genetic analysis of exoenzyme production by this organism. Generally useful ... sensitivity of indicator plates for detection of exoenzyme activities are presented.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1985, p. 1335-1337 0099-2240/85/051335-03$02.00/0 Copyright C) 1985, American Society for Microbiology
Vol. 49, No. 5
Simple Screening Method for Identification of Nonpleiotropic Mutants for Exoenzyme Production KAREN E. ROSE, H. E. HEATH, AND GARY L. SLOAN*
Department of Microbiology, The University of Alabama, University, Alabama 35486 Received 26 October 1984/Accepted 27 February 1985
A differential medium that distinguishes between pleiotropic and nonpleiotropic mutants for exoenzyme production has been developed for Staphylococcus simulans biovar staphylolyticus. The medium will facilitate genetic analysis of exoenzyme production by this organism. Generally useful strategies for increasing the sensitivity of indicator plates for detection of exoenzyme activities are presented.
that heat-killed lyophilized cells of Micrococcus lysodeikticus at a final concentration of 0.4 mg/ml of medium gave optimal sensitivity for detection of this enzyme. The protease produced by S. staphylolyticus is capable of hydrolyzing a wide variety of protein substrates (G. L. Sloan, unpublished data). Casein in skim milk was determined to be the best protein substrate for use in the indicator medium. As previously reported (11), the protease is activated by reducing agents such as dithiothreitol, suggesting that it is a sulfhydryl protease. Our preliminary attempts to optimize proteolysis by colonies of S. staphylolyticus showed that addition of the optimal concentrations of casein (0.5% skim milk) and dithiothreitol (0.15%) increased the size of the zone of casein hydrolysis but did not provide the clear zones needed for unambiguous identification of nonpleiotropic mutants in a medium containing multiple substrates. The lack of clarity of these zones presumably was due to the presence of the initial hydrolysis products (paracaseins), which are relatively insoluble (6, 7). We supposed that the hydrolysis was incomplete owing to diffusion of the protease, which is a small protein (Sloan, unpublished data). We found that the use of a slightly increased concentration of agar (1.75% rather than 1.5%) produced significantly clearer zones of casein hydrolysis, apparently owing to decreased diffusion of the protease. However, this higher agar concentration did not markedly affect zones of cellular lysis due to the endopeptidase or the hexosaminidase. When the three substrates were combined into a single indicator medium, we found that the use of a clear agar underlay below the screening medium improved the sharpness of the zones and enhanced our ability to differentiate between wild-type and mutant colonies. The underlay also decreased the amounts of skim milk and lyophilized cells used per plate. To prepare the multisubstrate indicator medium, 6 g of NZ Amine E (Sheffield Products, Norwich, N.Y.), 0.5 g of NaCl, 0.25 g of K2HPO4, 1.75 g of Bacto-Agar (Difco Laboratories, Detroit, Mich.), 0.03 g of lyophilized S. aureus FDA 209P cells, 0.04 g of lyophilized M. lysodeikticus cells, and 0.15 g of dithiothreitol were dissolved in 90 ml of deionized water. The mixture was boiled to melt the agar, thoroughly mixed, and autoclaved for 15 min at 121°C. Bacto skim milk (0.5 g) was dissolved in 10 ml of deionized water and autoclaved for 5 min at 121°C. The skim milk solution was then added to the agar medium and the mixture was cooled to 50°C, and approximately 10 ml of the screening medium was poured into sterile plastic petri dishes containing approximately 10
Our study of the regulation of exoenzyme production by Staphylococcus simulans biovar staphylolyticus (S. staphylolyticus [12]) has been hampered by difficulties in isolating nonpleiotropic mutants for the production of three exoenzymes: a staphylolytic endopeptidase, a micrococcolytic hexosaminidase, and a sulfhydryl protease. Mutants selected for the loss of one of these three activities invariably have lacked the other two as well (5). Such pleiotropic alterations in exoenzyme production also have been reported for Staphylococcus aureus (1, 2, 8, 13, 17), Serratia marcescens (9, 14), Pseudomonas aeruginosa (3, 15), Bacillus subtilis (16), and Aeromonas hydrophila (4). In this communication, we describe a simple screening method for the isolation of S. staphylolyticus mutants with nonpleiotropic defects in exoenzyme production. This procedure allows the detection of putative nonpleiotropic mutants without the needless assay of many mutants that possess multiple defects in exoenzyme production. The method is based on hydrolysis of multiple insoluble substrates in a single indicator medium. With appropriate modifications, this technique could also be used to study production of a wide variety of exoenzymes by other microorganisms.
