Eur. J. Biochem. 97, 541 -545 (1979)

Methylation of Basic Proteins in Ribosomes from Wild-Type and Thiostrepton-Resistant Strains of Bacillus megaterium and Their Electrophoretic Analysis

Michael CANNON and Eric CUNDLIFFE

Department of Biochemistry, University of London King's College, and Department of Biochemistry, University of Leicester

(Received January 22, 1979)

Ribosomes, radioactively labelled in vivo with both [l-'4C]methionine and [methyl-3H]methio- nine, have been isolated from both wild-type and thiostrepton-resistant strains of Bacillus megaterium and their constituent proteins separated by two-dimensional gel electrophoresis. Ribosomes from the wild-type strain possess one basic protein that is extensively methylated. In contrast no such protein can be detected in ribosomes from the thiostrepton-resistant strain.

Ribosomes from Escherichia coli have been exten- sively studied and electrophoretic analysis has shown them to possess 53-55 individual proteins [l-31 several of which are subject to post-translational modification. In the 50-S particle, for example, at least six proteins contain methylated amino acids [4-61 and this situation has been found to apply particularly to ribosomal protein L11 [4,7]. Furthermore, eukary- otic ribosomes are also methylated in vivo; in HeLa cells for example [8], and in Saccharomyces cerevisiae [9,10] where methylation of one protein, numbered L15 by Cannon et al. [9], is again clearly apparent (cf. [4,7]). Because of this striking similarity between E. coli ribosomal protein L11 and S. cerevisiue ribo- somal protein L15 it may be that these two proteins are in some way homologous.

Ribosomal protein L11 has also featured pronii- nently in studies involving inhibition of bacterial protein synthesis by the antibiotic thiostrepton. This drug prevents the binding of the various protein factors which enter and leave the ribosome during the different stages of protein synthesis and also inhibits the hydrolysis of GTP usually associated with these events (for a review see [ I l l ) . Thiostrepton binds to bacterial 50-S ribosomal subunits [12,13] and recon- stitution studies have indicated that protein L11 figures predominantly in forming the ribosomal recep- tor site for the drug [I31 although other ribosomal proteins are undoubtedly implicated (for a review see [ll]). This type of work has invariably involved studies in vitro with E. coli ribosomes since this bac- terium, along with other gram-negative organisms, is impermeable to the drug.

__ Enzyme. Deoxyribonuclease 1 or DNase 1 (EC 3.1.4.5).

In contrast to the situation for E. coli, intact cells of Bacillus megaterium are freely permeable to thio- strepton. The cells are strongly inhibited by the anti- biotic and we have been able to isolate a thiostrepton- resistant strain of this gram-positive organism. To date, ribosomes from B. megaterium have not been fully characterized and the present communication describes an analysis, by two-dimensional gel electro- phoresis, of ribosomal proteins from both wild-type and drug-resistant strains. In addition cells have been labelled in vivo with radioactive methionine and we have determined which of the ribosomal proteins are subject to methylation.

MATERIALS AND METHODS

Bacterial Strains and Growth Conditions

B. megaterium (strain KM) was transferred from freeze-dried samples and cultured on nutrient agar plates. A strain of B. meguterium (designated PD1) and resistant to thiostrepton was isolated by plating out wild-type cells on nutrient agar plates containing 10 pg/ml thiostrepton. The drug-resistant strain was selected after incubation of the plates at 37°C for 2 days and stored as freeze-dried ampoules. Both wild-type and drug-resistant strains were maintained on nutrient agar plates containing 3 pg/ml thiostrepton and were subcultured every 7 days.

The strains were each grown into late logarithmic phase in 500 ml of M9 salts medium which contained per litre K2HP04 (7 g), KH2P04 (3 g), trisodium citrate (0.5 g), MgS04 . 7H20 (0.1 g), (NH4)2S04 (1 g), glucose (10 g) and NH4Cl (5 g). In addition,

542 Methylation of Ribosomal Proteins in Bacillus nzegarrrizrm

the medium contained 15 pCi of [l-'4C]methionine and 150 pCi of [rnethyl-3H]methionine. Cells were harvested by centrifugation and were washed once with 10 mM Tris-HC1, pH 7.6, Containing 10 mM magnesium acetate, 50 mM NH4CI and 3 mM 2-mer- captoethanol. Cells were stored frozen at - 70 "C. Under the above growth conditions the generation time of the wild-type strain was 50 rnin whereas that of the thiostrepton-resistant mutant was 2 h.

