Bacillus suhtilis strains. Antonie ran Leeuwenhoek 37,. RIVA, S., POLSINELLI, M. & FALASCHI, A. (1968). A new phage of Bacillus subtilis with infectious DNA.
Journal of General Microbiology (1983), 129, 3499-3504.
3499
Printed in Great Britain
Bacillus subtilis Mutation Blocking Irreversible Binding of Bacteriophage SPPl By M A R I O A. SANTOS,'y2 H E R M i N I A D E L E N C A S T R E 1 * 3 * A N D LUIS J . Laboratbrio de Genktica Molecular, Instituto Gulbenkian de CiZncia, Oeiras, Portugal Faculdade de CiZncias de Lisboa and Universidade Nova de Lisboa
(Received 23 May 1983; revised 19 June 1983) ~
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Mutants of Bacillus subtilis 168 were isolated which allow adsorption but not infection by bacteriophage SPPl. The adsorbed phages can be subsequently recovered in active form. From ten other bacteriophages tested, only the SPP1-related phages 41c, 22a, p15 and SF6 failed to plate on the mutant cells. The mutation responsible for this behaviour (pha-2) was mapped by PBSl transduction, showing 95% cotransduction with ald-1.
INTRODUCTION
Adsorption of most B. subtilis phages has been shown to depend on the presence of glucosylated teichoic acid in the cell surface (Young, 1967; Yasbin et al., 1976). Three classes of bacterial mutants defective in the glucosylation of teichoic acid have been identified (Young, 1967) and the genes involved (gtaA,gtaB and gtaC) have been mapped in the chromosome of B. subtilis 168 (Young et al., 1969). A mutation conferring resistance to phage SPOl (pha-1)was also mapped, but the corresponding mutants were not further characterized (Lepesant-Kejzlarovh et al., 1975). Membrane receptors have been proposed to account for the adsorption features of B. subtilis phages $29 (Jacobson & Landman, 1977) and PBSZ-1 (Steensma, 1981). In addition to membrane components, both these phages require the presence of glucosylated teichoic acid in the wall of the host as a primary receptor site. SPPl is one of the few phages known to infect gta strains with high efficiency (Jacobson & Landman, 1975). In an attempt to elucidate the nature of SPPl receptors, mutants of B. subtilis 168 resistant to this phage were isolated and studied. Their mutation, which we designate pha-2, maps far from gta loci, and allows reversible binding of SPPl as well as normal infection by SPOl, SP02, $105 and other phages. METHODS
Bacterial strains. The strains used are listed in Table 1. They were all derivatives of Bacillus subtilis 168 trPC2. Phage strains and methods ofpropagation. The strains used are listed in Table 1. SPPl lysates were prepared by O.l), exponentially growing cultures of B. subtilis 168T' in M-broth. Phage infecting, at low input multiplicity (I stocks with titres of approximately 10" p.f.u. mI--l were obtained after 7 h incubation at 37 "Cwith shaking. The same procedure was used for phages 41c, 22a, p15 and SF6. Bacteriophages SPO1, SF5, SF7 and 19 were collected from plates of confluent lysis by placing the soft agar in TBT buffer (2 ml per plate), mixing, and then centrifuging for 10 min at 10000g.$105 and SP02 lysates were prepared by induction of lysogenic strains with mitomycin C, as described by Rutberg e f al. (1969). PBSl-transducing lysates were prepared in the isogenic SPPl mutants by the method of Karamata & Gross (1970) using PABY as growth medium. Media. M-broth and M soft agar were as described by Yasbin & Young (1974) except for the supplements: MgC1, and MnC1, were omitted and the concentration of CaCI, was increased to 10 mM. Minimal medium for mutagenesis was as described by Paveia & Archer (1980). GM1 and GM2 (Yasbin et al., 1973) were used for the development of competence. TBT buffer was prepared as reported by Biswal et al. (1967). PAB was antibiotic medium no. 3 (Difco). PABY was PAB supplemented with 0.5% (w/v) yeast extract. Selective medium for 0022-1287/83/0001-1268$02.00 0 1983 SGM
