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FEMS MicrobiologyLetters 70 (1990) 91-94 Published by Elsevier 91 FEMSLE 04048 The patterns of extracellular protein formation by spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus B. AI-Ani, M. A b o s h k i w a , R.E. Glass a n d G. C o l e m a n Department of Biochemistry. Nottingham University Medical School. Queen's Medical Centre. Nottingham. U.K. Received5 December 1989 Received28 February 1990 Accepted 15 March 1990 Key words: Staphylococcus aureus; Extracellular protein; Rifampicin-resistant mutants 1. S U M M A R Y Spontaneous!y-occurring rifampicin-resistant mutants of Sataphylococcus aureus were isolated on 4 ~ ( w / v ) Tryptone Soya Agar containing 4 and 40 times the m.i.c, for rifampicin. A number of colonies were selected at each rifampicin concentration and were grown aerobically in 3~ ( w / v ) Tryptone Soya Broth for 24 h at 37°C. In the case of S. aureus RN4220 all the mutants grew to bacterial densities up to approximately 1.7 times more than the parent organism. The corresponding levels of extracellular protein secretion varied over a 5-fold range, all the mutants being less productive than the parent. By contrast, mutants of the wild-type Wood 46 strain achieved bacterial densities of only 4 5 - 8 3 ~ that of the parent whilst exoprotein secretion showed a smaller 1.7-fold variation. However, widely-differing patterns of exoproteins were revealed by SDS-polyacryla- Cc~,'~.spondence m: Dr. G. Coleman, Department of Biochem- istry, Nottingham University Medical School, Clifton Bouleyard, Nottingham NG7 2UH, U.K. mide gel electrophoresis of the parevt and mutant organisms of both strains. 2. I N T R O D U C T I O N Biochemical studies on the control of extracellular protein formation in the Gram-positive organism Staphylococcus aureus showed a characteristic pattern of secretion in which massive production of exoprotein occurred after the end of exponential growth. This was accounted for in terms of more resources being available for increased exoprotein formation when cell growth decreased [1]. More recently, Recsei et al. [2] demonstrated the existence of a trans-active positive control element which had a pleiotropic effect on the production of a number of exoproteins. This finding was confirmed by Jargon et al. [3] who showed a similar effect exerted at the level of transcription which was growth-rate dependent. These data supported the conclusion that a key factor in the regulation is the affinity of R N A polymerase for specific promoters [1]. 0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties Glass and coworkers [4] have carried out a detailed genetic analysis of the role of the/~-subunit of E. coli RNA polymerase in DNA transcription and they have demonstrated that single amino acid substitutions can result in altered promoter selectivity [5]. They isolated spontaneouslyoccurring amber mutants which arose by single base changes in a small defined region of the RNA polymerase/i-subunit gene. The isolation of spontaneously-occurring rifampicin resistant mutants of S. aureus [6] provides a convenient means of generating organisms with altered RNA polymerases. The effects of these changes on the patterns and characteristics of extracellular protein formation have been examined. tone soya agar containing 10 /tg ml -~ chioramphenicol where growth was observed in all cases. Mutants of S. aureus (Wood 46) were isolated in exactly the same manner the only difference being the omission of chlorampbenicol. 3. MATERIALS AND METHODS 3. 4. Bacterial density determination The method of Stormonth and Coleman [8] was employed. 3.1. Organisnts Staphylococcus aureus RN4220, a mutant organism derived through a series of steps from NTCC 8325, was obtained from dr. Richard Novick. It was transformed by electroporation with plasmid pCK1 which carries a chloramphenicol resistance gene [7]. This provided a convenient "marker" to ensure that Rif-r mutants were derived from the parent organism and not from contaminants. The wild-type organism S. aureus (Wood 46) (NCTC 7121) was also used. 3.2. Isolation of mutants 3~ (w/v) Tryptone Soya Broth (Oxoid) containing 10/tg m l - i chloramphenicol (Sigma) was inoculated with the parent organism (S. aureus RN4220+pCK1) and incubated overnight at 37°C. Aliquots of the culture, 0.1 ml containing approximately l0 s bacteria, were spread evenly on 4~ (w/v) Tryptone Soya Agar (Oxoid) containing 0.08 or 0.8 /tg ml -~ rifampicin (Lepetit, Milan, Italy), that is, 4 and 40 times, respectively, the m.i.c. After incubation for 24 h at 37°C, a small number of mutant colonies appeared on each plate which were purified on the same rifampicin-containing medium and a single colony selected in each case. All the isolated mutants were checked for chloramphenicol resistance on 4% (w/v) Tryp- 3. 