Isolation and characterization of temperature-sensitive mutants of

MGG

Molec, gen. Genet. 149, 297-302 (1976)

© by Springer-Verlag 1976

Isolation and Characterization of Temperature-Sensitive Mutants of Escherichia coil with Altered Ribosomal Proteins Katsumi Isono and Johanna Krauss Max-Planck-Institut ftir Molekulare Genetik, Abt. Wittmann, Ihnestr. 63-73, D-1000 Berlin 33

Yukinori Hirota National Institute of Genetics, Mishima, Japan

Summary. The ribosomal proteins of temperaturesensitive mutants of Escherichia coli isolated independently after mutagenesis with nitrosoguanidine were analyzed by two-dimensional gel electrophoresis. Out of 400 mutants analyzed, 60 mutants (15%) showed alterations in a total of 22 different ribosomal proteins. The proteins altered in these mutants are $2, $4, $6, $7, $8, S10, S15, S16, S18, L1, L3, L6, L10, L l l , L14, L15, L17, L18, L19, L22, L23 and L24. A large number of them (25 mutants) have mutations in protein $4 of the small subunit, while four mutants showed alterations in protein L6 of the large subunit. The importance of these mutants for structural and functional analyses of ribosomes is discussed.

1. Introduction The ribosome of Escherichia coli consists of three RNA molecules and fifty-four different proteins. Many of the genes coding for these ribosomal components are clustered at two major loci: at 72 rain and at 88 min of the new E. coIi genetic map (Bachmann et al., 1976). A few of them, however, are located away from these clusters. For example, the gene for protein S18 of the small subunit was shown to be located at 94 rain (Bollen et al., 1973; de Wilde et al., 1974). Likewise, the genes for $2 (4 min) and $20 (0.5 rain) were found to be located distantly from the two major clusters (B6ck et al., 1974; Nashimoto and Uchida, 1975; Friesen et al., 1976; Takata, 1976). More recently, the genes for S15 and L21 were detected in the neighborhood but separated from the 72 min cluster (Takata, personal communication). The locations of the genes for proteins S1, $6, S16, $21, L9, L19, L20, L27, L28, L31, L32, L33 and

L34 are so far not known. These facts suggest that apparently there exist quite a few transcriptional units of ribosomal protein cistrons. The question, therefore, arises how they are regulated in vivo. In an attempt to explore studies on the regulatory mechanisms in the synthesis and function of ribosomes in vivo, we undertook isolation of temperaturesensitive mutants of E. coli with altered ribosomal proteins. Since its molecular weight has been computed to be 2.5-2.8 x 109 daltons (Cooper and Helmstetter, 1968; Ohtsubo et al., 1974; Bachmann et al., 1976), E. coli DNA is potentially capable of coding for polypeptides with a total of 1.3-1.5 x 106 amino acids, if all regions of the molecule are expressed. This value would be equivalent to 5000~6000 cistrons, if we assume the average molecular weight of E. coli proteins to be some 20,000 daltons. The regions of DNA coding for various RNAs (ribosomal as well as transfer RNAs) are less than 0.5% and can be neglected for this calculation. We have isolated 5000 temperature-sensitive mutants; a number which has already reached the total number of E. coli genes estimated above. Moreover, since these mutants received rather heavy mutagenesis, most of them are expected to carry multiple mutations both in the genes which are indispensible for growth in a rich medium at high temperatures and had been selected in our isolation procedure as well as in those genes dispensible for growth in a rich medium which were not selected. It would be quite likely, therefore, to find relevant mutants by examining randomly the ribosomal proteins of each mutant in this collection. Below we describe the results of the isolation and characterization of sixty temperature-sensitive mutants of E. coli harboring mutations in a total of twenty-two different ribosomal proteins.

K. Isono et al. : Ribosomal Protein Mutants of E. coli

298

2. Material and Methods

Table 1. Temperature-sensitivemutantswith altered 30S ribosomal proteins

a) Isolation of Mutants An E. coli K-12 strain, PA3092 (F-, thr, leu, trp, his, thyA, argH, thi, lacY, malA, mtl, xyl, tonA, supE, str) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine as described (Adelberg et al., 1965). Temperature-sensitive mutants of independent origin which grew at 30°C but not at 40°C on L-agar plate (Lenox, 1955) were isolated by a replica plating technique (Lederberg and Lederbcrg, 1952). Five thousand mutants were thus isolated and stocked. This mutant-collection served as the source of mutants with altered ribosomal proteins. The detailed nature of this collection will be described elsewhere.

