INFECTION AND IMMUNITY, Nov. 1991, p. 4097-4102 0019-9567/91/114097-06$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 59, No. 11
Insertion of Tn916 in Neisseria meningitidis Resulting in Loss of Group B Capsular Polysaccharide DAVID S. STEPHENS,l.2.3* JOHN S. SWARTLEY,2'3 SOPHIA KATHARIOU,4t AND STEPHEN A. MORSE4 Departments of Medicine1 and Microbiology and Immunology,2 Emory University School of Medicine, Atlanta, Georgia 30303, Laboratories of Microbial Pathogenesis, VA Medical Center, Atlanta, Georgia 30033,3 and Division of Sexually Transmitted Diseases Laboratory Research, Centers for Disease Control, Atlanta, Georgia 303334 Received 20 May 1991/Accepted 29 August 1991
We recently found that the 16.4-kb conjugative transposon Tn916 could be introduced into Neisseria meningitidis by transformation and that it appeared to transpose to many different sites in the chromosome of recipient meningococci. In order to identify transposon-induced alterations of specific meningococcal virulence determinants, a library of meningococcal Tetr transformants containing Tn916 was made and screened for those altered in the production of group B capsular polysaccharide. A capsule-defective mutant, M7, was identified by using monoclonal and polyclonal antisera to group B polysaccharide in immunoblot and agar antiserum procedures. Growth of M7 was similar to that of the parent strain. M7 produced no group B capsular polysaccharide by rocket immunoelectrophoresis, and the mutation was stable during laboratory passage. The capsule-defective phenotype was linked to Tetr, as demonstrated by immunoblot and Southern blot analysis of progeny Tetr transformants (transformants of the parent strain obtained with DNA from M7). A capsule-deficient mutant, 08, was identified by using a similar approach. Analysis of the Tn916 insertions in M7 and 08 indicated that a significant portion of the transposon on either side of the tetM determinant had been lost. The ability of Tn916 to generate defined, stable mutations in meningococcal virulence determinants is demonstrated by our study.
The pathogenesis of Neisseria meningitidis infections is poorly understood at the genetic level. This problem is due, in part, to the lack of a system for genetic analysis of the meningococcus (36). Recently, we found that the 16.4-kb conjugative transposon Tn916 could be introduced into N. meningitidis by transformation using the suicide vectors pAM120 and pAM170 (17). After being introduced, Tn916 appeared to transpose as a single copy to many different sites in the chromosome of recipient meningococci and was stably integrated. These results suggested that Tn916 could be used as a genetic tool for the study of N. meningitidis. Demonstration that Tn916 mutagenesis can inactivate specific meningococcal virulence determinants is crucial to the development of this system. In this report, we describe the creation of a library of meningococcal transformants containing Tn9O6, the screening of this library for transformants altered in the production of group B capsular polysaccharide, and the characterization of capsule-defective and capsule-deficient Tn9O6 meningococcal transformants. MATERIALS AND METHODS Bacterial strains and growth media. Strain NMB (CDC 8201085) is a group B meningococcal strain that was isolated in Pennsylvania in 1982 from the cerebrospinal fluid of a patient with meningitis. Additional relevant phenotypic characteristics of this strain have been previously described (17). Strain 269BSpecr is a spontaneous spectinomycinresistant derivation of group B meningococcal strain 269B (17, 32). Escherichia coli strains CG120 and CG170, containing the plasmids pAM120 and pAM170, respectively, were Corresponding author. t Present address: Department of Microbiology, University of Hawaii, Honolulu, HI 96822. *
obtained from D. B. Clewell (University of Michigan, Ann Arbor, Mich.). pAM120 and pAM170 were derived from pGL101 into which Tn9O6 was cloned (13). Strains of N. meningitidis were grown under aerobic conditions on GC base agar (Difco, Detroit, Mich.) supplemented with 1% (vol/vol) IsoVitaleX enrichment (BBL, Cockeysville, Md.) (GcIso agar) at 37°C in an atmosphere containing 3% CO2. Meningococci were also grown in either GC broth (24) with 1% IsoVitaleX or in a chemically defined medium (24). E. coli was grown in Luria-Bertani broth (28) at 37°C with aeration. Tetracycline (Sigma Chemical Co., St. Louis, Mo.) was added at a final concentration of 5 ,ug/ml for N. meningitidis (17) and 4 pug/ml for E. coli (13). Meningococcal transformants arising from pAM120 or pAM170 were confirmed as resistant at a concentration of 20 ,ug of tetracycline per ml. Spectinomycin (Sigma) was added at a final concentration of 100 ,ug/ml. Meningococcal strains and transformants of strain NMB were stored at -70°C in Trypticase soy broth and 20% glycerol. Chromosomal DNA preparations. Chromosomal DNA was prepared by a modification of the method of Dowson et al. (9). Confluent growth from a single Gclso agar plate was resuspended in 2 ml of 10 mM Tris-HCl containing 10 mM EDTA (pH 7.5). Two microliters of lysozyme (10 mg/ml) (Sigma) was then added, and the solution was gently mixed and then incubated at room temperature for 10 min. Next, 2 ml of 50 mM Tris-HCl containing 10 mM EDTA and 2% (vol/vol) Triton X-100, pH 8.0, was added, and the resultant mixture was incubated for 10 min at room temperature. Two microliters of RNase A (10 mg/ml, boiled) (Sigma) was then added, and the preparation was incubated at 37°C for 30 min. Two microliters of proteinase K (10 mg/ml) (Sigma) was then added, and the mixture was incubated at room temperature for 30 min. At this stage, proteins were removed from the viscous lysate by extracting once with Tris-EDTA (TE)4097
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saturated phenol, twice with TE-saturated phenol-chloroform, and twice with TE-saturated chloroform. The nucleic acids were then precipitated with ethanol, resuspended in
