APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1985, p. 438-440 Vol. 50, No. 2 0099-2240/85/080438-03$02.OO/0 Copyright © 1985, American Society for Microbiology Possible Role of Released from Spore Cell Wall of griseus ISTVAN SZAB6,* AGNES BENEDEK, AND GYORGY BARABAS Institute ofBiology, University Medical School, H4012 Debrecen, Hungary Received 6 December 1984/Accepted 6 May 1985

Vegetative mycelia and spores of the investigated high- and low-producer strains of Streptomyces griseus bound significant amounts (4%) of streptomycin, which could be removed by increasing ionic strength. The release of from the spores was easier when the spores were germinating. This phenomenon is considered to play an ecological role. We suppose that the streptomycin released during the germination process may protect the young hyphae from the different growing in the microenvironment of the Streptomyces spores.

The biological function of has been the subject removed from the surface of the solid medium with glass of much speculation and many different hypotheses. First, beads and washed twice with distilled water. The number of Waksman supported the classification of streptothricin as a spores was determined in a Buerker chamber after ultrasonic metabolic waste product (15, 16). Krassilnikov attributes treatment (3 min in distilled water; cooling with ice bath; antibiotics with importance in the competition of the mi- MSE Ultrasonic Power Unit No. 3000, 2 kHz, at 1.5 A). The crobes fighting against each other (8). According to Gottlieb, ultrasonic treatment served to remove the vegetative however, antibiotics do not exist in the , at least not in a hyphae. detectable amount (5). This contradicts the former theory. Killing of spores. Killing of spores was carried out by Foster believes that these special substances are shunt treating the spores (109/cm3) with ethylene oxide for 3 h and products accumulating by certain environmental effects (4). -y-rays ('OCo) for 90 min with a dose of 0.5 Mrad. The loss of There is some evidence for the possible biological function viability was complete. of the peptide antibiotics in sporulation (10-12), but the fact Isolation of spore wall. The spores, suspended in distilled that there exist antibiotic-negative mutants which sporulate water, were disintegrated in cell homogenizer MSK B. as well as the parental strains makes this suggestion uncer- Braun by glass beads (diameter, 0.10 to 0.11 mm, no. 54140) tain. Therefore, it was suggested recently by Piret and six times for 0.5 min each (cooled with dry ice), and their Demain (9) that the antibiotic synthesis and sporulation may walls were isolated as described previously (2). be two independent processes regulated by a common Measurement of inhibition of growth. B. control mechanism. They supposed that the antibiotics may cereus spores were spread on the surface of the bouillon agar either interact with the spore after sporulation, when the plate (see Materials and Methods), and holes of 3-mm antibiotics may activate or inhibit germination or outgrowth, diameter were made on the agar slab. A suspension (20 j,l) of or protect the spore during germination or outgrowth. A living or killed spores (5 x 108 spores) of S. griseus 52-1 were germination inhibitor antibiotic found by Hirsch and Ensign pipetted into the holes. After overnight incubation at 27°C, (6) and gramicidin S (9) are examples to support the former the diameters of the inhibition zones were measured and theory. Our findings revealed here support the latter one. compared with those of authentic standards. Searching for the biological role of the aminoglycoside SM liberation experiments. The 48-h-old mycelium of S. antibiotics, we found that streptomycin and neomycin were griseus 52-1 cultivated in soybean liquid media (13), the present in the cell wall of Streptomyces griseus and Strep- 14-day-old spores of S. griseus 52-1 and 811 grown on tomyces fradiae, respectively (2, 3, 14). We introduced a soybean solid media (13), and the isolated spore wall of S. model to study the release of streptomycin from the spores griseus 52-1 were incubated with different concentrations of of S. griseus strains to try to answer the question. NaCl (0.015 to 1.0 M) dissolved in 0.066 M phosphate buffer (pH 7.6) at 5°C overnight. After incubation, mycelia and MATERIALS AND METHODS spores were centrifuged at 5,000 x g for 20 min at 4°C. The Abbreviations. SM, Streptomycin. spore wall was centrifuged at 30,000 x g for 30 min at 4°C. Strains and cultivation. S. griseus 52-1 and S. griseus 811 SM content of