b) Preparation of Ribosomes and Ribosomal Proteins Mutants were grown at 30°C in a rich medium containing per liter of distilled water: Bactotryptone, 10 g; yeast extract, 10 g; KHzPO~, 5.6 g; K2HPO4.3 H20, 37.9 g; t h i a m i n e HC1, 10 mg; thymidine, 20 mg. Cells were harvested at early stationary phase and kept frozen until use. Frozen cells were disrupted by grinding with twice their weights of alumina (Alcoa), and ribosomes were extracted with 8 ml of Tris-HC1 buffer, pH 7.5, containing 60 m M NH4C1, 10 m M MgCla and 6 m M 2-mercaptoethanol. After lowspeed centrifugation, ribosomes were sedimented by centrifugation at 45,000 rpm for 3.5 h in a Spinco Ti 50 rotor, resuspended in 1 ml of the above buffer and clarified by low-speed centrifugation. Ribosomal proteins were extracted with 67% acetic acid (Hardy et al., 1969), dialyzed extensively against 2% acetic acid and lyophilized.

3. Results and Discussion 400 mutants were cultured, their ribosomes prepared and the ribosomal proteins analysed by two-dimensional gel electrophoresis. The electrophoretic profile of the ribosomal proteins of the parental strain, PA 3092, is shown in Figure 1 which serves as control. Figures 2 and 3 show the mutational alterations detected in the proteins of the small and of the large ribosomal subunit, respectively, of some of the temperature-sensitive mutants we have isolated. Distinct changes were observed in one protein or, in some cases in two proteins (e.g. in mutants JE 101 and JE 159). Altogether, the proteins altered in these mutants are $2, $4, $6, $7, $8, S10, S15, S16 and S18 of the small subunit (Fig. 2) and L1, L3, L6, L10, Lll, L14, L15, L17, L18, L19, L22, L23 and L24 of the large subunit (Fig. 3). The number of mutants thus identified to have alterations in the ribosomal proteins is sixty (see Tables 1 and 2), which is about 15% of the total number of temperature-sensitive mutants analysed. This percentage is much higher than expected on the basis that the number of genes coding for the ribosomal proteins is fifty-three and comprises about 1% of the total genes of E. coli. Possible reasons for this may be that we selected only those mutations which

Protein

Mutant

$2 $4

JE JE JE JE JE JE JE JE JE JE JE JE

$6 $7 $8 S10 S15 S16 S18

932 16", JE 57, JE 110, JE 216, JE 221, JE 228, 235 ~, JE 236, JE 241, JE 265 b, JE 359, JE 369 b, 398b, JE 617U, JE 621,JE 626, JE 659, JE 660, JE 702, 709, JE 753, JE 772, JE 930, JE 931, JE 959 210 101 a, JE 163 937" 159~,JE 904 132 101 a 16a, JE 190

" Mutants JE 16, JE 101, JE 159 and JE 937 are double mutants b These mutants harbor an additional alteration in either protein $5 or protein LI 1 (see text)

Table 2. Temperature-sensitivemutantswith altered 50S ribosomal proteins Protein

Mutant

LI L3 L6 L10 Lll L14 L15 L17 L18 L19 L22 L23 L24

JE JE JE JE JE JE JE JE JE JE JE JE JE

a

217, JE 224 68 64, JE 143, JE 169, JE 271 283 104, JE 937 a 234, JE 645, JE 717 92, JE 159~,JE 294 393 253 274, JE 942 55 179, JE 288 357

Mutants JE 159 and JE 937 are double mutants

had occurred in the genes essential for growth in a rich medium and also that we used nitrosoguanidine as mutagen which has been shown to induce multiple mutations primarily at the D N A replication point (Cerdfi-Olmedo etal., 1968; Guerola etal., 1971). However, the mutations resulting in two altered proteins in one mutant (for example, JE 16 and JE 937; see Tables 1 and 2) do not map in a small region of the bacterial chromosome as expected from the action of nitrosoguanidine. Tables 1 and 2 summarize the mutational alterations in the temperaturesensitive mutants we have examined. JE 210 (Fig. 2c) has a rather drastic alteration in protein $6. This protein has been shown to have six glutamic acid residues in one row at its C-terminus (Hitz et al., 1975). It is likely that mutant JE 210 has a mutation in the C-terminal region of protein

299

Fig. 1. Two dimensional gel electrophoresis of 70S ribosomal proteins of the parental strain PA 3092. Nomenclature of each protein is according to Wittmann et al. (1971). The protein L34 has migrated out

Fig. 2a-g. Two-dimensional gel electrophoresis of 70S ribosomal proteins of temperature-sensitive mutants with alterations in 30S ribosomal proteins. Only the portions of electropherograms containing altered proteins are shown. Arrows indicate the proteins altered by mutations (see Table 1) and circles indicate the positions of corresponding wild type proteins, a JE 932; b JE 235; c JE 210; d JE 101 ; e JE 159; f JE 132; g JE 190