sterile distilled water, and stored at 4°C. The concentration of DNA was determined spectrophotometrically at a wavelength of 260 nm. Transformation. Plasmid preparations of pAM120 and pAM170 used in these experiments were obtained from E. coli strains CG120 and CG170, either as described by Birnboim and Doly (2) or in large-scale preparations (28). Isolated colonies of meningococci, grown overnight on GC base agar, were used as recipients for genetic transformation. Competence of N. meningitidis was confirmed by transforming strain NMB to spectinomycin resistance with DNA from 269BSpecr. The semiquantitative transformation assay of Janik et al. (16) was used as previously described (17). Southern blots. Restriction enzymes were used according to the instructions of the supplier (Bethesda Research Laboratories, Gaithersburg, Md.). Plasmid pAM120 (13) was 32P-labeled by nick translation (28). Alternatively, the random priming method of Feinberg and Vogelstein (10) was used for radiolabeling pAM120. The probe was stored at -20°C until it was ready to use. Restriction endonucleasedigested DNA was electrophoresed on 0.4 to 0.8% agarose gels in Tris-acetate EDTA buffer (28). Transfer of DNA to nitrocellulose (Schleicher and Schuell, Keene, N.H.) in 20x SSC (3.0 M NaCl-0.3 M sodium citrate, pH 7.0) was performed as described by Southern (31). The filters were prehybridized in a rotating water bath at 42°C for at least 4 h in a solution containing the following: 50% formamide (deionized), 5 x Denhardt's solution (28), 5 x SSC, 0.1% sodium dodecyl sulfate (SDS), 100 ,ug of freshly denatured calf thymus DNA per ml, and 200 ,ug of yeast tRNA per ml. At the end of this incubation, an amount of freshly denatured probe (boiled 3 to 4 min) corresponding to -1 x 107 cpm was added directly to the prehybridization mix. The hybridizing filter was then allowed to incubate in the rotating water bath at 42°C overnight. The hybridized filters were washed five times at 42°C in 0.2x SSC and 0.1% SDS. After the washes, autoradiographs were obtained by exposing Kodak X-Omat film (Eastman Kodak Co., Rochester, N.Y.) to the filters for 6 to 72 h at -70°C with
intensifying screens. Colony immunoblot screening assays. Three microliters of suspension from frozen stocks of strain NMB and of tetracycline-resistant transformants of NMB was placed as drops on GcIso agar without and with tetracycline, respectively, and grown overnight. Individual colonies were transferred to nitrocellulose discs (Schleicher and Schuell), placed on agar, and left overnight at 4°C. The discs were then placed in -50 ml of Tris-Tween blocking buffer (0.01 M Tris, 0.15 M NaCl
[pH 8.0], 0.05% [vol/vol] Tween 20 [Sigma], 3% [vol/vol] serum albumin) and gently agitated on a rotating platform (Tekpro; American Hospital Supply) for 60 min. The blocking buffer was removed and replaced with the antibody solution (1:500 dilution of antibody in blocking buffer), and the disc was agitated for 60 min. Murine immunoglobulin M (IgM) monoclonal antibody 2-2-B (37) (kindly supplied by Wendell Zollinger, Walter Reed Army Institute of Research) or polyclonal (horse) antiserum (kindly supplied by Carl Frasch, Bureau of Biologics), both specific for group B capsular polysaccharide, were the bovine
antibodies used. The nitrocellulose disc was then washed three times for 10 min with Tris-Tween buffer without bovine serum albumin. Binding of IgM monoclonal antibody 2-2-B was detected with a goat anti-mouse IgM-IgG antibody
INFECT. IMMUN.
conjugated to alkaline phosphatase (28). A rabbit anti-horse IgG antibody conjugated to alkaline phosphatase was used to detect polyclonal horse antiserum. After being incubated with the second antibody for 1 h, the filter was washed three times for 10 min with Tris-Tween buffer, pH 8.0. NBT-BCIP substrate (0.025 g of Nitro Blue Tetrazolium [NBT; Sigma] in 250 pAl of N,N-dimethylformamide [DMF] and 250 >1l of ethanol; 0.025 g of 5-bromo-4-chloro-3-indolyl phosphate [BCIP; US Biochemical] in 150 RI of DMF and 350 RIl of ethanol; 198 RI of the NBT solution and 99 pAl of the BCIP solution were then added to 30 ml of alkaline phosphatase buffer [100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2]) was added for 5 to 15 min. Antiserum agar technique. Meningococcal colonies were examined after growth on antiserum agar plates, as described by Craven et al. (7) and Kuzemenska et al. (21). Three microliters of the suspension of frozen stock of strain NMB and frozen stock of selected transformants was grown from drops on Gclso agar containing the polyclonal (horse) antiserum to group B capsular polysaccharide. A 1:10 dilution of antiserum to agar was used. After 18 h of growth, the isolated colonies were examined with a binocular dissecting microscope (Bausch and Lomb, St. Louis, Mo.). Precipitin bands were detected with incident light. In some experiments, the surrounding precipitin band was highlighted by staining the colonies with 0.1% Coomassie blue. Isolation of meningococcal capsular polysaccharide and rocket immunoelectrophoresis. Group B meningococcal capsular polysaccharide was extracted from both the tetracycline-resistant transformants and parent strain NMB with Cetavlon (hexadecyltrimethylammonium bromide). Parent or transformant cells were grown in 200 ml of Gc broth plus 1% IsoVitaleX to a reading of 100 Klett units (4 to 5 h). Cetavlon was added (final concentration of 0.1%), and the capsular polysaccharide was extracted for 30 min at room temperature with slow shaking. The suspension was centrifuged at 10,000 x g for 20 min at 4°C. Three volumes of cold ethanol were added to the supernatant, and the mixture was stirred at 4°C for 10 min and placed on ice for 1 h. After centrifugation at 10,000 x g for 20 min at 4°C, the pellet was extracted twice with phenol and resuspended in 1 ml H20. Rocket immunoelectrophoresis using 1% SeaKem agarose gels (SeaKem LE Agarose; FMC) was carried out as described by Andrews et al. (1). Three percent polyclonal group B antiserum (see above) was added to the agarose gels and stabilized with 2% polyethylene glycol 8000. Electrophoresis was performed at 80 V for 2.5 h at room temperature. The gels were pressed onto a glass plate soaked in water, dried with a portable hair dryer, stained with 0.25% Coomassie brilliant blue R250, and destained in methanolacetic acid in water. Peak heights were measured on a dry surface of the glass plate (1). Biosafety. These studies were approved by the Emory University Biosafety Committee and the CDC Office of Biosafety and were conducted by utilizing laboratory practices, safety equipment, and facilities recommended for Biosafety Level 2. RESULTS Library of Tn916 transformants. Transformation of the group B meningococcal strain NMB was performed with 1 ,g of the plasmid vector pAM120 or pAM170. We have previously shown (17, 34) that only Tn916 integrated into the meningococcal genome following transformation with pAM120 or pAM170 and that the location of the transposon
VOL. 59, 1991
Tn916-GENERATED MENINGOCOCCAL CAPSULE MUTANTS
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FIG. 1. (A) Immunoblot of Tn916-derived Tetr transformants of meningococcal strain NMB probed with an IgM monoclonal antibody specific for group B capsular polysaccharide. The capsule-deficient mutant M7 is indicated with an asterisk. (B) Growth of M7 (*) on the Gclso agar plate used for the immunoblot of panel A. Growth-deficient transformants (A) were also noted.
