ANTIMICROBIAL AGzNTS AND CHEMOTHERAPY, Dec. 1975, p. 651-656 Copyright 0 1975 American Society for Microbiology
Vol. 8, No. 6 Printed in U.S.A.
Genetic and Biochemical Characterization of a Ribosomal Mutant of Bacillus subtilis Resistant to Sporangiomycin M. BAZZICALUPO,* B. PARISI, G. PIRALI, M. POLSINELLI, AND F. SALA Cattedra di Genetica, Universitlz di Firenze, Florence,* Laboratorio di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche, Pavia, and Laboratorio Richerche Lepetit S.p.A., Milan, Italy
Received for publication 6 August 1975
The antibiotic sporangiomycin affects the growth ofBacillus subtilis by inhibiting protein synthesis. Mutants of B. subtilis resistant to sporangiomycin have been isolated. One of these, PB 1690, has been further studied. The analysis of subcellular fractions from the mutant has shown that the biochemical effect of the mutation is an alteration of a site on the 60S ribosomal subunit responsible for the binding of the antibiotic: the mutant ribosomes do not bind sporangiomycin and are capable of carrying out phenylalanine polymerization in the presence of sporangiomycin. The resistance mutation maps on the chromosomal region where the ribosomal markers map. The mutant strain is also resistant to the action of the chemically related antibiotic thiostrepton. Treatment of B. subtilis ribosomes with LiCl results in the detachment of a group of proteins including the one responsible for sporangiomycin resistance. Active ribosomes can be reconstructed by mixing "split proteins" and "core particles" of either parental or mutant origin. The fate of the mutant protein can now be followed by assaying reconstructed ribosomes for capacity to bind sporangiomycin and for resistance to the action of the antibiotic in the reactions for phenylalanine polymerization.
Sporangiomycin is a sulfur-containing peptide antibiotic (molecular weight ca. 1,800) chemically related to the thiostrepton group antibiotics, which include thiostrepton, siomycin, and thiopeptin (9, 18). It affects the growth of cultures of Bacillus subtilis and other gram-positive bacteria (18) by inhibiting protein synthesis (14), whereas gram-negative bacteria, as well as eukaryotic organisms, are not affected (18). Sporangiomycin binds in vitro to the 50S ribosomal subunits of B. subtilis and of Escherichia coli in a 1:1 molar ratio, thus inhibiting the polyuridylic acid [poly(U)]-directed phenylalanine polymerization (14). Ribosomes prepared from chloroplasts but not from the cytoplasm of the green alga Chlamydomonas reinhardii are also inhibited (unpublished data). On the other hand sporangiomycin does not interfere with the activity catalyzed on the cytoplasmic ribosomes of Saccharomyces cerevisiae (14). These observations suggest that sporangiomycin is a specific inhibitor of the prokaryotic-type mechanism for protein synthesis, the in vivo resistance of gram-negative bacteria possibly being due to impermeability of the cell envelope or to the presence of an
antibiotic inactivating system. As described for the thiostrepton group antibiotics (1, 3, 8, 10, 11, 13, 19, 20), sporangiomycin inhibits the ribosome-dependent reactions catalyzed by the elongation factors EF-T and EF-G and not the initiation reactions (14). In addition it has been reported that thiostrepton inhibits peptide chain termination in E. coli (2). Thus we can hypothesize that one antibiotic molecule binds to a ribosomal uni- or multimolecular site playing a central role in the machinery for protein synthesis. In this report we describe the isolation and characterization of a mutant of B. subtilis resistant to sporangiomycin whose ribosomes have lost the capacity to bind the antibiotic in vivo and in vitro. MATERIALS AND METHODS Materials. Sporangiomycin and [35S]sporangiomycin were kindly supplied by R. J. White of Gruppo Lepetit (specific activity, 10.3 XCi/tmol), Milan, Italy. Thiostrepton was kindly supplied by L. Delcambe of I.C.I.A., Liege, Belgium. The antibiotics were dissolved in dioxane; the solvent, used at concentrations less than 2%, did not impair the in vivo and in vitro experiments. [14C]phenylalanine, specific activity 464 juCi/,umol, was from 651
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ANTiMICROB. AGzNTs CHzMOTHzR.
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NEN biochemicals. Poly(U) was from Miles Laboratories. Bacterial growth and selection of mutants. Mutants resistant to sporangiomycin were selected by plating spores of B. subtilis SB 25, hikBtrpC2 (12), on agar plates containing the complex organic PS medium (15) and 0.08 pg of sporangiomycin per ml. The frequency of resistant mutants was around 6 x 10-8. Growth of wild-type cells was inhibited at an antibiotic concentration of 0.01 pg/ml, whereas all isolated mutants were resistant to 3 jpg of sporangiomycin per ml. The mutant used in the present work has been classified as PB 1690. Liquid cultures were prepared on a rotary shaker at 37 C in 500-ml Erlenmeyer flasks containing 100 ml of the complex PS medium (15) or the chemically defined medium previously described (16). Large quantities of cells were prepared in a 20-liter fermenter at 37 C under strong aeration. Transduction. Transduction was performed with PBS1 phage following the procedure described by Hoch et al. (6). Assay of antibiotic incorporation into intact cells. [35S]sporangiomycin (0.5 ,ug/ml) was added to cultures in the early log phase of growth in the chemically defined medium. After 5 min the cells were harvested by centrifugation and washed twice with the same culture medium. Part of the suspension was used to determine the radioactivity associated with intact cells; part was used to prepare the supernatant fraction at 30,000 x g (16) where the radioactivity associated with the ribosomes was determined (14). Preparation of cell fraction and assays. Ribosomes, polymerizing enzymes, and E. coli 30s and 5OS ribosomal subunits were prepared as described (16). B. subtilis ribosomal core particles were prepared by treating NH4Cl-washed ribosomes with different concentrations of LiCl as described (14). Reconstitution of ribosomes from core particles and split proteins was performed as described for E. coli ribosomes (14), except that KCI was substituted by NH4Cl in all buffers used. [14Clphenylalanyl-transfer ribonucleic acid was prepared as described (16). Poly(U)-dependent synthesis of polyphenylalanine was assayed as described (16). Binding [35S]sporangiomycin to ribosomes, centrifugation of ribosomes in 5 to 25% linear sucrose gradients followed by fraction collection, assay of their optical density at 254 nm, and radioactivity count were performed as described (14).