300


Fig. 3a-j. Two-dimensional gel electrophoresis 70S ribosomal proteins of temperature-sensitive mutants with alterations in 50S ribosomal proteins (see Table 2). a JE 224; b JE 68; c JE 169; d JE 717; e JE 393; f J E 253; g JE 942; h JE 55; i JE 179; j JE 357

$6 and has lost some of the consecutive g l u t a m i c acid residues, thus its $6 having become less acidic. This deduction is based on its resemblance to a mutant whose protein $6 has lost four of the six consecutive glutamic acid residues at its C-terminus (Hitz et al., 1975) and which migrates on the gel electropherogram to a similar position as that of mutant JE 210. The location of the gene coding for protein $6 is so far not established, and JE 210 will be useful for genetic studies along this line. As shown in Table 1, 25 out of 60 mutants so far analyzed showed alterations in protein $4 which are schematically illustrated in Figure 4. Protein $4 was found to cover a large region of 16S R N A when it binds to it, and it plays a key role in the assembly of the small subunit. Streptomycin-dependent mutants often yield streptomycin-independent "rever-

tants" with drastic alterations in protein $4 in a very similar fashion to that shown in Figure 4 (Hasenbank et al., 1973). A mutated protein $4 which has also an activity to suppress streptomycin-dependence and was derived from a mutant originally selected for temperature-sensitiveness was found to cause an impairment in the assembly at a non-permissive temperature of the 30S subunit (Olsson et al., 1974). Our preliminary experiments showed that some of the temperature-sensitive mutants with altered protein $4 had a distinct defect in the ribosome assembly at 42 ° C. A similar case was reported for a temperature-sensitive mutant with altered protein $4 (Rosset et al., 1971 ; Feunteun et al., 1974a, b). Among the mutants with altered large subunit proteins, four mutants harbored mutations in protein L6. JE 169 (Fig. 3 c) is one of them. This protein has


$3

©

OL 2

L3

0

o

®io0O 0

0

S5+Lll

®, - o ok

Fig. 4. Schematic illustration of altered protein $4 of various temperature-sensitive mutants. 25 mutants with alterations in protein $4 (see Table 1) were classified into 12 groups according to t h e migration of protein $4 on two-dimensional electropherograms. The spots a through 1 indicate the positions of mutant $4 of: a JE 702; b JE 16 and JE 57; c JE 236; d JE 241; e JE221, JE 626 and JE930; f JE216, JE359 and JE931; g JE 398; h JE 617 and JE 659; i JE 228; j JE 753; k JE 110, JE 621 and JE 660; I JE 235, JE 265, JE 369, JE 709, JE 772 and JE 959

been shown to be an R N A - N n d i n g protein (St6ffler et al., 1971; Garrett et al., 1974) and thus m a y play an i m p o r t a n t role in the assembly of the large subunit. In fact, the assembly of the large subunit in one of these m u t a n t s was f o u n d to be impaired at 4 2 ° C (M611er and Isono, unpublished). Apparently, the assembly of the 30S subunit in this m u t a n t remains unaltered. This is the first case of a ribosomal m u t a n t with a m u t a t i o n in a large subunit protein affecting the assembly of the large subunit. The gene for protein L1 has been reported to be located at the 88 min region of the E. coli genetic m a p (Lindahl et al., 1975). So far we have obtained two different m u t a n t s h a r b o r i n g mutations in this protein (Table 2). In one of them, JE 224, protein L1 has b e c o m e slightly m o r e basic (Fig. 3a), while in another, JE 217, it has become m o r e acidic (not shown). In a " r e v e r t a n t " f r o m a special type o f strept o m y c i n - d e p e n d e n t mutant, this protein was f o u n d to be apparently missing (Dabbs, 1976). Since this is also one of the R N A - b i n d i n g proteins (St6ffler et al., 1971; G a r r e t t et al., 1974), functional as well as structural analysis o f the m u t a n t ribosomes is to be of interest. There are five m u t a n t s with altered protein $4 which, at the same time, have a change in either protein $5 or protein L l l . EJ 235 (Fig. 2b) is one of them. The protein $4 of this m u t a n t is drastically

301 changed. C o n c o m i t a n t l y , either $5 or L l l (these two proteins overlap in the electropherogram) is altered to migrate slightly faster in b o t h dimensions of the two-dimensional electrophoresis. By separating subunits, it became clear that it was protein $5 that had been partially altered. The spot o f this protein on two-dimensional electropherograms was elongated or sometimes split into two portions, one of which migrated faster in both directions o f electrophoresis than the other that remained at the wild type $5 position (data not shown). The nature of this change in protein $5 is n o t yet known. In addition to these m u t a n t s described above, we have isolated several other m u t a n t s with alterations in ribosomal protein $3, L5, L20, L29, L30 and L33 which have been not fully characterized yet. One of t h e m apparently lacks protein L33. Except for a few cases, m o s t o f the m u t a n t s with altered ribosomal proteins so far isolated by other workers are those resistant to, or dependent on, antibiotics k n o w n to inhibit the protein synthesis. As a result, the proteins altered in these m u t a n t s are confined to only several of the ribosomal proteins. The temperature-sensitive mutants described in this paper will be of great potential usefulness not only to establish the genetic loci for individual ribosomal proteins but also to investigate the role of each protein in the function as well as in the assembly of the ribosome in vivo. Genetical, physiological and biochemical characterization of these mutants are n o w in progress and the details will be published elsewhere. Acknowledgement. We thank Dr. H.G. Wittmann for critical comments on the manuscript and Mr. A.G. Cumberlidge for excellent technical assistance.