in the genome was variable. Tetracycline-resistant transformants were obtained at a frequency of 1.6 x 10-6 to 1 x 10-8 per recipient. Differences from experiment to experiment in the amount of the closed circular form of the transposon or changes in competence factors (34) may account for the range in frequency. These frequencies yielded 2 to -100 transformants per transformation in our assay. Multiple transformations were performed to decrease sibling selection. Over 1,000 Tetr transformants from >150 transformations were selected, passed once on GcIso agar with tetracycline, and stored at -70°C. We have previously shown that the tetracycline resistance of the transformants is stable during laboratory passage (17). Identification of Tn916 transformants altered in the production of group B capsular polysaccharide. Initial screening was performed by using colony immunoblots (Fig. 1). Five hundred and sixty-two individual transformants from the library were probed with IgM monoclonal antibody 2-2-B. This monoclonal antibody is specific for meningococcal group B capsular polysaccharide (37). Transformants which differed from the parent strain in reactivity with the anticapsular monoclonal antibody were identified. The differences were confirmed by repeating the assay three or more times. Transformants that were obviously growth deficient on agar were excluded. By using this approach, two putative capsular polysaccharide overproducers and six putative capsular polysaccharide-deficient transformants were identified. Two of the six capsular polysaccharide-deficient phenotypes did not react with monoclonal antibody 2-2-B. In the other four, reactivity was reduced compared with that of the parent strain. It was postulated that the capsule-deficient mutants might be altered in the amount of capsular polysaccharide produced or altered in the expression of the 2-2-B epitope. To distinguish between these possibilities, the mutants were also studied by using polyclonal group B meningococcal capsular antiserum in an agar antiserum procedure (7, 21). Capsular polysaccharide was identified as a precipitin halo
surrounding colonies of parent strain NMB. No precipitin halo was noted in the M7 mutant or in a second capsuledeficient mutant, 08. Colonies of M7 and 08 exhibited a rough or cracked colony phenotype and were opaque (Op+ +) when examined by incident light (33). Similar findings have previously been reported for spontaneous mutants of group B meningococci incapable of producing capsular polysaccharide (23). Growth of M7 and 08 in a chemically defined medium did not differ significantly from growth of randomly selected transformants or from growth of parent strain NMB. Similar results were also obtained with Gclso broth. Capsular polysaccharide production. To determine the amount of capsular polysaccharide produced by the mutants, capsular polysaccharide was extracted with Cetavlon from parent strain NMB, from mutants M7 and 08, and from other transformants arising from pAM120. Rocket immunoelectrophoresis, using polyclonal antiserum to group B capsule, was performed (Fig. 2). No group B capsular polysaccharide from the M7 mutant was detected by rocket immunoelectrophoresis. The 08 mutant produced a small amount of capsular material. Tn916-containing transformants, which had normal capsule expression in our immunologic screening assays, produced amounts of capsular polysaccharide similar to those produced by parent strain NMB. Thus, the M7 and 08 mutants were defective in the expression of group B capsular polysaccharide. Linkage of capsule-deficient phenotype and tetracycline resistance. To study the possibility that M7 or 08 mutants were spontaneous capsule-deficient mutants (8, 23), linkage of the phenotype and tetracycline resistance was tested by transforming parent strain NMB with DNA from M7. Tetracycline-resistant transformants were obtained at a frequency of 10-3 to 10-4 per recipient. The tetracycline-resistant transformants (>1,000) obtained with M7 or 08 DNA were identical to the parent M7 or 08 mutants in the lack of
4100 1
INFECT. IMMUN.