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FIG. 1. Growth curves of the parental strain and of the resistant mutant PB 1690 in the presence or absence of sporangiomycin. Bacteria were grown in the complex PS liquid medium. Addition of sporangiomycin (0.5 pg/mi) to the culture was at the times indicated by the arrow. Symbols: 0, parental strain; U, parental strain plus sporangiomycin; 0, mutant strain; 0, mutant strain plus sporangiomycin.
duced rate of growth: its generation time is about 90 min, whereas the generation time of the parental strain is about 30 min. Similar behavior has been observed when the experiment was repeated in the chemically defined liquid medium (data not shown). A slow growth of the mutant is also evident on both solid media. In the presence of 0.08 ,ug of sporangiomycin per ml, the growth of the mutant strain in liquid cultures is not affected, whereas the growth of the parental strain is completely inhibited. Further tests on agar plates have shown that antibiotic concentrations as high as 5 pyg/ml are required to inhibit completely the growth of the mutant strain, whereas the parental strain is inhibited by 0.01 j.g of sporangiomycin per ml. Similar results have been obtained when the same concentrations of the chemically related antibiotic thiostrepton were present in the agar cultures of the mutant and wild-type cells, indicating a crossRESULTS resistance between the two antibiotics. Mechanism of resistance to sporangiomycin. Effect of sporangiomycin on bacterial growth. One of the sporangiomycin-resistant In a preliminary experiment in which [35S]spomutants of B. subtilis (PB 1690), isolated after rangiomycin was added to growing liquid culthe procedure outlined above, has been geneti- tures, it was noticed that the amount of anti~ biotic associated with the mutant strain, cally and biochemically characterized. Growth curves at 37 C were determined for assayed by counting the radioactivity present the wild type and the mutant strain in the in the washed cells, was about 35% of the complex PS liquid medium (Fig. 1). The mutant parental strain. The experiment illustrated in Fig. 2 was perstrain is characterized by a considerably re-
SPORANGIOMYCIN RESISTANCE IN B. SUBTILIS
VOL. 8, 1975
formed to establish the fate of sporangiomycin when it enters the cells. [35S]sporangiomycin was fed to growing cultures, and ribosomes were extracted and fractionated on sucrose gradients. The gradient profiles show that [35S]_ sporangiomycin is associated with the 50S ribosomal subunit of the parental strain, whereas no radioactivity is found on ribosomes of the mutant cells. The results suggest that we may be dealing with a mutant modified in the
A
B
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500 F
I) 0.2
u
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n 0.
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o
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o
20 30 0 10 F RACTION (ML)
40
20 30
FIG. 2. Binding [35S]sporangiomycin to ribosomal particles of the parental and the mutant PB 1690. [35S]sporangiomycin (0.5 pg/ml) was supplied to cultures in the early log phase of growth, and association of radioactivity with the ribosomal particles was assayed by sucrose gradient of the supernatant fraction at 30,000 x g as described in the text. (A) Parental strain; (B) mutant strain.
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ribosomal site responsible for the binding of sporangiomycin. This hypothesis has been further investigated in an experiment where ribosomal preparations isolated from the parental and mutant strain were assayed using the poly(U)-dependent polyphenylalanine synthesizing system. The data illustrated in Fig. 3A show that ribosomes from the mutant strain are resistant to the action of the antibiotic. Almost complete inhibition of the parental system is achieved when the concentration of the antibiotic in the reaction mixture reaches a 1:1 molar ratio with the ribosomes, whereas at the same concentration the system prepared from the mutant strain retains about 65% of its activity. Furthermore the data show that the resistance of the antibiotic is linked to the ribosomal fraction, whereas the polymerizing enzymes fraction isolated from the mutant strain does not confer any detectable resistance to the susceptible ribosomes. In addition (Fig. 3B), the polymerizing system from PB 1690 is also resistant to thiostrepton, a peptide antibiotic whose chemical structure is similar to that reported for sporangiomycin (9). Cross-resistance between sporangiomycin and thiostrepton was also observed in vivo as described above, thus substantiating the assumption that to related chemical structures of the thiostrepton group antibiotics corresponds an identical mechanism of inhibition. Binding of [35S]sporangiomycin to ribosomes has been tested on isolated ribosomal fractions. B
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FIG. 3. Effect of sporangiomycin (A) and of thiostrepton (B) on the reaction for poly(U)-dependent phenylalanine polymerization in cell extracts from the parental and from the mutant PB 1690. Symbols: 0, parental ribosomes plus parental elongation factors; 0, parental ribosomes plus mutant elongation factors; *, mutant ribosomes plus parental elongation factors; 0, mutant ribosomes plus mutant elongation factors. Assay conditions were as described in the text. Ribosomes and elongation factors were used at concentrations of300 and 340 pglml, respectively. Incubation was for 30 min at 30 C. In the four control experiments without the antibiotics, 212, 189, 221, and 213 pmol of phenylalanine per mg of ribosome were polymerized.