3. Growth of the organisms 3~ (w/v) Tryptone Soya Broth, 50 ml batches in 250 ml conical flasks, was inoculated with the rifampicin-resistant mutants and the parent organisms and incubated at 37 ° C in a Gyrotary incubator-shaker (New Brunswick Scientific Co. Inc., New Jersey, U.S.A.). After 24 h samples were taken for assay of bacteria density, total exoprotein and for SDS-polyacrylamide gel electrophoresis. 3.5. Extracellular protein estimation The protein content of culture supernatant fractions was estimated by the method of Scdmak and Grossberg [9] as described by Coleman et al. [101. 3.6. SDS-polyacrylamide gel electrophoresis This was carried out as described by Laemmli [11]. Samples were prepared, in each case, by precipitating the exoprotein from 1 ml aliquots of culture supernatant fraction with 0.1 mi 100~ (w/v) trichloroacetic acid. The precipitates were dissolved in 95 /tl sample buffer plus 5 /tl of a saturated solution of Tris (Sigma) and 15/tl was introduced into each sample well. 4. RESULTS AND DISCUSSION Three rifampicin-resistant mutants isolated on 4% (w/v) Tryptone soya agar containing 4 times the m.i.c, and three isolated in the presence of 40 times the m.i.c, for rifampicin were chosen at random, in each case. They were incubated aerobically in 3% (w/v) Tryptone soya broth for 24 h at 37°C after which time growth had ceased. The bacterial densities and extracelhilar protein levels in the culture media were determined in each case and compared with the values obtained for the parent organisms. It can be seen in Table 1 that the parent organism, S. aureus RN4220 + pCK1 achieved the highest level of exoprotein from the lowest bacterial density. The exoprotein formed per mg of bacterial dry weight was 127/~g, which was 1.6 times higher than that of mutant 2 which produced more exoprotein than any of the other mutants from the lowest mutant bacterial density. Mutant 1 achieved the highest bacterial density and, within the limits of experimental error, the equal lowest amount of exoprotein. However, the other mutants did not all conform to the same pattern. Mutants 3 and 5 which were isolated in the presence of different rifampicin concentrations achieved the same bacterial density and the same exoprotein levels. Further, they produced the same SDS-polyacrylamide gel electrophoresis pattern (Fig. 1). This suggested that they were identical or that the mutation in each was in a region which produced the same effect. The parent produced a characteristic SDS-polyacrylamide gel electro- Table 1 Bacterial densitiesand extracellular protein levelsin a culture of Staphylococcus aureu~ RN4220, transformed with plasmid pCKI, and a number of rifampicin-resistantmutants after 24 h growth at 37°C in 3% (w/v) Tryptone soya broth Rifamplein-resistammutants 1-3 wereisolatedon 40 times the m.i.c, and mutants 4-6 on 4 times the m.i.c, of rifampicin.All values were the mean of dupUcate assays with a standard deviation of leas than 5:5% Oqpmism Parent Mutant I Mutant 2 Mutant 3 Mutant 4 Mutant 5 Mutant 6 Bacterial density (ms dry wt. ml- l ) 2.01 3.37 2.30 2.83 2.47 2.86 2.69 Exoprotein Exoprotein formed per mg bacteria (~g) (~gml - t ) 253 56 180 102 54 106 69 ! 27 17 78 36 22 37 26 w W w M P 1 2 N 3 4 ..... 5 6 Fig. 1. SDS-polyacrylamidegel electrophoresispatterns of the extracellular proteins produced in a 24 h culture of Staphylococcus aureus RN4220. transformedwith plasmid pCKI (lane P) and a number of rifampicin-resistantmutants (lanes 1-6). as in Table i, g-own in 3% (w/v) Tryptone soya broth at 37°C. Exoproteins from the same volume of culture supernatant fractions were loaded in adjacent lanes. Molecular weight marker proteins (MW-SDS-70L;Sigma)wereseparated in lane M. from top to bottom. 66. 45. 36. 29. 24. 