STEPHENS ET AL. 2
3 4
5
6
7
8
9
10 11 12
13
a
b 2
1
2
3
4
4
_
4
I 4
Ah
,. 5
.4 kb
w
1
FIG. 2. Rocket immunoelectrophoresis of capsular polysaccharide extracted with Cetavlon from meningococcal strain NMB and Tn916-derived mutants of NMB. Lanes: 1, 2, 12, and 13, blank controls; 5 and 9, capsule-deficient mutants M7 and 08, respectively; 7, parent strain NMB; 3, 4, 6, 8, 10, and 11, other Tn916derived mutants that do not differ from the parent strain in the amount of capsule expressed.
reaction with the monoclonal or polyclonal antisera to group B capsular polysaccharide on colony immunoblots. Southern hybridization also demonstrated the linkage and stability of the M7 mutation. As shown in Fig. 3, the original M7 mutant and three of the progeny Tetr transformants (NMB as a recipient of M7 chromosomal DNA) had identical profiles on Southern blots probed with pAM120. The high frequency of transformation to Tetr (10-3 to 10-4), the identical immunoblot profiles with both monoclonal and polyclonal antisera, and the identical Southern blot profiles indicated that the M7 and 08 mutants were stable and transferred at high frequency, presumably by homologous recombination. Analysis of Tn916 insertion in M7 and 08 mutants. Southern blot data indicated that Tn916 had inserted as a single copy in the M7 and 08 mutants. However, in contrast to the majority of Tetr transformants previously reported (17), Tn916 in M7 and 08 (Fig. 4) has undergone a major alteration. 1
2
3 4 5 6
4
6S
ob
4
2 4 1_
FIG. 4. Autoradiograms of Southern blot hybridizations of chromosomal DNA digested with HaeIII or Sau3A and probed with 32P-labeled pAM120. (a) Agarose gel (0.6%). Lanes: 1, HaeIII digest of NMB DNA; 2, HaeIII digest of M7 DNA; 3, 1-kb ladder. (b) Agarose gel (0.85%). Lanes: 1, 1-kb ladder; 2, Sau3A digest of 08 DNA; 3, Sau3A digest of 120A1 DNA.
Eight fragments are predicted to result from a Sau3A digest of Tn916 (6) (Fig. 5), and they are present in a pAM120generated transformant of NMB, 120A1, containing the entire transposon (Fig. 4b). In M7, a single Sau3A site was present within the Tn916 insert, resulting in 2.4- and 3.5-kb hybridizing fragments (data not shown). A single Sau3A site was also present within the Tn916 insert of 08 (Fig. 4b), although the sizes of the hybridizing fragments, 1.8 and 3.1 kb, were different from those of M7. We postulated that this was the Sau3A site within the tetM determinant (6), which suggested that a significant portion of the transposon on either side of tetM had been deleted in the M7 and 08 mutants. Results obtained by other Southern blot data using BstXI (Fig. 3) or HaeIII (Fig. 4a) support this hypothesis. If Tn916 were intact in M7, two hybridizing bands would be seen with BstXI-digested DNA (one at -4 kb and the other at >12.5 kb), and a single band of no less than 16.4 kb would be seen Tn 916
C
x
12
|1
^
kb6
Sa
0
Sa
Sa
Sa..Sa
Sa. Sa e
-L tel M
5
Tn 916 In M7 capsule defective mutant Sa
-om tetM
FIG. 3. Autoradiogram of Southern blot hybridization (0.6% agarose gel) of chromosomal DNA digested with BstXI and probed with 32P-labeled pAM120. Lanes: 1, radiolabeled 1-kb ladder; 2, NMB chromosomal DNA; 3, M7 chromosomal DNA; 4 to 6, Tetr transformants obtained by transformation of NMB with M7 DNA.
Sa
=
Sau 3A site
kb FIG. 5. Schematic representation of Tn916 (6) and of Tn9J6 in the capsule-defective meningococcal mutant M7.
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Tn916-GENERATED MENINGOCOCCAL CAPSULE MUTANTS
with HaeIII-digested DNA, since there is no HaeIII site in the Tn916 sequence (6). The results are quite different. In the BstXI digest, there was a single band of approximately 10.5 kb (Fig. 3), and in the HaeIII digest there was a single band of 3.8 kb (Fig. 4a). These results, combined with the Sau3AI restriction data, demonstrate that there has been a major deletion of Tn916 in the M7 mutant (Fig. 5). The data suggest that much of the transposon, with the exception of the tetM determinant, has been lost. Since there is no longer a BstXI site, the upstream genes, responsible for the excision and integration of the transposon (27), are presumably missing, further stabilizing the insert. DISCUSSION Tn916 is a large, 16.4-kb transposon (5, 6, 13-15, 30) which contains the antibiotic resistance determinant tetM (22). Excision and insertion of Tn916 involves a novel recombination mechanism (4, 27, 29). Tn916 and the other transposons of this family (3) have been used to study the genes of grampositive bacteria. Recently, transposition of these conjugative transposons was also shown to occur in E. coli and Haemophilus influenzae (13, 18). In addition, the tetM determinant of Tn916 has been found and is expressed in clinical isolates of N. meningitidis, Neisseria gonorrhoeae, and commensal Neisseria spp. (19). This event appears to have occurred by the insertion of tetM into the 24.5-MDa conjugative plasmid present in N. gonorrhoeae. Subsequently, this plasmid has been transmitted in vivo to other Neisseria species (20, 25). In commensal Neisseria spp., the tetM determinant appears to have inserted into the chromosome. We investigated the possibility that conjugative transposons of the Tn916 class, which carry tetM, might be used to develop a system for generating random mutations in the meningococcal chromosome (17, 34). We have found that Tn916 can be introduced into N. meningitidis by transforming it with plasmids pAM120 and pAM170, which contain a functional copy of Tn916. This transposon delivery system was selected on the basis of the inability of the vector plasmid to replicate in pathogenic Neisseria species. Tn916 inserted at a frequency of ca. 10-7 per recipient CFU. The presence of single copies of Tn916 in most transformants and the finding that the location of the transposon was variable suggested that Tn916 could be used as a genetic tool for the study of N. meningitidis. Recently, Nassif et al. (26) have shown that a derivative of TnJS45, a closely related conjugative transposon, can also generate variable insertions in N. meningitidis and N. gonorrhoeae. Whether Tn916 insertion in the meningococcal chromosome is a completely random event remains to be determined, but analysis of some 40 transformants by Southern hybridization (17, 31a) does not provide evidence for "hot spots" of insertion. Tn916 is stably incorporated in the meningococcal chromosome. Tn916-carrying transformants retained resistance to tetracycline during laboratory passage and upon overnight growth on medium without tetracycline (reversion rate,