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The results of Fig. 4A and E show that no significant amount of the antibiotic is associated with the subunit of the mutant strain, whereas parental 50S subunits bind sporangiomycin in a 1:1 molar ratio. This is in accord with the results obtained when sporangiomycin was added to growing cells (Fig. 2). Fractionation of ribosomal proteins. B. subtilis ribosomes have been described to be very unstable to manipulation under standard laboratory techniques (4, 17); this has been demonstrated to be due to instability of the 3aS subunit when it is separated from the 50S subunit (16, 17). However, in our case, the problem of 30S instability is not relevant, since sporangiomycin interacts with the 50S subunit and the B. subtilis 305 subunit can be functionally substituted by 30S E. coli ribosomal particles. Methods have been refined for E. coli ribosomes, for the detachment of ribosomal proteins by treatment with high concentrations of LiCl, and for the reconstitution of active ribosomes from the isolated components (7). We tested whether similar techniques were also effective for B. subtilis ribosomes. As shown in Fig. 4B and F, treatment of ribosomes with 2.5 M LiCl for 16 h results in the detachment of a A
B
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P84690 CORK
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4.
group of proteins (split proteins) from the parental and mutant ribosomes, leaving core particles. The number and characteristics of the split proteins have not yet been investigated. Ribosomes were reconstituted by mixing core particles with split proteins (Fig. 4C, D, G, H). The results of Fig. 4D show that split proteins obtained from the parental ribosomes confer to ribosomal core particles of mutant source the capacity to bind [35S]sporangiomycin. On the other hand, the property of binding the antibiotic is partially lost when parental core particles are mixed with mutant split proteins (Fig. 4H). The amount of [35S]sporangiomycin bound to the 50S subunit in the gradients of Fig. 4 has been calculated and the data are reported in Table 1. The reported values indicate that only 50 to 60% of ribosomes have been involved in the exchange phenomenon of the altered protein. This can be expected since gradient profiles (Fig. 4B, F) show that the preparation of core particles is not homogenous; part of the subunits retain a high sedimentation value. This was the case also when LiCl concentration was increased or when incubation time was
prolonged (unpublished data). CS19, coR,90 CO REc P8 4690 spLr sB 25 SPLIT it,, T SOS
00
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FQACTION8 (ML) FIG. 4. Binding of [35SJsporangiomycin to B. subtilis ribosomes (A and E), to core particles obtained by treatment with 2.5 MLiCl (B and F), or to ribosomes reconstructed by core particles and split proteins (C, D, G and H). Experimental details in the text. 5 to 25% sucrose gradients were in 0.5 mM magnesium acetate.
SPORANGIOMYCIN RESISTANCE IN B. SUBTILIS
VOL. 8, 1975
TABLE 1. Binding of sporangiomycin to the intact, degraded, or reconstructed 50S ribosomal subunit from the parental SB25 and the mutant PB 1690a Sporangiomycin
Ribosomes from:
to 1 mol of ~ bound 50S subunit
(mol)
PB 1690 PB 1690 core PB 1690 core PB 1690 core SB 25 SB 25 core SB 25 core + SB 25 core +
+ PB 1690 split + SB 25 split
SB 25 split PB 1690 split
0.02 0.01 0.01 0.58 1.09 0.49 0.91 0.46
the right of cysA14, that is, in the region where other markers of ribosomal proteins map (21). Although highly unlikely, we can't rule out (with our data) the order spg adeA cysA.
DISCUSSION A mutant of B. subtilis resistant to sporangiomycin has been isolated. The experimental TABLE 2. Resistance to the action of sporangiomycin of ribosomes reconstructed from core particles and split proteins in the poly(U-directed phenylalanine polymerizationa Act in the presence of sporangiomycin (% of
Ribosomes from: The values have been obtained from the data of Fig. 4 by computing the moles of sporangiomycin that migrate with the larger ribosomal subunit in 1. SB 25 the sucrose gradients. 2. PB 1690 SB 25 core + SB 25 split The preparations of the reconstituted ribo- 3. 4. SB 25 core + SB 1690 split somes have been tested for susceptibility to 5. PB 1690 core + SB 25 split sporangiomycin in the in vitro system for poly- 6. PB 1690 core + PB 1690 split a
phenylalanine synthesis. Both parental and mutant core particles are resistant to the action of the antibiotic when mixed with split proteins obtained from the mutant ribosomes (Table 2). The above observations clearly show that the split proteins fraction contains one or more proteins which are responsible for resistance to sporangiomycin. Mapping of resistance to sporangiomycin. The mutation to sporangiomycin resistance of PB 1690 has been mapped using PBS1 transduction. The strain PB 1690 was used as donor; recipient strain was PB 30409 adeA16 cysA14 Leu8 (requiring cysteine, adenine, and leucine for growth). Transductants selected for prototrophy to adenine were then checked for resistance to sporangiomycin and for prototrophy to cysteine. The results reported in Table 3, from which the map distance of Fig. 5 was calculated, show that the sporangiomycin marker is located to
655
control) 0.7 34.0 0.5 23.1 0.6 25.5
a Poly(U)-directed phenylalanine polymerization was assayed as described in the text. The sporangiomycin concentration was 1 ,ug/ml. The values reported in the table are the percentage ofthe polymerizing activity of control systems assayed in the absence of the antibiotic. The control systems incorporated 204, 157, 26.3, 35.6, 26.1, and 27.6 pmol of phenylalanine/mg of ribosomes in experiments 1 to 6, respectively. Activity of core particles alone was substracted from all the calculated values. Ribosome concentration was 300 ,ug/ml. E. coli 30S ribosomal subunits (80 j,g/ml) were present in all tests. ade
cys
A 16
A14
spg
99
95
FIG. 5. Location of the sporangiomycin marker on the B. subtilis chromosome. The map distance is calculated according to Nester and Lederberg (12).
TABLE 3. Location of the mutation to sporangiomycin resistancea Strains
oftade Number transductants
Recombinant classes
Deduced order
ade+ cys+ spgr 13 adeA,6 cysAA14 spgr ade+ cyss+pg8 40 ade+ cys spgr 0 Recipient strain: PB 3409 adeA 16 cysA14 ade+ cys- spgs 505 leu8spg8 a cys, ade, his, trp: requirement for cysteine, adenine, histidine, and tryptophan, respectively; spgr and spg8: resistance and selectivity to sporangiomycin. The colonies ade+ selected on minimal medium were reisolated on the same selective medium and then checked for cys+ and spgr. The resistance to sporangiomycin was checked on nutrient agar (PS medium) containing 0.3 ,ug of sporangiomycin per ml. Donor strain: PB 1690 his2 trp2 spgr
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AN?IMIcRoB. AGENTS CUMOTHR.