Preparation of Ribosomes

Frozen cells were mixed with 2.5 times their wet weight of alumina and ground for several minutes at 0-4 "C. The paste was extracted with 5 mM Tris-HCI, pH 7.4, containing 10 mM magnesium acetate and 86 mM KCl (4 ml for every 1 g of original wet weight of the cells). Deoxyribonuclease (obtained from Boeh- ringer, Mannheim, F.R.G. and adjusted to a final concentration of 10 &ml) was added and the mixture incubated at 30 "C for 15 min. Sodium deoxycholate (final concentration 0.5 %) was then added and a further incubation carried out for 15 rnin at 30°C. This procedure effectively liberates ribosomes from the intracellular membranes of B. megaterium. The extracts were centrifuged at lOOO0xg for 15 min in an MSE 18 centrifuge and the supernatant fraction retained and then further cleared by centrifugation at 20000 x g for 20 min. The 'crude extract' thus obtained was layered in 2-ml batches over 10 ml of 5 mM Tris-HC1 buffer, pH 7.4, containing 30 mM magnesium acetate, 1 M NH4Cl and 25% (w/v) sucrose, and the tubes centrifuged in a Spinco Ti 50 rotor at 39000 rev./min for 18 h. The supernatant liquid was discarded and the ribosome pellet retained.

Preparation of Ribosomal Proteins

Ribosomes were resuspended (final concentration 2 mg/ml) in 0.1 M MgC12 solution and extracted for 1 h at 0°C with 2 vol. glacial acetic acid. The resultant precipitate was removed by centrifugation at 10000 x g for 15 min and the supernatant liquid mixed with 5 vol. acetone to precipitate protein. The precipitate was left at - 20 "C for 2 h and then collected after centrif- ugation at 10000 x g for 20 min. Protein precipitates were dried under vacuum and dissolved in 200 p1 of a solution containing 8 M urea and 10 mM dithio- threitol. The ribosomal proteins (approx. 1 mg) were analyzed and assayed for radioactivity as described previously [9].

Radioactive Compounds

[l-'4C]Methionine (57 Ci/mol) and [~e thy l -~H] - methionine (6 Ci/mmol) were obtained from The Radiochemical Centre (Amersham, Bucks, U.K.).

RESULTS AND DISCUSSION

Although ribosomes from E. coli and, to a lesser extent, S. cerevisiue are well characterized, those from B. megaterium are not. Accordingly we decided to analyze ribosomes from B. megaterium by two-dimen- sional gel electrophoresis and determine which, if any, of the individual proteins is subject to methylation in vivo.

The gel separation patterns for proteins from 70-S ribosomes from wild-type cells of B. megaterium is illustrated in Fig. 1 A. Only the basic proteins are separated under our experimental conditions and approximately 26 spots can be located on the gel with the majority of these being we11 stained. This gel is represented diagrammatically in Fig. 1 B with the individual protein spots numbered as indicated. This numbering system is arbitrary and must not be com- pared directly with, for example, the published num- bering systems commonly used to illustrate the protein patterns from E. coli [14] and S. cerevisiae ribosomes [9,15]. The present numbering system is used merely to allow Fig. 1 and the data of Table 1 to be related. Clearly, however, the total number of protein spots detected here for B. megaterium ribosomes is low compared with that obtained using E. coli ribosomes (for references see [16,17]). It is, however, very likely that several of the spots on the gel in Fig. 1 represent two or more proteins that have failed to separate under the experimental conditions used. A more rigorous characterization of the exact number of ribosomal proteins involved in forming the B. mega- terium ribosome will ultimately require the separation and analysis of individual ribosomal subunits.