the supernatant was measured by the agar- were cultivated in filtered liquid and solid soybean medium plate method. The standard solutions contained the same as described previously for S. griseus 52-1 (13). concentration of NaCl as the different samples. Autolysis Bouillon medium. Bouillon medium was composed of0.3% was inhibited by the low temperature. Difco meat extract, 0.5% Bacto-Peptone, 0.1% glucose, 2% Electrophoresis of the released antibiotic. An agar disc cut agar, and 0.052 to 0.234% NaCl (pH 7.4). out of the inhibition zone (diameter of 3 mm) was placed on Antibiotic assay. Antibiotic assay was performed by an Whatman 3MM paper and left there for 30 min to let the agar hole diffusion method with Bacillus subtilis ATCC 6633. antibiotic diffuse into the paper. Paper electrophoresis was The growth medium was described previously (1). carried out with two different buffers. Buffer A, formic Isolation of spores. S. griseus 52-1 and 811 were grown on acid-acetic acid-water (0.02:0.08:1.0 [by volume]) (pH 1.9), 2% soybean medium containing 2% agar (13). Spores were was run for 60 min at 18.6 V - cm-'. Buffer B, 0.3 M ammonium formate containing 0.1 M sodium chloride (pH * Corresponding author. 7.6), was run for 60 min at 18.6 V * cm-'. The antibiotic

438 VOL.VOL.50,50,19851985~~~~~~~ECOLOGICALROLE OF STREPTOMYCIN 439

curve demonstrates the heterogeneous nature of the binding sites. There exist very weak binding sites and relatively 801; A strong sites as well; the latter needs almost an ionic strength of 0.3 to remove the SM molecules. The maximum amount of SM bound to the cell wall was 4% (wt/wt) (Fig. 1A). 60X Since the ionic strength of the solid and liquid soybean medium (see Materials and Methods) is 0.13 and that of the 40- soil usually is not higher, the binding between SM and the cell wall may exist in nature and may protect the strain when the antibiotic is released. This effect may be significant in germinating spores, when the young hyphae are most sensi- " tive to the environmental effects (i.e., products of other live I microbes). Therefore, the spores were also investigated. B Figure lB shows the SM binding capacity of the spores. A comparison of Fig. lA and B shows that the total SM content is almost the same (4%, related to cell wall dry weight), but there is a difference in the strong binding sites (in the ionic strength range of 0.1 to 0.3); there are fewer of these in the spores. a S. griseus 52-1 is a relatively high SM producer compared :0 I with the wild strains isolated from nature. It produces about 100 pLg of SM per ml in soybean medium. Therefore, the E ionic strength dependence of the binding of SM to the cell 0 wall was determined in S. griseus 811 too, which produces 0. GP only a few micrograms of SM per milliliter under the same &. w conditions. The total amount of SM bound to the spores was Ull) than was in the it can be seen in C less it high producer. But, 10Y0 Fig. iC that the excess amount of SM with the high-producer strain shown in Fig. lB came from the saturation of the weak b01 binding sites. There is practically no difference in the quan- tity of SM bound to the strong binding sites. The saturation 60[ of the weak binding sites needs high concentration of the antibiotic, which can be performed only by high-producer

40 strains. In the former case, one-half of the total SM can be liberated at an ionic strength of 0.1, while in this case only one-fourth was liberated; that is, there is no difference in the 20 quantity of the strongly bound SM, which is probably biologically important. 000 0.06 016 0.24 0.32 Q40 0.48 As was shown earlier (2, 14), it was the cell wall of the Ionic strength mycelium that bound SM; therefore, we tried to investigate this phenomenon with the spore wall. Similar to results with FIG. 1. Ionic strength-dependent stability of the binding between the spores of the S. griseus 811 low-producer strain, SM mycelium (A), spores of S. griseus 52-1 (B), and spores of S. griseus bound predominantly to the strong binding sites; moreover, 811 (C) and SM. A 10-mg (dry weight) portion of 48-h-old mycelium some amount of SM remained bound even at an ionic of 52-1 was incubated in 2 ml of NaCi of different S. griseus new arose or became concentrations. Symbols: x-x, bound SM expressed as the percent- strength of 0.3. Probably binding sites age of the totally bound antibiotic at an ionic strength of 0.0 0-4, accessible during the isolation. The amount of the totally SM content referring to the dry weight of the mycelium. The bound SM was much lower than it was with