BAZZICALUPO ET AL.
evidence shows that the mutation affects a ribosomal protein of the 50S subunit. Indeed, the large subunit of the mutant cells has lost the capacity to bind the antibiotic; mutant ribosomes are resistant to the action of sporangiomycin when assayed in vitro in the poly(U)dependent phenylalanine polymerization reactions; a protein fraction can be isolated from mutant ribosomes. This fraction confers sporangiomycin resistance to the susceptible ribosome. The resistance mutation maps in the same chromosomal region of the other ribosomal protein markers. Furthermore, the resistant strain B. subtiis PB 1690 is also resistant to thiostrepton, a chemically related antibiotic, thus further substantiating the hypothesis that these antibiotics inhibit protein synthesis by an identical mechanism. The thiostrepton group antibiotics, to which sporangiomycin belongs, act on a ribosomal site functionally very important since they prevent the regular functions of EF-T and EF-G on the ribosomes (1, 3, 8, 10, 13, 14, 19, 20). The protein alteration in the mutant cells has not yet been identified. It appears important to determine whether the mutant ribosomal protein is itself involved in EF-T and/or EF-G functioning or whether it is present in a position where it affects the active ribosomal site. The study of a series a B. subtilis ribosomal mutants and the identification of the altered protein molecules should help in the identification and characterization of the proteins involved in this site and of their functional significance. LITERATURE CITED 1. Bodley, J. W., L. Lin, and J. H. Highland. 1970. Studies on translocation. VI. Thiostrepton prevents the formation of a ribosome-G factor-guanine nucleotide complex. Biochem. Biophys. Res. Commun. 41:14061411. 2. Brot, N., W. P. Tate, C. T. Caskey, and H. Weinbach. 1974. The requirement for ribosomal protein L7 and L12 in peptide-chain termination. Proc. Natl. Acad.
Sci. U.S.A. 71:89-92. 3. Cannon, M., and K. Burns. 1971. Modes of action of
erythromycin and thiostrepton as inhibitors of protein synthesis. FEBS Lett. 18:1-5. 4. Doi, R. H. 1971. Bacillus subtilis protein synthesizing system, p. 67-88. In J. A. Last a-id A. I. Laskin (ed.), Protein biosynthesis in bacterial sytk mas. M. Dekker, Inc., New York. 5. Goldthwaite, C., D. Dubnau, and I. Smith. 1970.
Genetic mapping of antibiotic resistance markers in Baciluw subtilis. Proc. Natl. Acad. Sci. U.S.A. 66M96-103. 6. Hoch, J. A., M. Parat, and C. Anagnostopoulos. 1967. Transformation and transduction in recombinationdefective mutants of Bacillus subtilis. J. Bacteriol. 93:1925-1937. 7. Itoh, T., E. Otaka, and S. Asawa. 1968. Release of ribosomal protein from Escherichia coli ribosomes with high concentrations of lithium chloride. J. Mol. Biol. 33:109-122. 8. Kinoshita, T., Y. F. Liou, and N. Tanaka. 1971. Inhibition by thiopeptin of ribosomal functions asocated with T and G factors. Biochem. Biophys. Res. Commun. 44:859-863. 9. Miyairi, N., T. Miyoshi, H. Aoki, M. Kobsaka, H. Ikushina, K. Kinugita, H. Sakai, and H. Imanaka. 1970. Studies on thiopeptin antibiotics. I. Characteristic of thiopeptin B. J. Antibiot. 23:113-119. 10. Modolell, J., B. Cabrer, A. Parmeggiani, and D. Vazquez. 1971. Inhibition by siomycin and thiostrepton of both aminoacyl-tRNA and factor G binding to ribosomes. Proc. Natl. Acad. Sd. U.S.A. 68: 1796-1800. 11. Modolell, J., D. Vazquez, and R. E. Monro. 1971. Ribosomes, G-factor and siomycin. Nature (London) New Biol. 230:109-119. 12. Nester E. N., and J. Lederberg. 1961. Linkage of genetic units of Bacillus aubtilw in DNA tansormation. Proc. Natl. Acad. Sci. U.S.A. 47:52-55. 13. Pestka, S. 1970. Thiostrepton: a ribosomal inhibitor of translocation. Biochem. Biophys. Res. Commun. 40: 667-673. 14. Pirali, G., G. C. Lancini, B. Parisi, and F. Sala. 1972. Interaction of sporangiomycin with the bactel ribosome. J. Antibiot. 25:561-568. 15. Riva, S., M. Polsinelli, and A. Falaschi. 198. A new phage of Bacillus subtilis with infectious DNA having separable strands. J. Mol. Biol. 35:347-356. 16. Sala, F., M. Bazzicalupo, and B. Parisi. 1974. Protein synthesis in Bacillus subtilis: differential effiet of potassium ions on peptide chain initiation and elongation. J. Bacteriol. 119:821-829. 17. Takeda, M., and F. Lipmann. 1966. Comparison of amino acid polymerization in Bacillus subtilis and Escherichia coli cell-free systems: hybridization of their ribosomes. Proc. Natl. Acad. Sci. U.S.A. 56: 1875-1882. 18. Thiemann, J. E., C. Coronelli, H. Pagani, G. Beretta, G. Tamoni, and V. Arioli. 1968. Antibiotic production by new form genera of the Actinomycetales. I. Sporangiomycin an antibacterial agent isolated from Planomonospora parontospora var. nov. J. Antibiot.
21:525-531. 19. Watanabe, S., and K. Tanaka. 1971. Effect of siomycin on the acceptor site of Escherichia coli ribosomes. Biochem. Biophys. Res. Commun. 46:728-734. 20. Weisblum, B. and V. Demohn. 1970. Inhibition by thiostre of the formation of a ribosome-bound leotide complex. FEBS Lett. 11:149-152. guanin 21. Young, . , and G. A. Wilson. 1972. Genetics of Ba( lus tiis and other gram-positive sporulating bacilli, p. 77-106. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Micro} :ology, Washington, D.C.
Vol. 8, No. 6 Printed in U.S.A.