For all our experiments ribosomes were prepared from cells previously labelled in vivo with both [1-14C]- methionine and [methyl-3H]methionine. This proce- dure allows the 3H/'4C ratio for each isolated protein to be determined and this ratio can then be compared directly with that obtained for the total ribosomal protein population. A significant increase over this latter ratio, for any one protein, is taken to indicate methylation [4]. The results of our analysis are shown in Table 1. Ribosomes from B. megaterium clearly possess one basic protein (number 11) that is more heavily methylated than any other protein present on the gel (Fig. 1) although other proteins (Le. numbers 1, 3, 9, 12 and 17) may also contain methylated amino acids. Elsewhere we have shown that this protein (number 11) is immunologically homologous to ribo- somal protein L11 of E. coli and it is accordingly designated 'BM-L11' [18]. A similar pattern of methy- lation is also apparent in ribosomes from S. cerevisiae [9,10] although the apparent extent of methylation does, however, differ significantly in the three cases now considered. Thus, for S. cerevisiae, a protein designated L15 [9] shows a fourfold increase in the

M. Cannon and E. Cundliffe 543

B

26 25 m * 28 0

Fig. 1. Separation of ribosomal proteins from 70-S ribosomes of B. megaterium (wild-type strain) by two-dimensional gel electro- phoresis. The experimental conditions are described in Materials and Methods. (A) Electrophoretic separation of proteins. (B) Dia- grammatic representation of the numbering system used to identify individual proteins. Migration in the first dimension was from left to right and in the second from top to bottom

Table 1. Levels of incorporation of [ 1-14C]methionine and [methyl- 'H]methionine into proteins found in 70-S ribosomes from wild-type B. megaterium B. megateriurn (wild-type) was grown in the presence of both [l-14C]methionine and [methyl-3H]methionine. Cells were harvested and 70-S ribosomes isolated. Ribosomal proteins were prepared and analyzed by two-dimensional gel electrophoresis. The protein spots were located by staining and then excised and counted for both 3H and I4C. All experimental details are outlined in Materials and Methods. The 3H/14C ratio for the total unfractionated ribo- somal protein was 3.8: 1. An asterisk marks the proteins which are considered to have methyl groups present in excess over methionine. The criterion used for this is that the average d3H/14C for the two protein preparations (wild-type source and mutant source) should be greater than + 0.5

Protein 3H I4C 3H/14C LI 'H/I4C incorporated incorporated ratio

counts/min

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

400 1868 2838 1046 900 700 62

436 860 812

2522 440

1638 668

1400 1200 430

1182 650 646 400 180 810 128 688 72

171 152

86 454 650 308 264 200

15 98

196 186 354 94

400 168 374 320 96

332 200 170 118 50

226 36

204 20 50 45

4.65 4.11 4.31 3.40 3.41 3.50 4.13 4.45 4.39 4.36 7.12 4.68 4.09 3.98 3.74 3.75 4.48 3.56 3.25 3.80 3.39 3.60 3.58 3.55 3.37 3.60 3.42 3.38

+ 0.85* + 0.31 + 0.57* - 0.40 - 0.39 - 0.30 + 0.33 + 0.65 + 0.59* + 0.56 + 3.32* + 0.88* + 0.29 + 0.18 - 0.06 - 0.05 + 0.68* - 0.24 - 0.55

0.00 - 0.41 ~ 0.20 - 0.22 - 0.25 - 0.43 - 0.20 - 0.38 ~ 0.42

3H/14C ratio relative to that of the unfractionated ribo- somal protein and in E. coli the corresponding increase for protein L11 is approximately 50 % greater than the control [4]. Protein BM-L11 from B. megaterium is intermediate in this respect with a corresponding increase of twofold over the control for the 3H/'4C ratio (see Table 1).

Intact cells of B. rnegaterium are strongly inhibited by thiostrepton and this has allowed us to produce a thiostrepton-resistant strain of the organism (desig- nated PD1). Ribosomes from this mutant were iso- lated and proteins prepared and analyzed by two- dimensional gel electrophoresis. The separation ob-

tained is illustrated in Fig.2 with the numbering system for the individual proteins based exactly upon that used in Fig. 1 B. Clearly the two patterns (cf. Fig.1 and 2) are identical except in one area of the gels. Thus, the spot arrowed in Fig. 1 B is apparently absent in Fig.2B although in the latter case a small 'additional' spot can be observed just above the expected position of the 'missing' wild-type protein. As indicated from the data of Table 1 this 'missing' protein is the one (BM-L11) that is heavily methylated. Determination of the 3H/'4C ratios for the ribosomal proteins of the thiostrepton-resistant mutant confirms the above picture. Thus, it can be seen from Table 2