3500
M. A . SANTOS, H . DE LENCASTRE AND L. J . ARCHER
Table 1. Bacterial and phage strains Bacterial strain 168T+ BR151 RUB808 RUB813 BDlll QB936 IL6 IGCg 101 IGCg 102 IGCgl03 I GCg 104 IGCglO5 IGCgl06 IGCgl07 IGCglO8 IGCglW IGCgllO IGCgl11 Phage strain SPPI 41c 22a P15 SPO1 4105 SP02 SF5 SF6 SF7 I9 PBSl
Genotype
i
Origin
Prototroph metBlO Iys3 trpC2 F. E. Young metBlO fys3 trpC2 gtaA gtaA (4105) C. Anagnostopoulos thrA cysB3 trpC2 leuA8 aroG932 ald-1 trpC2 R. A. Dedonder D. Dean trpC2 (SP02)
metBlO lys3 pha-2
Origin
T. A. Trautner 0. E. Landman 0. E. Landman 0. E. Landman F. E. Young F. E. Young D. Dean H. Y. Steensma H. Y. Steensma H. Y. Steensma H. Y. Steensma C. Anagnostopoulos
Prepared as described in Methods
Reference Riva et al. (1 968) Zsigray et af. (1973) Jacobson & Landman (1975) Jacobson & Landman (1975) Okubo et al. (1964) Reilly (1965) Okubo & Romig (1965) Rima & Steensma (1971) Steensma & Block (1979) Steensma & Duermeyer (1979) Rima & Steensma (1971) Takahashi (1961)
transductants was MM (Anagnostopoulos & Spizizen, 1961) solidified with 1.5% (wlv) agar. Carbon sources and auxotrophic requirements were added to a final concentration of 0.1 % (w/v) and 20 pg ml- I , respectively. NA (nutrient agar, Difco) was the solid rich medium used. Chemicals. Pancreatic deoxyribonuclease Type I (DNAase), mitomycin C, and N-methyl-N’-nitro-Nnitrosoguanidine were obtained from Sigma. Isolation of phage-resistant mutants. Mutagenesis with nitrosoguanidine was as described by Paveia & Archer (1980). Plates were prepared by embedding 0.1 ml of the mutagenized culture of B. subtilis 168T+ in M soft agar together with 0.1 ml of a SPPl lysate with a titre of about 1O1O p.f.u. ml- Isolated colonies were streaked twice on M soft-agar plates containing phage (lo9 p.f.u. per plate) to check for resistance, and further streaked on NA plates. Eleven of the isolated resistant mutants were made isogenic by congression into B. subtilis BR151, and numbered IGCglOl to IGCgl11 (Table 1). Competent cultures were prepared according to Yasbin et al. (1973) and transformed by saturating amounts of DNA extracted from the SPPIRmutants as described by Barat et a f . (1965). SPPl transformants were selected among Trp+ transformants. Determination of adsorption efficiencies. To 1-0ml bacilli in M-broth, 0.01 ml phage (approximately 1.0 x lo7 p.f.u.) was added for an input multiplicity 5 0.1. In control tubes, 0.01 ml phage was added to 1.0 ml broth. After 10 min incubation at 37 “C, the mixtures were centrifuged at 10000g for 10 min and the supernatants were assayed for unadsorbed phage. Determination of pfating efficiencies. The procedure described by Lencastre & Archer (1980) was used. SPPIR mutants and B. subtilis 168T+ were used as indicator strains. Rewrsibiliry of adsorption. Reversibility of adsorption was studied essentially as described by Jacobson & Landman (1977). Bacterium-phage complexes were allowed to form for 10 min in M-broth. At this time, ‘zero time’, samples were drawn for assay of unadsorbed phage and the remaining cells diluted in the same medium. After 20 min, samples were drawn again and assayed similarly for unadsorbed phage. All values were corrected by subtracting the percentage of input phage remaining unadsorbed at ‘zero time’.