Genetic and Biochemical Characterization of a Ribosomal Mutant of Bacillus subtilis Resistant to Sporangiomycin M. BAZZICALUPO,* B. PARISI, G. PIRALI, M. POLSINELLI, AND F. SALA Cattedra di Genetica, Universitlz di Firenze, Florence,* Laboratorio di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche, Pavia, and Laboratorio Richerche Lepetit S.p.A., Milan, Italy
Received for publication 6 August 1975
The antibiotic sporangiomycin affects the growth ofBacillus subtilis by inhibiting protein synthesis. Mutants of B. subtilis resistant to sporangiomycin have been isolated. One of these, PB 1690, has been further studied. The analysis of subcellular fractions from the mutant has shown that the biochemical effect of the mutation is an alteration of a site on the 60S ribosomal subunit responsible for the binding of the antibiotic: the mutant ribosomes do not bind sporangiomycin and are capable of carrying out phenylalanine polymerization in the presence of sporangiomycin. The resistance mutation maps on the chromosomal region where the ribosomal markers map. The mutant strain is also resistant to the action of the chemically related antibiotic thiostrepton. Treatment of B. subtilis ribosomes with LiCl results in the detachment of a group of proteins including the one responsible for sporangiomycin resistance. Active ribosomes can be reconstructed by mixing "split proteins" and "core particles" of either parental or mutant origin. The fate of the mutant protein can now be followed by assaying reconstructed ribosomes for capacity to bind sporangiomycin and for resistance to the action of the antibiotic in the reactions for phenylalanine polymerization.
Sporangiomycin is a sulfur-containing peptide antibiotic (molecular weight ca. 1,800) chemically related to the thiostrepton group antibiotics, which include thiostrepton, siomycin, and thiopeptin (9, 18). It affects the growth of cultures of Bacillus subtilis and other gram-positive bacteria (18) by inhibiting protein synthesis (14), whereas gram-negative bacteria, as well as eukaryotic organisms, are not affected (18). Sporangiomycin binds in vitro to the 50S ribosomal subunits of B. subtilis and of Escherichia coli in a 1:1 molar ratio, thus inhibiting the polyuridylic acid [poly(U)]-directed phenylalanine polymerization (14). Ribosomes prepared from chloroplasts but not from the cytoplasm of the green alga Chlamydomonas reinhardii are also inhibited (unpublished data). On the other hand sporangiomycin does not interfere with the activity catalyzed on the cytoplasmic ribosomes of Saccharomyces cerevisiae (14). These observations suggest that sporangiomycin is a specific inhibitor of the prokaryotic-type mechanism for protein synthesis, the in vivo resistance of gram-negative bacteria possibly being due to impermeability of the cell envelope or to the presence of an
antibiotic inactivating system. As described for the thiostrepton group antibiotics (1, 3, 8, 10, 11, 13, 19, 20), sporangiomycin inhibits the ribosome-dependent reactions catalyzed by the elongation factors EF-T and EF-G and not the initiation reactions (14). In addition it has been reported that thiostrepton inhibits peptide chain termination in E. coli (2). Thus we can hypothesize that one antibiotic molecule binds to a ribosomal uni- or multimolecular site playing a central role in the machinery for protein synthesis. In this report we describe the isolation and characterization of a mutant of B. subtilis resistant to sporangiomycin whose ribosomes have lost the capacity to bind the antibiotic in vivo and in vitro. MATERIALS AND METHODS Materials. Sporangiomycin and [35S]sporangiomycin were kindly supplied by R. J. White of Gruppo Lepetit (specific activity, 10.3 XCi/tmol), Milan, Italy. Thiostrepton was kindly supplied by L. Delcambe of I.C.I.A., Liege, Belgium. The antibiotics were dissolved in dioxane; the solvent, used at concentrations less than 2%, did not impair the in vivo and in vitro experiments. [14C]phenylalanine, specific activity 464 juCi/,umol, was from 651
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NEN biochemicals. Poly(U) was from Miles Laboratories. Bacterial growth and selection of mutants. Mutants resistant to sporangiomycin were selected by plating spores of B. subtilis SB 25, hikBtrpC2 (12), on agar plates containing the complex organic PS medium (15) and 0.08 pg of sporangiomycin per ml. The frequency of resistant mutants was around 6 x 10-8. Growth of wild-type cells was inhibited at an antibiotic concentration of 0.01 pg/ml, whereas all isolated mutants were resistant to 3 jpg of sporangiomycin per ml. The mutant used in the present work has been classified as PB 1690. Liquid cultures were prepared on a rotary shaker at 37 C in 500-ml Erlenmeyer flasks containing 100 ml of the complex PS medium (15) or the chemically defined medium previously described (16). Large quantities of cells were prepared in a 20-liter fermenter at 37 C under strong aeration. Transduction. Transduction was performed with PBS1 phage following the procedure described by Hoch et al. (6). Assay of antibiotic incorporation into intact cells. [35S]sporangiomycin (0.5 ,ug/ml) was added to cultures in the early log phase of growth in the chemically defined medium. After 5 min the cells were harvested by centrifugation and washed twice with the same culture medium. Part of the suspension was used to determine the radioactivity associated with intact cells; part was used to prepare the supernatant fraction at 30,000 x g (16) where the radioactivity associated with the ribosomes was determined (14). Preparation of cell fraction and assays. Ribosomes, polymerizing enzymes, and E. coli 30s and 5OS ribosomal subunits were prepared as described (16). B. subtilis ribosomal core particles were prepared by treating NH4Cl-washed ribosomes with different concentrations of LiCl as described (14). Reconstitution of ribosomes from core particles and split proteins was performed as described for E. coli ribosomes (14), except that KCI was substituted by NH4Cl in all buffers used. [14Clphenylalanyl-transfer ribonucleic acid was prepared as described (16). Poly(U)-dependent synthesis of polyphenylalanine was assayed as described (16). Binding [35S]sporangiomycin to ribosomes, centrifugation of ribosomes in 5 to 25% linear sucrose gradients followed by fraction collection, assay of their optical density at 254 nm, and radioactivity count were performed as described (14).