Vol. 59, No. 11
Insertion of Tn916 in Neisseria meningitidis Resulting in Loss of Group B Capsular Polysaccharide DAVID S. STEPHENS,l.2.3* JOHN S. SWARTLEY,2'3 SOPHIA KATHARIOU,4t AND STEPHEN A. MORSE4 Departments of Medicine1 and Microbiology and Immunology,2 Emory University School of Medicine, Atlanta, Georgia 30303, Laboratories of Microbial Pathogenesis, VA Medical Center, Atlanta, Georgia 30033,3 and Division of Sexually Transmitted Diseases Laboratory Research, Centers for Disease Control, Atlanta, Georgia 303334 Received 20 May 1991/Accepted 29 August 1991
We recently found that the 16.4-kb conjugative transposon Tn916 could be introduced into Neisseria meningitidis by transformation and that it appeared to transpose to many different sites in the chromosome of recipient meningococci. In order to identify transposon-induced alterations of specific meningococcal virulence determinants, a library of meningococcal Tetr transformants containing Tn916 was made and screened for those altered in the production of group B capsular polysaccharide. A capsule-defective mutant, M7, was identified by using monoclonal and polyclonal antisera to group B polysaccharide in immunoblot and agar antiserum procedures. Growth of M7 was similar to that of the parent strain. M7 produced no group B capsular polysaccharide by rocket immunoelectrophoresis, and the mutation was stable during laboratory passage. The capsule-defective phenotype was linked to Tetr, as demonstrated by immunoblot and Southern blot analysis of progeny Tetr transformants (transformants of the parent strain obtained with DNA from M7). A capsule-deficient mutant, 08, was identified by using a similar approach. Analysis of the Tn916 insertions in M7 and 08 indicated that a significant portion of the transposon on either side of the tetM determinant had been lost. The ability of Tn916 to generate defined, stable mutations in meningococcal virulence determinants is demonstrated by our study.
The pathogenesis of Neisseria meningitidis infections is poorly understood at the genetic level. This problem is due, in part, to the lack of a system for genetic analysis of the meningococcus (36). Recently, we found that the 16.4-kb conjugative transposon Tn916 could be introduced into N. meningitidis by transformation using the suicide vectors pAM120 and pAM170 (17). After being introduced, Tn916 appeared to transpose as a single copy to many different sites in the chromosome of recipient meningococci and was stably integrated. These results suggested that Tn916 could be used as a genetic tool for the study of N. meningitidis. Demonstration that Tn916 mutagenesis can inactivate specific meningococcal virulence determinants is crucial to the development of this system. In this report, we describe the creation of a library of meningococcal transformants containing Tn9O6, the screening of this library for transformants altered in the production of group B capsular polysaccharide, and the characterization of capsule-defective and capsule-deficient Tn9O6 meningococcal transformants. MATERIALS AND METHODS Bacterial strains and growth media. Strain NMB (CDC 8201085) is a group B meningococcal strain that was isolated in Pennsylvania in 1982 from the cerebrospinal fluid of a patient with meningitis. Additional relevant phenotypic characteristics of this strain have been previously described (17). Strain 269BSpecr is a spontaneous spectinomycinresistant derivation of group B meningococcal strain 269B (17, 32). Escherichia coli strains CG120 and CG170, containing the plasmids pAM120 and pAM170, respectively, were Corresponding author. t Present address: Department of Microbiology, University of Hawaii, Honolulu, HI 96822. *
obtained from D. B. Clewell (University of Michigan, Ann Arbor, Mich.). pAM120 and pAM170 were derived from pGL101 into which Tn9O6 was cloned (13). Strains of N. meningitidis were grown under aerobic conditions on GC base agar (Difco, Detroit, Mich.) supplemented with 1% (vol/vol) IsoVitaleX enrichment (BBL, Cockeysville, Md.) (GcIso agar) at 37°C in an atmosphere containing 3% CO2. Meningococci were also grown in either GC broth (24) with 1% IsoVitaleX or in a chemically defined medium (24). E. coli was grown in Luria-Bertani broth (28) at 37°C with aeration. Tetracycline (Sigma Chemical Co., St. Louis, Mo.) was added at a final concentration of 5 ,ug/ml for N. meningitidis (17) and 4 pug/ml for E. coli (13). Meningococcal transformants arising from pAM120 or pAM170 were confirmed as resistant at a concentration of 20 ,ug of tetracycline per ml. Spectinomycin (Sigma) was added at a final concentration of 100 ,ug/ml. Meningococcal strains and transformants of strain NMB were stored at -70°C in Trypticase soy broth and 20% glycerol. Chromosomal DNA preparations. Chromosomal DNA was prepared by a modification of the method of Dowson et al. (9). Confluent growth from a single Gclso agar plate was resuspended in 2 ml of 10 mM Tris-HCl containing 10 mM EDTA (pH 7.5). Two microliters of lysozyme (10 mg/ml) (Sigma) was then added, and the solution was gently mixed and then incubated at room temperature for 10 min. Next, 2 ml of 50 mM Tris-HCl containing 10 mM EDTA and 2% (vol/vol) Triton X-100, pH 8.0, was added, and the resultant mixture was incubated for 10 min at room temperature. Two microliters of RNase A (10 mg/ml, boiled) (Sigma) was then added, and the preparation was incubated at 37°C for 30 min. Two microliters of proteinase K (10 mg/ml) (Sigma) was then added, and the mixture was incubated at room temperature for 30 min. At this stage, proteins were removed from the viscous lysate by extracting once with Tris-EDTA (TE)4097
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STEPHENS ET AL.