544 Methylation of Ribosomal Proteins in Bacillus megatrr-ium

Table 2. Levels of incorporation of [I-i4C]metl~ionine and [methyl- 'Hlmethionine into proteins found in 70-S rihosomes.from thiostrep- ton-resistant B. megaterium All experimental conditions are described under Table 1 except that the thiostrepton-resistant strain (PDl) of B. megaterium was used. The 'H/I4C ratio for the totdl unfractionated ribosomal protein was 3.85: 1

~~

Protein 'H 14C 3H/14C d 'H/I4C incorporated incorporated ratio

1 2 3 4 5 6 7 8 9

10 I l a 12 13 14 15

280 1152 2050 800 600 400

49 304 600 600 280 360

1400 548

1000

~

counts/min - ~-

62 276 450 222 156 108

13 76

130 I40 64 80

310 134 244

4.52 + 0.67* 4.37 + 0.32 4.55 + 0.70* 3.60 - 0.25 3.85 0.00 3.70 - 0.15 3.17 - 0.08 4.00 + 0.15 4.61 + 0.76% 4.28 f 0.43 4.38 + 0.53 4.50 i 0.65* 4.52 + 0.67 4.09 + 0.24 4.10 + 0.25

1 2 7 16 800 200 4.00 + 0.15

18 590 150 3.93 + 0.08 19 530 152 3.49 - 0.36 20 480 140 3.43 - 0.42 21 300 78 3.85 0.00

l?#Z 2 0 22 100 29 3.45 - 0.40 23 504 140 3.60 - 0.25 24 101 30 3.37 - 0.48

2 8 2 7 OZ4 0

0 4 5 0 17 342 76 4.50 + 0.65% 358z; =6 a 12

'"@-14 2 0 26 c 15 0

1 7 0 16

0 8 23 * B 0 25 400 110 3.64 - 0.21

26 50 15 3.33 - 0.52 Fig. 2. Separation (8 rihosomul proteins from 70-S ribosomes of 27 110 34 3.23 - 0.62 B. megaterium ithiostrepton-resistant strain) hy two-dimmsional 28 100 30 3.33 - 0.52 gel electrophoresis. The experimental conditions are described in the legend to Fig. 1, (A) Electrophoretic separation of proteins. (B) Diagrammatic representation of the numbering system used to identify individual proteins

that not one of the ribosomal proteins of mutant PDI is significantly methylated by comparison with BM- L11. This negative finding also applies to the small 'additional' spot referred to above and identified in Table 2 as protein number l l a . This protein has very weak staining properties as is apparent from Fig. 2A. Furthermore it is possible that this same protein is indeed present on Fig. 1 A with its appearance being masked by the presence of the dense staining protein

We conclude from these data that ribosomes from the thiostrepton-resistant mutant of B. megateviunz (PDI) are devoid of a protein that is methylated in ribosomes of the wild type. Elsewhere [18] we have demonstrated rigorously that the mutant PD1 and other similar thiostrepton-resistant strains of B. mega- teriurn are lacking a protein (BM-Lll) that is immuno-

BM-Lll.

logically homologous with ribosomal protein L11 from E. coli. The data reported here confirm this homology to the extent that proteins L11 and BM-L1 1 are the most heavily methylated proteins in their respective ribosomes. By comparison with E. coli ribosomes [13] we should expect protein BM-L11 to be a vital component of the receptor site for thio- strepton in ribosomes of wild-type B. megatevium.

Other thiostrepton-resistant bacterial strains have in fact been obtained from Bacillus subtilis and it has been claimed that they have altered ribosomes [19]. Again a protein that is homologous with E. coli ribo- somal protein L11 is involved here. In contrast a strain of E. roli (resistant to the thiostrepton-related com- pound thiopeptin) and isolated following mutagenesis has been claimed [20] to contain an altered form of ribosomal protein L5 although it is not clear whether

M. Cannon and E. Cundliffe 545

or not additional components may be modified in this case. It does appear, however, that the mutation con- ferring resistance to thiostrepton described in the present work is of a completely different type from many other mutations conferring resistance to other antibiotics (e.g. the strA mutation, see [21]) since our mutant is actually devoid of a ribosomal protein. Further work will be required with our mutant to characterize the antibiotic-resistant lesion more fully.