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TIM^E (M^INUTES)
FIG. 1. Growth curves of the parental strain and of the resistant mutant PB 1690 in the presence or absence of sporangiomycin. Bacteria were grown in the complex PS liquid medium. Addition of sporangiomycin (0.5 pg/mi) to the culture was at the times indicated by the arrow. Symbols: 0, parental strain; U, parental strain plus sporangiomycin; 0, mutant strain; 0, mutant strain plus sporangiomycin.
duced rate of growth: its generation time is about 90 min, whereas the generation time of the parental strain is about 30 min. Similar behavior has been observed when the experiment was repeated in the chemically defined liquid medium (data not shown). A slow growth of the mutant is also evident on both solid media. In the presence of 0.08 ,ug of sporangiomycin per ml, the growth of the mutant strain in liquid cultures is not affected, whereas the growth of the parental strain is completely inhibited. Further tests on agar plates have shown that antibiotic concentrations as high as 5 pyg/ml are required to inhibit completely the growth of the mutant strain, whereas the parental strain is inhibited by 0.01 j.g of sporangiomycin per ml. Similar results have been obtained when the same concentrations of the chemically related antibiotic thiostrepton were present in the agar cultures of the mutant and wild-type cells, indicating a crossRESULTS resistance between the two antibiotics. Mechanism of resistance to sporangiomycin. Effect of sporangiomycin on bacterial growth. One of the sporangiomycin-resistant In a preliminary experiment in which [35S]spomutants of B. subtilis (PB 1690), isolated after rangiomycin was added to growing liquid culthe procedure outlined above, has been geneti- tures, it was noticed that the amount of anti~ biotic associated with the mutant strain, cally and biochemically characterized. Growth curves at 37 C were determined for assayed by counting the radioactivity present the wild type and the mutant strain in the in the washed cells, was about 35% of the complex PS liquid medium (Fig. 1). The mutant parental strain. The experiment illustrated in Fig. 2 was perstrain is characterized by a considerably re-
SPORANGIOMYCIN RESISTANCE IN B. SUBTILIS
VOL. 8, 1975
formed to establish the fate of sporangiomycin when it enters the cells. [35S]sporangiomycin was fed to growing cultures, and ribosomes were extracted and fractionated on sucrose gradients. The gradient profiles show that [35S]_ sporangiomycin is associated with the 50S ribosomal subunit of the parental strain, whereas no radioactivity is found on ribosomes of the mutant cells. The results suggest that we may be dealing with a mutant modified in the
A
B
S S
0.4F
1000
50s
9 a-
'-' o-a
0.3
500 F
I) 0.2
u
4 0
n 0.
0
4 at
o
0
o
20 30 0 10 F RACTION (ML)
40
20 30
FIG. 2. Binding [35S]sporangiomycin to ribosomal particles of the parental and the mutant PB 1690. [35S]sporangiomycin (0.5 pg/ml) was supplied to cultures in the early log phase of growth, and association of radioactivity with the ribosomal particles was assayed by sucrose gradient of the supernatant fraction at 30,000 x g as described in the text. (A) Parental strain; (B) mutant strain.
653
ribosomal site responsible for the binding of sporangiomycin. This hypothesis has been further investigated in an experiment where ribosomal preparations isolated from the parental and mutant strain were assayed using the poly(U)-dependent polyphenylalanine synthesizing system. The data illustrated in Fig. 3A show that ribosomes from the mutant strain are resistant to the action of the antibiotic. Almost complete inhibition of the parental system is achieved when the concentration of the antibiotic in the reaction mixture reaches a 1:1 molar ratio with the ribosomes, whereas at the same concentration the system prepared from the mutant strain retains about 65% of its activity. Furthermore the data show that the resistance of the antibiotic is linked to the ribosomal fraction, whereas the polymerizing enzymes fraction isolated from the mutant strain does not confer any detectable resistance to the susceptible ribosomes. In addition (Fig. 3B), the polymerizing system from PB 1690 is also resistant to thiostrepton, a peptide antibiotic whose chemical structure is similar to that reported for sporangiomycin (9). Cross-resistance between sporangiomycin and thiostrepton was also observed in vivo as described above, thus substantiating the assumption that to related chemical structures of the thiostrepton group antibiotics corresponds an identical mechanism of inhibition. Binding of [35S]sporangiomycin to ribosomes has been tested on isolated ribosomal fractions. B
A leo
V
a I
43
Iu
_ Is~ I I
0
L-
I0
5PO"H6IoMYCjr4
15
(p.g/.1)
0
Os
7HIOSTOZEPTO4
(pW/*M
FIG. 3. Effect of sporangiomycin (A) and of thiostrepton (B) on the reaction for poly(U)-dependent phenylalanine polymerization in cell extracts from the parental and from the mutant PB 1690. Symbols: 0, parental ribosomes plus parental elongation factors; 0, parental ribosomes plus mutant elongation factors; *, mutant ribosomes plus parental elongation factors; 0, mutant ribosomes plus mutant elongation factors. Assay conditions were as described in the text. Ribosomes and elongation factors were used at concentrations of300 and 340 pglml, respectively. Incubation was for 30 min at 30 C. In the four control experiments without the antibiotics, 212, 189, 221, and 213 pmol of phenylalanine per mg of ribosome were polymerized.
654
ANTIMICROB. AGENTS CHECMOTHER.
BAZZICALUPO ET AL.