To develop a medium containing multiple substrates for the differentiation of mutants for exoenzyme production, we first optimized our ability to identify mutants for each enzyme. We previously have reported the use of lawns of viable S. aureus cells to identify hyperproducing, hypoproducing, and nonproducing mutants for the endopeptidase produced by S. staphylolyticus (5). However, because of the competition between the developing S. staphylolyticus colonies and the S. aureus cells in the lawns, it takes approximately 4 days to identify colonies with alterations in the ability to produce this enzyme. Heat-killed S. aureus cells were substituted for the viable cells that were used in the previously described medium (5) and were found to be more susceptible to lysis by the endopeptidase based on the size of zones of bacteriolysis. In addition, the absence of competition for nutrients allowed identification of variants within 2 days. In attempts to standardize the technique, lyophilized cells which could be weighed and added to the medium before autoclaving were tested and found to be even more susceptible to lysis by the enzyme. A final concentration of 0.3 mg of lyophilized S. aureus cells per ml of medium gave optimal sensitivity for detecting the endopeptidase. Similar studies for detection of the hexosaminidase showed *
Corresponding author. 1335
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APPL. ENVIRON. MICROBIOL.
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FIG. 1. Differentiation of S. staphylolyticus variants producing different combinations of exoenzymes. The composition and preparation of the screening medium were as described in the text. (A) Wild-type organism which produced all three exoenzymes and therefore had a clear zone of cell lysis and casein hydrolysis; (B) endopeptidase-negative variant which had a slightly turbid zone due to the presence of unlysed staphylococcal cells; (C) endopeptidase-negative, hexosaminidase-negative variant which had a moderately turbid zone due to the presence of unlysed staphylococcal and micrococcal cells; (D) pleiotropic nonproducer of the three exoenzymes.
ml of a clear agar underlay (1.5 g of Bacto agar per 100 ml of deionized water). Figure 1 shows that spontaneous or N-methyl-N'-nitro-Nnitrosoguanidine-induced variants of S. staphylolyticus (10) with different eyoenzyme-producing capabilities can be identified easily with the screening medium. The wild-type organism produced all three exoenzymes and, therefore, had a clear zone of cell lysis and casein hydrolysis surrounding each colony (Fig. 1A). A variant that had a slightly turbid zone around each colony (Fig. 1B) was found on subsequent assay of culture supernatant (10, 11) to have lost the ability to produce active endopeptidase, although it still produced active protease and hexosaminidase. These additional assays were required to identify the specific enzyme defect, because the medium did not allow a differentiation among the three types of single mutants. A variant that had a moderately turbid zone surrounding each colony (Fig. 1C) was derived from the organism shown in Fig. 1B. Assay of culture supernatant for the three exoenzymes, as previously described (10, 11), revealed that the organism shown in Fig. 1C produced neither active endopeptidase nor active hexosaminidase but did produce active protease. No zone was observed on the screening medium for a mutant which, based on quantitative assays, did not produce any of the three exoenzyme activities (Fig. 1D). The small size of colonies lacking protease activity presumably was due to decreased availability of nutrients from the medium. To carry out genetic studies on exoenzyme production by bacteria, a screening method is needed by which rare nonpleiotropic mutants can be identified easily in the presence of more common pleiotropic mutants. We have developed such a technique for use in studies on exoenzyme production by S. staphylolyticus and are now using it in