Finally, we note that the organism Streptomyces U Z U Y ~ U S which produces thiostrepton but is itself totally resistant to the drug modifies its own ribosomes in a mechanism involving specific methylation of 23-S ribosomal RNA [22]. It is, therefore, of particular relevance that the normal target site for thiostrepton on ribosomes from several bacterial species involves a different macromolecule (i.e. protein L11 in E. coli or its homologous form in other bacteria) that is itself methylated.

REFERENCES

1. StoHer, G. (1974) in Ribosomes (Nomura, M., Tissieres, A. & Lengyel, P., eds) pp. 615-667, Cold Spring Harbor Labo- ratory, New York.

2. Pettersson, I.. Hardy, S. J. S. & Liljas, A. (1976) FEBS Lett. 64, 135- 138.

3. Chu, F., Caldwell, P., Samuels, M., Weissbach, H. & Brot, N. (1977) Biochem. Biophys. Res. Commun. 76, 593 -601.

4.

5.

6.

I. 8.

9.

10.

11.

12.

13.

14. 15. 16.

17.

18.

19.

20.

21.

22.

Chang, F. N., Chang, C. N. & Paik, W. K . (1974) J . Bucteriol.

Chang, C. N. & Chang, F. N. (1974) h'ature (Lond.) 251.

Chang, C. N. & Chang, F. W. (1975) Biochemistry, 14, 468-

Alix, J.-H. & Hayes, D. (1974) J . Mol. B i d . 86, 139-159. Chang, F. N., Navickas, I. J., Chang, C. N. & Dancis, B. M.

(1976) Arch. Biochem. Biophys. 172, 627-633. Cannon, M., Schindler, D. & Davies, J. E. (1977) FEBS Lett.

75, I 87 - 1 91 . Hernandez, F., Cannon, M. & Davies, J . E. (1978) FEBS Lett.

89,271 - 275. Cundliffe, E. (1979) in Antihiotics (Hahn, F E., ed.) vol. 5 ,

in the press, Springer-Verlag, New York. Sopori, M. L. & Lengyel, P. (1972) Biochem. Biophys. Res.

Commuii. 46, 238 - 244. Highland, J. H., Howard, G. A. & Gordon, J . (1975) Eur. .J.

Biochrm. 53,313-318. Hardy, S. J . S. (1975) Mol. Gen. Genet. 140, 253-274. Kruiswijk, T.&Planta, R. J.(1974)MoI. B i d . Rep. 1,409-415. Wittmann, H . G. (1974) in Ribosomes (Nomura, M., Tissiires,

A. & Lengyel, P., eds) pp. 93-114, Cold Spring Harbor Laboratory, New York.

Stoffler, G. & Wittmann, H. G. (1977) in Molecular Me~~fuw1s~rz.s of Protein Biosynthesis (Weissbach, H . Sr. Pestka, S., eds) pp. 117-202, Academic Press, New York.

Cundliffe, E., Dixon, P., Stark, M., Stoffler, G., Ehrlich, R., Geisser, M. & Cannon, M. (1979) J . Mol. Biol. in the press.

Pestka, S., Weiss, D., Vince, R., Wienen, B., Stoffler, G . & Smith, I. (1976) Mol. Gen. Genet. 144, 235-241.

Liou, Y. F., Yoshikawa, M. & Tanaka, N. (1975) Biochem. Biophys. Rex Commun. 65, 1096- 1101

Ozaki, M., Mizushima, S. & Nomura, M. (1969) Nature (Lond.) 222,333-339.

Cundiiffe, E. (1978) Nature (Lond.) 272, 792-795.

120, 651 -656.

131 -733.

477.

M. Cannon, Department of Biochemistry, University of London King's College, Strand, London, Great Britain, WC2R 2LS

E. Cundliffe, Department of Biochemistry, School of Biological Sciences, University of Leicester University Road, Leicester, Great Britain, LEI 7RH