saturated phenol, twice with TE-saturated phenol-chloroform, and twice with TE-saturated chloroform. The nucleic acids were then precipitated with ethanol, resuspended in
sterile distilled water, and stored at 4°C. The concentration of DNA was determined spectrophotometrically at a wavelength of 260 nm. Transformation. Plasmid preparations of pAM120 and pAM170 used in these experiments were obtained from E. coli strains CG120 and CG170, either as described by Birnboim and Doly (2) or in large-scale preparations (28). Isolated colonies of meningococci, grown overnight on GC base agar, were used as recipients for genetic transformation. Competence of N. meningitidis was confirmed by transforming strain NMB to spectinomycin resistance with DNA from 269BSpecr. The semiquantitative transformation assay of Janik et al. (16) was used as previously described (17). Southern blots. Restriction enzymes were used according to the instructions of the supplier (Bethesda Research Laboratories, Gaithersburg, Md.). Plasmid pAM120 (13) was 32P-labeled by nick translation (28). Alternatively, the random priming method of Feinberg and Vogelstein (10) was used for radiolabeling pAM120. The probe was stored at -20°C until it was ready to use. Restriction endonucleasedigested DNA was electrophoresed on 0.4 to 0.8% agarose gels in Tris-acetate EDTA buffer (28). Transfer of DNA to nitrocellulose (Schleicher and Schuell, Keene, N.H.) in 20x SSC (3.0 M NaCl-0.3 M sodium citrate, pH 7.0) was performed as described by Southern (31). The filters were prehybridized in a rotating water bath at 42°C for at least 4 h in a solution containing the following: 50% formamide (deionized), 5 x Denhardt's solution (28), 5 x SSC, 0.1% sodium dodecyl sulfate (SDS), 100 ,ug of freshly denatured calf thymus DNA per ml, and 200 ,ug of yeast tRNA per ml. At the end of this incubation, an amount of freshly denatured probe (boiled 3 to 4 min) corresponding to -1 x 107 cpm was added directly to the prehybridization mix. The hybridizing filter was then allowed to incubate in the rotating water bath at 42°C overnight. The hybridized filters were washed five times at 42°C in 0.2x SSC and 0.1% SDS. After the washes, autoradiographs were obtained by exposing Kodak X-Omat film (Eastman Kodak Co., Rochester, N.Y.) to the filters for 6 to 72 h at -70°C with
intensifying screens. Colony immunoblot screening assays. Three microliters of suspension from frozen stocks of strain NMB and of tetracycline-resistant transformants of NMB was placed as drops on GcIso agar without and with tetracycline, respectively, and grown overnight. Individual colonies were transferred to nitrocellulose discs (Schleicher and Schuell), placed on agar, and left overnight at 4°C. The discs were then placed in -50 ml of Tris-Tween blocking buffer (0.01 M Tris, 0.15 M NaCl
[pH 8.0], 0.05% [vol/vol] Tween 20 [Sigma], 3% [vol/vol] serum albumin) and gently agitated on a rotating platform (Tekpro; American Hospital Supply) for 60 min. The blocking buffer was removed and replaced with the antibody solution (1:500 dilution of antibody in blocking buffer), and the disc was agitated for 60 min. Murine immunoglobulin M (IgM) monoclonal antibody 2-2-B (37) (kindly supplied by Wendell Zollinger, Walter Reed Army Institute of Research) or polyclonal (horse) antiserum (kindly supplied by Carl Frasch, Bureau of Biologics), both specific for group B capsular polysaccharide, were the bovine
antibodies used. The nitrocellulose disc was then washed three times for 10 min with Tris-Tween buffer without bovine serum albumin. Binding of IgM monoclonal antibody 2-2-B was detected with a goat anti-mouse IgM-IgG antibody
INFECT. IMMUN.
conjugated to alkaline phosphatase (28). A rabbit anti-horse IgG antibody conjugated to alkaline phosphatase was used to detect polyclonal horse antiserum. After being incubated with the second antibody for 1 h, the filter was washed three times for 10 min with Tris-Tween buffer, pH 8.0. NBT-BCIP substrate (0.025 g of Nitro Blue Tetrazolium [NBT; Sigma] in 250 pAl of N,N-dimethylformamide [DMF] and 250 >1l of ethanol; 0.025 g of 5-bromo-4-chloro-3-indolyl phosphate [BCIP; US Biochemical] in 150 RI of DMF and 350 RIl of ethanol; 198 RI of the NBT solution and 99 pAl of the BCIP solution were then added to 30 ml of alkaline phosphatase buffer [100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2]) was added for 5 to 15 min. Antiserum agar technique. Meningococcal colonies were examined after growth on antiserum agar plates, as described by Craven et al. (7) and Kuzemenska et al. (21). Three microliters of the suspension of frozen stock of strain NMB and frozen stock of selected transformants was grown from drops on Gclso agar containing the polyclonal (horse) antiserum to group B capsular polysaccharide. A 1:10 dilution of antiserum to agar was used. After 18 h of growth, the isolated colonies were examined with a binocular dissecting microscope (Bausch and Lomb, St. Louis, Mo.). Precipitin bands were detected with incident light. In some experiments, the surrounding precipitin band was highlighted by staining the colonies with 0.1% Coomassie blue. Isolation of meningococcal capsular polysaccharide and rocket immunoelectrophoresis. Group B meningococcal capsular polysaccharide was extracted from both the tetracycline-resistant transformants and parent strain NMB with Cetavlon (hexadecyltrimethylammonium bromide). Parent or transformant cells were grown in 200 ml of Gc broth plus 1% IsoVitaleX to a reading of 100 Klett units (4 to 5 h). Cetavlon was added (final concentration of 0.1%), and the capsular polysaccharide was extracted for 30 min at room temperature with slow shaking. The suspension was centrifuged at 10,000 x g for 20 min at 4°C. Three volumes of cold ethanol were added to the supernatant, and the mixture was stirred at 4°C for 10 min and placed on ice for 1 h. After centrifugation at 10,000 x g for 20 min at 4°C, the pellet was extracted twice with phenol and resuspended in 1 ml H20. Rocket immunoelectrophoresis using 1% SeaKem agarose gels (SeaKem LE Agarose; FMC) was carried out as described by Andrews et al. (1). Three percent polyclonal group B antiserum (see above) was added to the agarose gels and stabilized with 2% polyethylene glycol 8000. Electrophoresis was performed at 80 V for 2.5 h at room temperature. The gels were pressed onto a glass plate soaked in water, dried with a portable hair dryer, stained with 0.25% Coomassie brilliant blue R250, and destained in methanolacetic acid in water. Peak heights were measured on a dry surface of the glass plate (1). Biosafety. These studies were approved by the Emory University Biosafety Committee and the CDC Office of Biosafety and were conducted by utilizing laboratory practices, safety equipment, and facilities recommended for Biosafety Level 2. RESULTS Library of Tn916 transformants. Transformation of the group B meningococcal strain NMB was performed with 1 ,g of the plasmid vector pAM120 or pAM170. We have previously shown (17, 34) that only Tn916 integrated into the meningococcal genome following transformation with pAM120 or pAM170 and that the location of the transposon
VOL. 59, 1991
Tn916-GENERATED MENINGOCOCCAL CAPSULE MUTANTS
4099
FIG. 1. (A) Immunoblot of Tn916-derived Tetr transformants of meningococcal strain NMB probed with an IgM monoclonal antibody specific for group B capsular polysaccharide. The capsule-deficient mutant M7 is indicated with an asterisk. (B) Growth of M7 (*) on the Gclso agar plate used for the immunoblot of panel A. Growth-deficient transformants (A) were also noted.