intact spores, 14-day-old spores of S. griseus 52-1 and 811 were collected, and 10 but considering the drastic disintegrating procedure of the mg (dry weight) was incubated with 2 ml (52-1) and 1 ml (811) of wall isolation, it may be understandable. To prove that the NaCl of different concentrations. spore wall can bind more SM than the intact spore, the isolated spore wall was saturated with an excess amount of activity was detected by the agar plate method with B. SM. An unexpectedly high amount of SM could be bound to subtilis ATCC 6633 as test organism. The paper was placed the isolated spore wall at an ionic strength of 0.01. This is on the surface of the agar layer (thickness, 2.1 mm; compoL- almost 25% of the spore wall dry weight, and one-third of sition as described in reference 1) for 30 min to let the this remained bound even at an ionic strength of 0.1 (not antibiotic diffuse into the agar. The plate was incubated at shown). Twelve percent of the spore dry weight was de- 370C overnight. tected as bound SM after saturation of the intact spores with this antibiotic at an ionic strength of 0.0. These results show RESULTS that the isolated spore wall can bind a significant amount of Release of SM from the cell walls of S. griseus. The 48-h-old SM and that the wall is probably the primary binding site in mycelium of S. griseus 52-1 was incubated in 10-2 M the spores. phosphate buffer (pH 7.6) containing different amounts of Release of SM during germination. The ability of the spores NaCl, and the concentration of the released SM was mea- to store SM can supply S. griseus with a sufficient amount of sured as described in Materials and Methods. The ionic antibiotic in the microenvironment to kill different bacteria strength dependence is shown in Fig. 1A. In the range of and can protect the germinating spores when the antibiotic is ionic strength 0.0 to 0.3, all of the SM was released. The released. The effect of the germination process upon the 440 SZAB6 ET AL. APPL. ENVIRON. MICROBIOL. liberation of SM was investigated in liquid medium. Samples microenvironment of the germinating spores in the soil is an were taken from the culture medium of the germinating open one, in which the released SM can diffuse. We made a spores at different times, and their SM contents were deter- model of it in which solid medium was used with holes mined. There was no difference in the SM concentration containing the spores. The model was simple, compared during germination, taking samples every hour. The ionic with that found in nature. strength of the medium was 0.13. The liberation of SM The following explanation of the above two processes in corresponded to the concentration measured at an ionic the closed and in the open system was given. In the solution strength of 0.13 with nongerminating spores. This means that (i.e., in liquid medium), there is antibiotic liberation from the the steady-state concentration of SM was not influenced by spore wall during germination, and the antibiotic rebinds to the germination. the wall of the young hyphae in the closed system, but it can A model was constructed which can simulate the events in only partially rebind in the open system (solid medium). A the soil (which is an open system, in contrast to the liquid part of it diffuses into the agar and interacts with B. cereus medium) during the germination of the S. griseus spores. B. cells growing around the Streptomyces spores. This phe- cereus spores were spread on an agar plate (bouillon agar nomenon may occur in nature as well, and the antibiotic- containing 0.009 to 0.04 M NaCl; ionic strength of the producing strain may make use of this advantage among medium, 0.018 to 0.08), and equal amounts of living, killed, competing microbes in the struggle for life. and antibiotic-free (the SM was removed by washing with 1 M NaCl and spores of an SM nonproducing strain were used LITERATURE CITED also) S. griseus spores were put into holes of the plate (the 1. Barabas, Gy., A. Ottenberger, I. Szab6, J. Erdei, and G. Szab6. germinating spores in the hole are in an open system). After 1978. The biological role of aminoglucoside antibiotics in overnight cultivation at 27°C, there was no inhibition zone Streptomycetes, p. 353-361. In M. Mordarski, W. Kurylowicz, around the holes containing the antibiotic-free germinating and J. Jeljaszewicz (ed.), Nocardia and streptomyces. Gustav spores, germinating spores of the SM nonproducing strain, Fischer Verlag, Stuttgart, Federal Republic of Germany. 2. Barabis, Gy., I. Szab6, A. Ottenberger, V. Zsolnai-Nagy, and G. and killed spores, which contained SM. (The killing of the Szab6. 