Adsorption mutation in Bacillus subtilis
350 1
Time (min) Fig. 1. Kinetics of adsorption of SPPl to B. subtilis 168T+ and IGCgl06. Cells of control (@) and mutant ( 0 )strains were incubated with phage at an input multiplicity of approximately 0.1. Adsorption was carried out in M-broth, at 37 "C. At the indicated time intervals, samples were removed and centrifuged for 10 min at 1OOOOg. The supernatant fractions were assayed for unadsorbed phage. Transduction procedure. The recipient cultures for transduction were grown in PABY. Early in the stationary phase, and after checking the mobility of the cells, 0.1 ml transducing lysate was added per ml recipient culture. The transduction mixture was incubated for 30 min at 37 "Cand suitable dilutions were plated on selective media. RESULTS
Non-productive SPPI adsorption SPPl -resistant mutants of B. subtilis 168T-t (control strain) were isolated after nitrosoguanidine mutagenesis, and made isogenic in strain BR151. While plating efficiencies of SPPl on the mutants were less than lo-* of those obtained on the control strain, the adsorption efficiencies were only slightly less (78 to 88% of control). We concluded, therefore, that the mutants allowed a non-productive adsorption of SPPl. Reversible SPPl adsorption In order to analyse the small difference in SPPl adsorption between mutant and control cells, we studied the kinetics of adsorption. The initial rate of phage adsorption to bacilli was very high and similar in both control and mutant strains (Fig. 1). After 40 s incubation, however, the adsorption rates in the two strains sharply diverged, approaching zero for the mutant. Adsorption in all mutants followed the same pattern. This suggested a purely reversible binding to the mutants, as in the case of phage T1 to Escherichia coli strain B/1 (Garen, 1954). To test this prediction, the release of adsorbed phage particles was determined by dilution of bacterium-phage complexes (Table 2). More than 92% of the adsorbed phages were released from the mutant cells, whereas only 0.3% were recovered from the control. The adsorption of SPPl to the mutants was, therefore, reversible.
Phage specifcity of the mutation The interaction of ten other phages with one of our mutant strains and RUB 808, a strain carrying the gtaA mutation, was investigated and compared with the control strain (Table 3). In addition to SPP1, phages 41c, 22a, p15 and SF6 were unable to infect IGCgl 10. Mutants strains of B. subtilis 168 isolated for resistance to phage 22a are also resistant to SPP1, 41c and p15 (Jacobson & Landman, 1975). Therefore, these phages must share a common host receptor site. It is also interesting to note that these five phages do not apparently require glucosylated teichoic acid for infection. SP02 was the only phage tested to achieve infection of both types of resistant mutants.
3502
M . A . SANTOS, H . DE LENCASTRE A N D L . J . ARCHER
Table 2. Reversibility of adsorption Phage adsorbed Strain
(%I
Phage released ("/,>
168T+ IGCgl02 IGCgl04 IGCglOS IGCg 106
99.9 88.0 78-0 90.0 80.0
0.3 92.0 94.9 94.4 98.0
Table 3. Sensitivity of three strains of B. subtilis to infection by 11 bacteriophages Dilutions of phage lysates were spotted on a lawn of cells : + indicates formation of individual plaques; - indicates that individual plaques did not form. Strain of B. subtilis: 7
Phage SPP 1 41c 22a Pl5 SF6 SPO1 4105 SF5 SF7 I9 SP02
168T
+
+ + + + + + + + + + +
1
RUB808 gtuA
IGCgllO phu2
-
+
Table 4. Mapping of pha-2 marker by transduction Donor*
Recipient*
IGCgl06 @ha-2)
BD111 (cysB rhrA)
Selected phenotype
Recombinant class
Thr+
cys + SPPl cys+ SPPlS cys- SPPlR cys- SPPlS Thr+ SPPIR Thr+ SPPls Thr- SPPl Thr- SPPls
cys +
* In donor
No. of colonies scored
Implied order
:: 63
1Cy.B thrA pha-2
37
and recipient strains only the relevant markers are indicated; see Table 1 for complete genotype.
Mapping of pha-2 locus We have scanned the chromosome of B . subtilis 168 by transducing several auxotrophic mutants with lysates of PBS 1 made on phage-resistant strains. Preliminary transduction experiments indicated that all mutants were linked to the thrA marker with an average coinheritance of 66%. Three-factor transduction crosses (Table 4) suggested the order cysB, thrA, pha-2, this observation being confirmed for every one of the strains. We have subsequently found, using QB936 as the recipient strain, that pha-2 is very closely linked to ald-1 with a mean cotransduction frequency of 95 %.