The results of Fig. 4A and E show that no significant amount of the antibiotic is associated with the subunit of the mutant strain, whereas parental 50S subunits bind sporangiomycin in a 1:1 molar ratio. This is in accord with the results obtained when sporangiomycin was added to growing cells (Fig. 2). Fractionation of ribosomal proteins. B. subtilis ribosomes have been described to be very unstable to manipulation under standard laboratory techniques (4, 17); this has been demonstrated to be due to instability of the 3aS subunit when it is separated from the 50S subunit (16, 17). However, in our case, the problem of 30S instability is not relevant, since sporangiomycin interacts with the 50S subunit and the B. subtilis 305 subunit can be functionally substituted by 30S E. coli ribosomal particles. Methods have been refined for E. coli ribosomes, for the detachment of ribosomal proteins by treatment with high concentrations of LiCl, and for the reconstitution of active ribosomes from the isolated components (7). We tested whether similar techniques were also effective for B. subtilis ribosomes. As shown in Fig. 4B and F, treatment of ribosomes with 2.5 M LiCl for 16 h results in the detachment of a A
B
PI460 RItO SOs
P84690 CORK
r0$
4.
group of proteins (split proteins) from the parental and mutant ribosomes, leaving core particles. The number and characteristics of the split proteins have not yet been investigated. Ribosomes were reconstituted by mixing core particles with split proteins (Fig. 4C, D, G, H). The results of Fig. 4D show that split proteins obtained from the parental ribosomes confer to ribosomal core particles of mutant source the capacity to bind [35S]sporangiomycin. On the other hand, the property of binding the antibiotic is partially lost when parental core particles are mixed with mutant split proteins (Fig. 4H). The amount of [35S]sporangiomycin bound to the 50S subunit in the gradients of Fig. 4 has been calculated and the data are reported in Table 1. The reported values indicate that only 50 to 60% of ribosomes have been involved in the exchange phenomenon of the altered protein. This can be expected since gradient profiles (Fig. 4B, F) show that the preparation of core particles is not homogenous; part of the subunits retain a high sedimentation value. This was the case also when LiCl concentration was increased or when incubation time was
prolonged (unpublished data). CS19, coR,90 CO REc P8 4690 spLr sB 25 SPLIT it,, T SOS
00
_9
A
t
r,
('I d
0
Wo
E sS
25
R1OSo0ES F
Sa 25
CoRE
FQACTION8 (ML) FIG. 4. Binding of [35SJsporangiomycin to B. subtilis ribosomes (A and E), to core particles obtained by treatment with 2.5 MLiCl (B and F), or to ribosomes reconstructed by core particles and split proteins (C, D, G and H). Experimental details in the text. 5 to 25% sucrose gradients were in 0.5 mM magnesium acetate.
SPORANGIOMYCIN RESISTANCE IN B. SUBTILIS
VOL. 8, 1975
TABLE 1. Binding of sporangiomycin to the intact, degraded, or reconstructed 50S ribosomal subunit from the parental SB25 and the mutant PB 1690a Sporangiomycin
Ribosomes from:
to 1 mol of ~ bound 50S subunit
(mol)
PB 1690 PB 1690 core PB 1690 core PB 1690 core SB 25 SB 25 core SB 25 core + SB 25 core +
+ PB 1690 split + SB 25 split
SB 25 split PB 1690 split
0.02 0.01 0.01 0.58 1.09 0.49 0.91 0.46
the right of cysA14, that is, in the region where other markers of ribosomal proteins map (21). Although highly unlikely, we can't rule out (with our data) the order spg adeA cysA.
DISCUSSION A mutant of B. subtilis resistant to sporangiomycin has been isolated. The experimental TABLE 2. Resistance to the action of sporangiomycin of ribosomes reconstructed from core particles and split proteins in the poly(U-directed phenylalanine polymerizationa Act in the presence of sporangiomycin (% of
Ribosomes from: The values have been obtained from the data of Fig. 4 by computing the moles of sporangiomycin that migrate with the larger ribosomal subunit in 1. SB 25 the sucrose gradients. 2. PB 1690 SB 25 core + SB 25 split The preparations of the reconstituted ribo- 3. 4. SB 25 core + SB 1690 split somes have been tested for susceptibility to 5. PB 1690 core + SB 25 split sporangiomycin in the in vitro system for poly- 6. PB 1690 core + PB 1690 split a
phenylalanine synthesis. Both parental and mutant core particles are resistant to the action of the antibiotic when mixed with split proteins obtained from the mutant ribosomes (Table 2). The above observations clearly show that the split proteins fraction contains one or more proteins which are responsible for resistance to sporangiomycin. Mapping of resistance to sporangiomycin. The mutation to sporangiomycin resistance of PB 1690 has been mapped using PBS1 transduction. The strain PB 1690 was used as donor; recipient strain was PB 30409 adeA16 cysA14 Leu8 (requiring cysteine, adenine, and leucine for growth). Transductants selected for prototrophy to adenine were then checked for resistance to sporangiomycin and for prototrophy to cysteine. The results reported in Table 3, from which the map distance of Fig. 5 was calculated, show that the sporangiomycin marker is located to
655
control) 0.7 34.0 0.5 23.1 0.6 25.5
a Poly(U)-directed phenylalanine polymerization was assayed as described in the text. The sporangiomycin concentration was 1 ,ug/ml. The values reported in the table are the percentage ofthe polymerizing activity of control systems assayed in the absence of the antibiotic. The control systems incorporated 204, 157, 26.3, 35.6, 26.1, and 27.6 pmol of phenylalanine/mg of ribosomes in experiments 1 to 6, respectively. Activity of core particles alone was substracted from all the calculated values. Ribosome concentration was 300 ,ug/ml. E. coli 30S ribosomal subunits (80 j,g/ml) were present in all tests. ade
cys
A 16
A14
spg
99
95
FIG. 5. Location of the sporangiomycin marker on the B. subtilis chromosome. The map distance is calculated according to Nester and Lederberg (12).
TABLE 3. Location of the mutation to sporangiomycin resistancea Strains
oftade Number transductants
Recombinant classes
Deduced order
ade+ cys+ spgr 13 adeA,6 cysAA14 spgr ade+ cyss+pg8 40 ade+ cys spgr 0 Recipient strain: PB 3409 adeA 16 cysA14 ade+ cys- spgs 505 leu8spg8 a cys, ade, his, trp: requirement for cysteine, adenine, histidine, and tryptophan, respectively; spgr and spg8: resistance and selectivity to sporangiomycin. The colonies ade+ selected on minimal medium were reisolated on the same selective medium and then checked for cys+ and spgr. The resistance to sporangiomycin was checked on nutrient agar (PS medium) containing 0.3 ,ug of sporangiomycin per ml. Donor strain: PB 1690 his2 trp2 spgr
558
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AN?IMIcRoB. AGENTS CUMOTHR.