1980. An enzymatic release of antibiotic containing spores with -y-ray did not influence the quantity and the peptidoglycan fragments from the streptomycin producing elution profile of SM.) An inhibition zone was observed only Streptomyces griseus. Can. J. Microbiol. 26:141-153. around the hole containing germinating spores (which bound 3. Barabis, Gy., and G. Szab6. 1977. Effect of penicillin on the same amount of SM as the killed spores) even at the streptomycin production by Streptomyces griseus. Antimicrob. lowest ionic strength (0.018). The antibiotic content of the Agents Chemother. 11:392-395. inhibition zone was eluted and identified by paper electro- 4. Foster, J. W. 1949. Chemical activities of fungi. Academic phoresis (see Materials and Methods) at two different pH Press, Inc., New York. values as SM. The migration distance was 5.4 cm in buffer 5. Gottlieb, D. 1976. The production and role of antibiotics in soil. J. Antibiot. 29:987-1000. system A and 4.7 cm in system B for both the antibiotic 6. Hirsch, C. F., and J. C. Ensign. 1978. Some properties of obtained from the inhibition zone and for that of the authen- Streptomyces viridochromogenes spores. J. Bacteriol. tic SM used as a standard. The diameter of the inhibition 134:1056-1063. zone was 7 mm at an ionic strength of 0.018. This value is in 7. Kalakoutskii, L. V., and L. M. Pouzharitskaja. 1973. In G. the range of the ionic strength of different , and thus, the Sykes and F. A. Skinner (ed.), Actinomycetales: characteristics model can be applied to nature. These results suggest that and practical importance, p. 168. Academic Press, Inc. (Lon- the antibiotic liberation is favored in the germinating spores. don), Ltd., London. 8. Krassilnikov, N. A. 1958. Soil, microorganisms and higher DISCUSSION plants, p. 365. Acad. Sci. USSR, Moscow. 9. Piret, J. M., and A. L. Demain. 1981. Role ofgramicidin S in the The ionic strength dependence of SM binding to the producer organism, Bacillus brevis, p. 243-245. In H. S. mycelium (Fig. 1A) and spore (Fig. 1B) demonstrated that Levinson, A. L. Sonenshein, and D. J. Tipper (ed.), Sporulation the binding can exist at the ionic strength of the natural soil, and germination. American Society for Microbiology, Washing- which is usually less than 0.1. The producer strain has ton, D.C. enough SM bound to the spore to give a sufficient antibiotic 10. Ristow, H., B. Schazschneider, and H. Rleinkauf. 1975. Effects concentration in the close vicinity of the spores (i.e., in the of the peptide antibiotics tyrocidine and the linear gramicidin on microenvironment) to protect them against other microorga- RNA synthesis and sporulation. Biochim. Biophys. Acta nisms if SM can be liberated. It was shown that SM bind to 414:1085-1092. 11. Sarkar, N., D. Langley, and H. Paulus. 1977. Biological function the cell wall of the spore. The release of SM from the spore of gramicidin: selective inhibition of RNA polymerase. Proc. wall is a result of the germination process in which the cell Natl. Acad. Sci. U.S.A. 74:1478-1482. wall is loosened by swelling and by nicks which lytic 12. Sarkar, N., and H. Paulus. 1972. Function of peptide antibiotics enzymes make. It was tempting to suppose that SM liberated in sporulation. Nature (London) New Biol. 239:228-230. during the germination process could protect the young 13. Szab6, G., Gy. Barabas, and T. V§lyi-Nagy. 1961. Comparison hyphae. However, when the spores germinated in liquid of Streptomyces griseus strains which produce streptomycin medium, the concentration of SM did not increase, i.e., it and those which do not. Arch. Microbiol. 40:261-274. remained bound. The question arises as to which of the cell 14. Szab6, I., Gy. Barabas, E. Misley, A. Ottenberger, and G. Szab6. structures binds the SM after germination. According to 1981. The binding site for aminoglycoside antibiotics on the electron microscopic investigations, the spore wall is disin- Streptomyces griseus cell wall, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A (Suppl. tegrated only partially during germination (7), and there is a 11):477-480. new peptidoglycan in the hyphae wall. Both may bind SM 15. Waksman, S. A. 1943. Production and activity of streptomycin. molecules; that is, the SM liberated from the spore wall may J. Bacteriol. 46:299. rebind to the wall of the young hyphae since there is a closed 16. Waksman, S. A. 1956. The role of antibiotics in natural pro- system in the liquid culture. In contrast to this system, the cesses. G. Microbiol. 2:1-14.