genetic mapping studies and also for the identification of bacterial strains receiving genes for exoenzyme production in recombinant DNA studies. Similar multisubstrate indicator media could easily be developed for other proteases and bacteriolytic enzymes as well as amylases, cellulases, hemolysins, nucleases, P-lactamases, lipases, phosphatases, and many other extracellular enzymes produced by bacteria. LITERATURE CITED 1. Altenberg, R. A. 1975. Membrane mutations and production of enterotoxin B and alpha hemolysin in Staphylococcus aureus. Can. J. Microbiol. 21:275-280. 2. Forsgren, A., K. Nordstrom, L. Philipson, and J. Sjoquist. 1971. Protein A mutants of Staphylococcus aureus. J. Bacteriol. 107:
245-250. 3. Gray, G. L., R. M. Berka, and M. L. Vasil. 1982. Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins. J. Bacteriol. 150:1221-1226. 4. Howard, S. P., and J. T. Buckley. 1983. Intracellular accumulation of extracellular proteins by pleiotropic export mutants of Aeromonas hydrophila. J. Bacteriol. 154:413-418. 5. Larrimore, S. A., S. B. Clark, J. M. Robinson, H. E. Heath, and G. L. Sloan. 1982. Coordinate production of three exoenzymes by Staphylococcus staphylolyticus. J. Gen. Microbiol. 128:15291535. 6. Lawrence, R. C., and W. B. Sanderson. 1969. A micro-method for the quantitative estimation of rennets and other proteolytic enzymes. J. Dairy Res. 36:21-29. 7. Martley, F. G., A. W. Jarvis, D. F. Bacon, and R. C. Lawrence. 1970. Typing of coagulase-positive staphylococci by proteolytic activity on buffered caseinate-ag4r, with special reference to bacteriophage nontypable strains. Infect. Immun. 2:439-442. 8. Omenn, G. S., and J. Friedman. 1970. Isolation of mutants of Staphylococcus aureus lacking extracellular nuclease activity.
VOL. 49, 1985 J. Bacteriol. 101:921-924. 9. Reid, J. D., and D. M. Ogrydziak. 1981. Chitinase-overproducing mutant of Serratia marcescens. Appl. Environ. Microbiol. 41:664-669. 10. Robinson, J. M., J. K. Hardman, and G. L. Sloan. 1979. Relationship between lysostaphin endopeptidase production and cell wall composition in Staphylococcus staphylolyticus. J. Bacteriol. 137:1158-1164. 11. Robinson, J. M., M. S. Keating, and G. L. Sloan. 1980. The characteristics of extracellular protein secretion by Staphylococcus staphylolyticus. J. Gen. Microbiol. 118:529-533. 12. Sloan, G. L., J. M. Robinson, and W. E. Kloos. 1982. Identification of "Staphylococcus staphylolyticus" NRRL B-2628 as a biovar of Staphylococcus simulans. Int. J. Syst. Bacteriol. 32:170-174. 13. Wadstrom, T. 1973. Bacteriolytic enzymes from staphylococci, p. 397-405. In J. Jeljaszewicz (ed.), Staphylococci and staphylo-
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coccal infections. S. Karger, Basel. 14. Winkler, U., and K. Timmis. 1973. Pleiotropic mutations in Serratia marcescens which increase the synthesis of certain exocellular proteins and the rate of spontaneous prophage induction. Mol. Gen. Genet. 124:197-206. 15. Wretlind, B., L. Sjoberg, and T. Wadstrom. 1977. Protease-deficient mutants of Pseudomonas aeruginosa: pleiotropic changes in activity of other extracellular enzymes. J. Gen. Microbiol. 103:329-336. 16. Yoneda, Y., K. Yamane, and B. Maruo. 1973. Membrane mutation related to the production of extracellular a-amylase and protease in Bacillus subtilis. Biochem. Biophys. Res. Commun. 50:765-770. 17. Yoshikawa, M., F. Matsuda, M. Naka, E. Murofushi, and Y. Tsunematsu. 1974. Pleiotropic alteration of activities of several toxins and enzymes in mutants of Staphylococcus aureus. J. Bacteriol. 119:117-122.