20.1 and 14.2 kDa. phoresis pattern which was the same in the presence and absence of plasrmd p C K I . The similarity between the patterns of exoproteins produced by mutants 3 and 5, as shown in Fig. 1, appeared to be extended to mutant 6 which, however, produced considerably less total exoprotein. The other rifampicin-resistant organisms were quite different from each other and from the parent organism. These differences cannot be ascribed to proteolytic degradation since the patterns and amounts of protein in the supernatant fractions remain unchanged even after prolonged incubation. in order to establish that the changes reported were due to spontaneous mutations resulting in rifampicin resistance and independent of the organism's origin a parallel study was carried out with a wild-type bacterium from a completely different source, namely, S . a u r e u s (Wood 46). This further study provided results which led to the same conclusions as those obtained with the mutant RN4220 strain. On the basis of the frequency of the occurrence of the spontaneously-occurring rifampicin-resistant mutants, the mutations would be expected to have arisen by single base changes resulting in single amino acid substitutions in the fl-subunit [5]. These substitutions have been shown to cause dramatic changes in the characteristics of secretion and patterns of formation of extracellular proteins by $. aureus. In E. coil single amino acid substitutions in the /~-subunit of R N A polymerase can result in altered promoter selectivity [5]. However, whilst our data suggest a role for the transcriptional apparatus in the regulation of expression of particular exoproteins whether this involvement is direct or indirect and where the regulatory target is located remains the subject of an ongoing investigation. S . aure~;s P.~,220 was chosen for this study due to its ability to be readily transformed to chloramphenicol resistance by plasmid pCK1. This choice seemed justified since its characteristics of total extracellular protein secretion were very similar to those of the widely-studied wild-type organism S. aureus (Wood 46). Thus, the parent organisms produced 127 and 139 lag exoprotein per mg bacteria, res~.ectiwly, after 24 h growth in 3% ( w / v ) Tryptone soya broth. It is interesting to note that Peng et al. [12] reported that S. aureus RN4220 was low in exoprotein gene expression (exp-) which is not in agreement with the similarity, quoted above, between RN4220 and Wood 46. The reason for this disagreement is not known, although, unexplained differences of this type have been recorded in the RN4220-related 8325-4 strain (Novick, personal communication). In the present case the apparent disagreement could be associated with differences in composition of the growth media. Thus, Peng et al. [12] used a 1% casamino a c i d / l % yeast extract/0.5% glucose-containing medium whereas in this work the orsanisms were grown in 3~ Tryptone soya broth which was shown by Coleman and Abbas-Ali [13] to support a lower growth rate but a higher exoprotein productivity than a richer casein hydrolysate/yeast extract medium. More recently, Coleman et al. [14] showed that the presence of glucose had a marked effect on exoprotein production by S. aureus strain VS. The addition of 1~ glucose to a washed suspension of the bacteria, from an overnight culture, in 3~0 Tryptone soya broth resulted in a 10-fold reduction of exoprotein formation to a level closer to that observed in an e x p - mutant. REFERENCES Ill Coleman,G. (1984) in Bacterial Protein Toxins (Alouf, J., Fchrenbach, F., Freer, J. and Jcljaszewicz,J., eds.), pp. 99-106, Academic Press, London. [21 Recse/, P., Kreiswirth, B., O'Reilly, M., Schlieven, P., Gruss, A. and Novick, R.P. (1986) MOl. Gen. Genet. 202, 58-61. [3] Janzon, L., Lofdahl, S. and Arvidson, S. (1986) FEMS Mi~robiol. Lctt. 33,193-198. [4] Nene, V. and Glass, R.E. (1984) Mol. Gen. Genet. 194, 166-172. [5] Glass, R.E., Jones, S.T., Nene. V., Nomure. 1"., Fuiiita. N. and lshihama, A. (1986) MOl. Gen. Genet. 203, 487-491. [6] Morrow, TO. and Harmon, S.A. (1979) J. Bacteriol. 137, 374-383. [7] Gasson, MJ. and Anderson, P.H. (1985) FEMS Microbiol. Leu. 30,193-196. [8] Stormonth, D.A. and Coleman, G. 0972) J. Gen. Microbiol, 71, 407-408. [9] Sedmak, J.J. arid Grossberg, S.E. (1977) Anal. Biochcm. 79, 544-552. [10] Coleman, G., Jakeman, C.M. and Martin, N. (1978) J. Gen. Mierobiol. 107,189-192. [11] Laenunll, U.K. (1970) Nature 227, 680-685. [12] Peng, H.oL,, N0vick, R.P., Kn:iswirth, q., Kornblum, J. and Scldievert,P. (1988) J. Baet¢fiol.170, 4365-4372. [13] Coleman,G. and Abbas-Afi, B. (1977) Infect. lmmun. 17, 278-281. [14] Coleman,G., Ab0shkiwa, M. and AI.Ani, B. (1989) FEMS Mier0biol. Lett. 61, 247-250.