BAZZICALUPO ET AL.
evidence shows that the mutation affects a ribosomal protein of the 50S subunit. Indeed, the large subunit of the mutant cells has lost the capacity to bind the antibiotic; mutant ribosomes are resistant to the action of sporangiomycin when assayed in vitro in the poly(U)dependent phenylalanine polymerization reactions; a protein fraction can be isolated from mutant ribosomes. This fraction confers sporangiomycin resistance to the susceptible ribosome. The resistance mutation maps in the same chromosomal region of the other ribosomal protein markers. Furthermore, the resistant strain B. subtiis PB 1690 is also resistant to thiostrepton, a chemically related antibiotic, thus further substantiating the hypothesis that these antibiotics inhibit protein synthesis by an identical mechanism. The thiostrepton group antibiotics, to which sporangiomycin belongs, act on a ribosomal site functionally very important since they prevent the regular functions of EF-T and EF-G on the ribosomes (1, 3, 8, 10, 13, 14, 19, 20). The protein alteration in the mutant cells has not yet been identified. It appears important to determine whether the mutant ribosomal protein is itself involved in EF-T and/or EF-G functioning or whether it is present in a position where it affects the active ribosomal site. The study of a series a B. subtilis ribosomal mutants and the identification of the altered protein molecules should help in the identification and characterization of the proteins involved in this site and of their functional significance. LITERATURE CITED 1. Bodley, J. W., L. Lin, and J. H. Highland. 1970. Studies on translocation. VI. Thiostrepton prevents the formation of a ribosome-G factor-guanine nucleotide complex. Biochem. Biophys. Res. Commun. 41:14061411. 2. Brot, N., W. P. Tate, C. T. Caskey, and H. Weinbach. 1974. The requirement for ribosomal protein L7 and L12 in peptide-chain termination. Proc. Natl. Acad.
Sci. U.S.A. 71:89-92. 3. Cannon, M., and K. Burns. 1971. Modes of action of
erythromycin and thiostrepton as inhibitors of protein synthesis. FEBS Lett. 18:1-5. 4. Doi, R. H. 1971. Bacillus subtilis protein synthesizing system, p. 67-88. In J. A. Last a-id A. I. Laskin (ed.), Protein biosynthesis in bacterial sytk mas. M. Dekker, Inc., New York. 5. Goldthwaite, C., D. Dubnau, and I. Smith. 1970.
Genetic mapping of antibiotic resistance markers in Baciluw subtilis. Proc. Natl. Acad. Sci. U.S.A. 66M96-103. 6. Hoch, J. A., M. Parat, and C. Anagnostopoulos. 1967. Transformation and transduction in recombinationdefective mutants of Bacillus subtilis. J. Bacteriol. 93:1925-1937. 7. Itoh, T., E. Otaka, and S. Asawa. 1968. Release of ribosomal protein from Escherichia coli ribosomes with high concentrations of lithium chloride. J. Mol. Biol. 33:109-122. 8. Kinoshita, T., Y. F. Liou, and N. Tanaka. 1971. Inhibition by thiopeptin of ribosomal functions asocated with T and G factors. Biochem. Biophys. Res. Commun. 44:859-863. 9. Miyairi, N., T. Miyoshi, H. Aoki, M. Kobsaka, H. Ikushina, K. Kinugita, H. Sakai, and H. Imanaka. 1970. Studies on thiopeptin antibiotics. I. Characteristic of thiopeptin B. J. Antibiot. 23:113-119. 10. Modolell, J., B. Cabrer, A. Parmeggiani, and D. Vazquez. 1971. Inhibition by siomycin and thiostrepton of both aminoacyl-tRNA and factor G binding to ribosomes. Proc. Natl. Acad. Sd. U.S.A. 68: 1796-1800. 11. Modolell, J., D. Vazquez, and R. E. Monro. 1971. Ribosomes, G-factor and siomycin. Nature (London) New Biol. 230:109-119. 12. Nester E. N., and J. Lederberg. 1961. Linkage of genetic units of Bacillus aubtilw in DNA tansormation. Proc. Natl. Acad. Sci. U.S.A. 47:52-55. 13. Pestka, S. 1970. Thiostrepton: a ribosomal inhibitor of translocation. Biochem. Biophys. Res. Commun. 40: 667-673. 14. Pirali, G., G. C. Lancini, B. Parisi, and F. Sala. 1972. Interaction of sporangiomycin with the bactel ribosome. J. Antibiot. 25:561-568. 15. Riva, S., M. Polsinelli, and A. Falaschi. 198. A new phage of Bacillus subtilis with infectious DNA having separable strands. J. Mol. Biol. 35:347-356. 16. Sala, F., M. Bazzicalupo, and B. Parisi. 1974. Protein synthesis in Bacillus subtilis: differential effiet of potassium ions on peptide chain initiation and elongation. J. Bacteriol. 119:821-829. 17. Takeda, M., and F. Lipmann. 1966. Comparison of amino acid polymerization in Bacillus subtilis and Escherichia coli cell-free systems: hybridization of their ribosomes. Proc. Natl. Acad. Sci. U.S.A. 56: 1875-1882. 18. Thiemann, J. E., C. Coronelli, H. Pagani, G. Beretta, G. Tamoni, and V. Arioli. 1968. Antibiotic production by new form genera of the Actinomycetales. I. Sporangiomycin an antibacterial agent isolated from Planomonospora parontospora var. nov. J. Antibiot.
21:525-531. 19. Watanabe, S., and K. Tanaka. 1971. Effect of siomycin on the acceptor site of Escherichia coli ribosomes. Biochem. Biophys. Res. Commun. 46:728-734. 20. Weisblum, B. and V. Demohn. 1970. Inhibition by thiostre of the formation of a ribosome-bound leotide complex. FEBS Lett. 11:149-152. guanin 21. Young, . , and G. A. Wilson. 1972. Genetics of Ba( lus tiis and other gram-positive sporulating bacilli, p. 77-106. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Micro} :ology, Washington, D.C.