in the genome was variable. Tetracycline-resistant transformants were obtained at a frequency of 1.6 x 10-6 to 1 x 10-8 per recipient. Differences from experiment to experiment in the amount of the closed circular form of the transposon or changes in competence factors (34) may account for the range in frequency. These frequencies yielded 2 to -100 transformants per transformation in our assay. Multiple transformations were performed to decrease sibling selection. Over 1,000 Tetr transformants from >150 transformations were selected, passed once on GcIso agar with tetracycline, and stored at -70°C. We have previously shown that the tetracycline resistance of the transformants is stable during laboratory passage (17). Identification of Tn916 transformants altered in the production of group B capsular polysaccharide. Initial screening was performed by using colony immunoblots (Fig. 1). Five hundred and sixty-two individual transformants from the library were probed with IgM monoclonal antibody 2-2-B. This monoclonal antibody is specific for meningococcal group B capsular polysaccharide (37). Transformants which differed from the parent strain in reactivity with the anticapsular monoclonal antibody were identified. The differences were confirmed by repeating the assay three or more times. Transformants that were obviously growth deficient on agar were excluded. By using this approach, two putative capsular polysaccharide overproducers and six putative capsular polysaccharide-deficient transformants were identified. Two of the six capsular polysaccharide-deficient phenotypes did not react with monoclonal antibody 2-2-B. In the other four, reactivity was reduced compared with that of the parent strain. It was postulated that the capsule-deficient mutants might be altered in the amount of capsular polysaccharide produced or altered in the expression of the 2-2-B epitope. To distinguish between these possibilities, the mutants were also studied by using polyclonal group B meningococcal capsular antiserum in an agar antiserum procedure (7, 21). Capsular polysaccharide was identified as a precipitin halo
surrounding colonies of parent strain NMB. No precipitin halo was noted in the M7 mutant or in a second capsuledeficient mutant, 08. Colonies of M7 and 08 exhibited a rough or cracked colony phenotype and were opaque (Op+ +) when examined by incident light (33). Similar findings have previously been reported for spontaneous mutants of group B meningococci incapable of producing capsular polysaccharide (23). Growth of M7 and 08 in a chemically defined medium did not differ significantly from growth of randomly selected transformants or from growth of parent strain NMB. Similar results were also obtained with Gclso broth. Capsular polysaccharide production. To determine the amount of capsular polysaccharide produced by the mutants, capsular polysaccharide was extracted with Cetavlon from parent strain NMB, from mutants M7 and 08, and from other transformants arising from pAM120. Rocket immunoelectrophoresis, using polyclonal antiserum to group B capsule, was performed (Fig. 2). No group B capsular polysaccharide from the M7 mutant was detected by rocket immunoelectrophoresis. The 08 mutant produced a small amount of capsular material. Tn916-containing transformants, which had normal capsule expression in our immunologic screening assays, produced amounts of capsular polysaccharide similar to those produced by parent strain NMB. Thus, the M7 and 08 mutants were defective in the expression of group B capsular polysaccharide. Linkage of capsule-deficient phenotype and tetracycline resistance. To study the possibility that M7 or 08 mutants were spontaneous capsule-deficient mutants (8, 23), linkage of the phenotype and tetracycline resistance was tested by transforming parent strain NMB with DNA from M7. Tetracycline-resistant transformants were obtained at a frequency of 10-3 to 10-4 per recipient. The tetracycline-resistant transformants (>1,000) obtained with M7 or 08 DNA were identical to the parent M7 or 08 mutants in the lack of
4100 1
INFECT. IMMUN.
