, S., P. Metz, H. Hof, and W. Goebel. 1987. Tn9J6induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J. Bacteriol. 169:1291-1297. 16. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. 17. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning, a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 18. Sasaki, Y., N. Taketomo, and T. Sasaki. 1988. Factors affecting transfer frequency of pAM,1 from Streptococcus faecalis to Lactobacillus plantarum. J. Bacteriol. 170:5939-5942. 19. Scott, J. R., P. A. Kirchman, and M. G. Caparon. 1988. An intermediate in transposition of the conjugative transposon Tn9O6. Proc. Natl. Acad. Sci. USA 85:4809-4813. 20. Smith, L. D., and H. Sugiyama. 1988. Botulism, the organism, its toxins, the disease. Charles C Thomas Publisher, Springfield, Ill. 21. Thompson, D. E., J. K. Brehm, J. D. Oultram, T.-J. Swinfield, C. C. Shone, T. Atkinson, J. Melling, and N. P. Minton. 1990. The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene. Eur. J. Biochem. 189:73-81. 22. Volk, W. A., B. Bizzini, K. R. Jones, and F. L. Macrina. 1988. Inter- and intrageneric transfer of Tn916 between Streptococcus faecalis and Clostridium tetani. Plasmid 19:225-259. 23. Whitmer, M. E., and E. A. Johnson. 1988. Development of improved defined media for Clostridium botulinum serotypes A, B, and E. Appl. Environ. Microbiol. 54:753-759. 24. Young, M., N. P. Minton, and W. L. Staudenbauer. 1989. Recent advances in the genetics of the clostridia. FEMS Microbiol. Rev. 63:301-326.