Adsorption mutation in Bacillus subtilis
3503
DISCUSSION
The adsorption of bacteriophages is classically considered to occur in two successive steps, one reversible, the other irreversible (Garen, 1954; Adams, 1959). Here we describe a bacterial mutation (pha-2), which blocks the adsorption process between those two steps. It allows adsorption of SPPl phages but such adsorbed particles can be fully recovered in free and active form. This in uiuo system mimics, therefore, the fully reversible adsorption of PL-1 phages to isolated cell walls of Lactobacillus casei (Watanabe et al., 1977) and of N-1 phages to purified walls of Micrococcus lysodeikticus (Lovett & Shockman, 1970). While gta mutations affect the adsorption of a wide range of distinct viruses (Yasbin et al., 1976), the differently located pha-2 mutation seems to be specific for a small group of closely related phages. In fact, SF6,41c, 22a, p15 and SPPl have been found to share several biological properties and to be inactivated by anti-SPPl serum (unpublished work). All other B. subtilis phages tested successfully infected pha-2 mutants. We believe that pha-2 mutants constitute a useful tool for the detailed study of SPPl adsorption process. Such study is currently in progress. We wish to thank Dr H. Y. Steensma for the generous gift of phages SF5, SF6, SF7 and I9 and Dr 0. E. Landman for the offer of phages 41c, 22a and p15.
REFERENCES
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ultraviolet sensitivity of Bacillus subtilis bacteriophage SP02 and its infectious DNA. Journal of Molecular Biology 14, 130- 142. OKUBO,S., STRAUSS, B. & STODOLSKY, M. (1964). The possible role of recombination in the infection of competent Bacillus subtilis by bacteriophage deoxyribonucleic acid. Virology 24, 552-562. PAVEIA, H. & ARCHER,L. J. (1980). Location of genes for arabinose utilization in the Bacillus subtilis chromosome. Broteria-GenPtica Z 76, 169-1 76. REILLY,B. E. (1965). A Study of the bacteriophages of Bacillus subtilis and their infectiousnucleic acids. Ph. D thesis, Western Reserve University, Cleveland, Ohio, U.S.A. RIMA,B. K . & STEENSMA, H. Y. (1971). Bacteriophages of Bacillus suhtilis : comparison of different isolation techniques and possible use for classification of Bacillus suhtilis strains. Antonie ran Leeuwenhoek 37, 425-434. RIVA,S., POLSINELLI, M. & FALASCHI, A. (1968). A new phage of Bacillus subtilis with infectious DNA having separable strands. Journal of Molecular Biology 35, 347-356. RUTBERG,L., HOCH, J. A. & SPIZIZEN,J. (1969). Mechanism of transfection with deoxyribonucleic acid from the temperate Bacillus bacteriophage $105. Journal of Virology 4, 50-57. STEENSMA, H. Y. (1981). Adsorption of the defective phage PBSZl to Bacillus subtilis 168 wt. Journal qf' General Virology 52, 93- 101. STEENSMA, H. Y. & BLOCK,J. (1979). Effect of calcium ions on the infection of Bacillus subtilis by bacteriophage SF6. Journal of General Virology 42, 305-314. STEENSMA, H. Y. & DUERMEYER, W. (1979). An enzyme-linked immunosorbent assay (ELISA) for PBSZ-1, a defective phage of Bacillus subtilis. Journal of General Virology 44, 741-746. TAKAHASHI, I. (1961). Genetic transduction in Bacillus subtilis. Biochemical and Biophysical Research Communications 5 , 171-175. WATANABE, K., TAKESUE, s. & ISHIBASHI, K . (1977).
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Reversibility of the adsorption of bacteriophage PL1 to the cell walls isolated from Lactobacillus casei. Journal of General Virology 34, 189-194. YASBIN,R. E. & YOUNG,F. E. (1974). Transduction in Bacillus subtilis by bacteriophage SPPl . Journal of Virology 14, 1343-1 348. YASBIN, R. E., WILSON,G. A. &YOUNG,F. E. (1973). Transformation and transfection in lysogenic strains of Bacillus subtilis 168. Journal of Bacteriology 113, 540-548. YASBIN,R. E., MAINO,V. C. &YOUNG,F. E. (1976). Bacteriophage resistance in Bacillus subtilis 168 W23 and interstrain transformants. Journal of Bacteriology 125, 112c1126.
YOUNG,F. E. (1967). Requirement of glucosylated teichoic acid for adsorption of phage in Bacillus subtilis 168. Proceedings of the National Academy of Sciences of’the United States of America 58, 23772384. YOUNG,F. E., SMITH, C. & REILLY,B. E. (1969). Chromosomal location of genes regulating resistance to bacteriophage in Bacillus subtilis. Journal of Bacteriology 98, 1087-1097. 0. E. (1973). ZSIGRAY, R. M., MIS, A. L. & LANDMAN, Penetration of a bacteriophage into Bacillus subtilis : blockage of infection by deoxyribonuclease. Journal of Virology 11, 69-77.