STEPHENS ET AL. 2
3 4
5
6
7
8
9
10 11 12
13
a
b 2
1
2
3
4
4
_
4
I 4
Ah
,. 5
.4 kb
w
1
FIG. 2. Rocket immunoelectrophoresis of capsular polysaccharide extracted with Cetavlon from meningococcal strain NMB and Tn916-derived mutants of NMB. Lanes: 1, 2, 12, and 13, blank controls; 5 and 9, capsule-deficient mutants M7 and 08, respectively; 7, parent strain NMB; 3, 4, 6, 8, 10, and 11, other Tn916derived mutants that do not differ from the parent strain in the amount of capsule expressed.
reaction with the monoclonal or polyclonal antisera to group B capsular polysaccharide on colony immunoblots. Southern hybridization also demonstrated the linkage and stability of the M7 mutation. As shown in Fig. 3, the original M7 mutant and three of the progeny Tetr transformants (NMB as a recipient of M7 chromosomal DNA) had identical profiles on Southern blots probed with pAM120. The high frequency of transformation to Tetr (10-3 to 10-4), the identical immunoblot profiles with both monoclonal and polyclonal antisera, and the identical Southern blot profiles indicated that the M7 and 08 mutants were stable and transferred at high frequency, presumably by homologous recombination. Analysis of Tn916 insertion in M7 and 08 mutants. Southern blot data indicated that Tn916 had inserted as a single copy in the M7 and 08 mutants. However, in contrast to the majority of Tetr transformants previously reported (17), Tn916 in M7 and 08 (Fig. 4) has undergone a major alteration. 1
2
3 4 5 6
4
6S
ob
4
2 4 1_
FIG. 4. Autoradiograms of Southern blot hybridizations of chromosomal DNA digested with HaeIII or Sau3A and probed with 32P-labeled pAM120. (a) Agarose gel (0.6%). Lanes: 1, HaeIII digest of NMB DNA; 2, HaeIII digest of M7 DNA; 3, 1-kb ladder. (b) Agarose gel (0.85%). Lanes: 1, 1-kb ladder; 2, Sau3A digest of 08 DNA; 3, Sau3A digest of 120A1 DNA.
Eight fragments are predicted to result from a Sau3A digest of Tn916 (6) (Fig. 5), and they are present in a pAM120generated transformant of NMB, 120A1, containing the entire transposon (Fig. 4b). In M7, a single Sau3A site was present within the Tn916 insert, resulting in 2.4- and 3.5-kb hybridizing fragments (data not shown). A single Sau3A site was also present within the Tn916 insert of 08 (Fig. 4b), although the sizes of the hybridizing fragments, 1.8 and 3.1 kb, were different from those of M7. We postulated that this was the Sau3A site within the tetM determinant (6), which suggested that a significant portion of the transposon on either side of tetM had been deleted in the M7 and 08 mutants. Results obtained by other Southern blot data using BstXI (Fig. 3) or HaeIII (Fig. 4a) support this hypothesis. If Tn916 were intact in M7, two hybridizing bands would be seen with BstXI-digested DNA (one at -4 kb and the other at >12.5 kb), and a single band of no less than 16.4 kb would be seen Tn 916
C
x
12
|1
^
kb6
Sa
0
Sa
Sa
Sa..Sa
Sa. Sa e
-L tel M
5
Tn 916 In M7 capsule defective mutant Sa
-om tetM
FIG. 3. Autoradiogram of Southern blot hybridization (0.6% agarose gel) of chromosomal DNA digested with BstXI and probed with 32P-labeled pAM120. Lanes: 1, radiolabeled 1-kb ladder; 2, NMB chromosomal DNA; 3, M7 chromosomal DNA; 4 to 6, Tetr transformants obtained by transformation of NMB with M7 DNA.
Sa
=
Sau 3A site
kb FIG. 5. Schematic representation of Tn916 (6) and of Tn9J6 in the capsule-defective meningococcal mutant M7.
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Tn916-GENERATED MENINGOCOCCAL CAPSULE MUTANTS
with HaeIII-digested DNA, since there is no HaeIII site in the Tn916 sequence (6). The results are quite different. In the BstXI digest, there was a single band of approximately 10.5 kb (Fig. 3), and in the HaeIII digest there was a single band of 3.8 kb (Fig. 4a). These results, combined with the Sau3AI restriction data, demonstrate that there has been a major deletion of Tn916 in the M7 mutant (Fig. 5). The data suggest that much of the transposon, with the exception of the tetM determinant, has been lost. Since there is no longer a BstXI site, the upstream genes, responsible for the excision and integration of the transposon (27), are presumably missing, further stabilizing the insert. DISCUSSION Tn916 is a large, 16.4-kb transposon (5, 6, 13-15, 30) which contains the antibiotic resistance determinant tetM (22). Excision and insertion of Tn916 involves a novel recombination mechanism (4, 27, 29). Tn916 and the other transposons of this family (3) have been used to study the genes of grampositive bacteria. Recently, transposition of these conjugative transposons was also shown to occur in E. coli and Haemophilus influenzae (13, 18). In addition, the tetM determinant of Tn916 has been found and is expressed in clinical isolates of N. meningitidis, Neisseria gonorrhoeae, and commensal Neisseria spp. (19). This event appears to have occurred by the insertion of tetM into the 24.5-MDa conjugative plasmid present in N. gonorrhoeae. Subsequently, this plasmid has been transmitted in vivo to other Neisseria species (20, 25). In commensal Neisseria spp., the tetM determinant appears to have inserted into the chromosome. We investigated the possibility that conjugative transposons of the Tn916 class, which carry tetM, might be used to develop a system for generating random mutations in the meningococcal chromosome (17, 34). We have found that Tn916 can be introduced into N. meningitidis by transforming it with plasmids pAM120 and pAM170, which contain a functional copy of Tn916. This transposon delivery system was selected on the basis of the inability of the vector plasmid to replicate in pathogenic Neisseria species. Tn916 inserted at a frequency of ca. 10-7 per recipient CFU. The presence of single copies of Tn916 in most transformants and the finding that the location of the transposon was variable suggested that Tn916 could be used as a genetic tool for the study of N. meningitidis. Recently, Nassif et al. (26) have shown that a derivative of TnJS45, a closely related conjugative transposon, can also generate variable insertions in N. meningitidis and N. gonorrhoeae. Whether Tn916 insertion in the meningococcal chromosome is a completely random event remains to be determined, but analysis of some 40 transformants by Southern hybridization (17, 31a) does not provide evidence for "hot spots" of insertion. Tn916 is stably incorporated in the meningococcal chromosome. Tn916-carrying transformants retained resistance to tetracycline during laboratory passage and upon overnight growth on medium without tetracycline (reversion rate,