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302 Cooper, S., Helmstetter, C.E.: Chromosome replication and the division cycle ofEseherichia coli B/r. J. molec. Biol. 31,519-540 (1968) Dabbs, E.R., Wittmann, H.G. : A strain of Eseherichia coli which gives rise to mutations in a large number of ribosomal proteins. Molec. gen. Genet. (in press) De Wilde, M., Michel, F., Broman, K. : The structural gene for ribosomal protein S18 in Eseherichia eoli. II. Mapping outside the ribosomal protein gene cluster at minute 84 on the genome. Molec. gen. Genet. 133, 329-333 (1974) Feunteun, J., Monier, R., Vola, C., Rosset, R. : Ribosomal assembly defective mutants ofEscherichia coli. Nuc. Acid Res. 1, 149-169 ( 1974 b) Feunteun, J., Rosset, R., Ehresman, C., Stiegler, P., Fellner, P.: Abnormal maturation of precursor 16S RNA in a ribosomal assembly defective mutant of E. colL Nuc. Acid Res. 1, 141-147 (1974 a) Friesen, J.D., Parker, J., Watson, R.J., Fiil, N.P., Pedersen, S." Isolation of a transducing phage carrying rpsT, the structural gene for ribosomal protein $20. Molec. gen. Genet. 144, 115118 (1976) Garrett, R.A., Mfiller, S., Spierer, P., Zimmermann, R.A. : Binding of 50S ribosomal subunit proteins to 23S RNA of Escherichia coli. J. molec. Biol. 88, 553-557 (1974) Guerola, N., Ingraham, J.L., Cerdfi-Olmedo, E.: Induction of closely linked multiple mutations by nitrosoguanidine. Nature (Lond.) New Biol. 230, 122-125 (1971) Hardy, S.J.S., Kurland, C.G., Voynow, P., Mora, G. : The ribosomal proteins of Escberichia coll. I. Purification of the 30S ribosomal proteins. Biochemistry 8, 28972905 (1969) Hasenbank, R., Guthrie, C., St6ffler, G., Wittmann, H.G., Rosen, L., Apirion, D.: Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence. Molec. gen. Genet. 127, 1 - 1 8 (1973) Hitz, H., Sch~ifer, D., Wittmann-Liebold, B.: Primary structure of ribosomal protein $6 from the wild type and a mutant of Escherichia coli. FEBS Letters 56, 259-262 (1975) Kaltschmidt, E., Wittmann, H.G. : Ribosomal proteins. VII. Twodimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Ann. Biochem. 36, 401412 (1970) Lederberg, J., Lederberg, E.M. : Replica plating and indirect selection of bacterial mutants. J. Bact. 63, 399406 (1952)

K. Isono et al. : Ribosomal Protein Mutants of E. coli Lenox, E.S. : Transduction of linked genetic characters of the host by bacteriophage P1. Virol. 1, 190-206 (1955) Lindahl, L., Jaskunas, S.R., Dennis, P.P., Nomura, M.: Cluster of genes in Escherichia coli for ribosomal proteins, ribosomal RNA, and RNA polymerase subunits. Proc. nat. Acad. Sci. (Wash.) 72, 2743-2747 (1975) Nashimoto, H., Uchida, H.: Late steps in the assembly of the 30S ribosomal proteins in vivo in a spectinomycin-resistant mutant of Escherichia coll. J. molec. Biol. 96, 443453 (1975) Ohtsubo, E., Lee, H.J., Deonier, R.C., Davidson, N.: Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coll. VI. Mapping of F14 sequences homologous to ~80drnetBJF and (o80dargECBH bacteriophages. J. molec. Biol. 89, 599-618 (1974) Olsson, M., Isaksson, L., Kurland, C.G.: Pleiotropic effects of ribosomal protein $4 studied in Escheriehia coli mutants. Molec. gen. Genet. 135, 191-202 (1974) Rosset, R., Vola, C., Feunteun, J., Monier, R.: A thermosensitive mutant defective in ribosomal 30S subunit assembly. FEBS Letters 18, 127-129 (1971) St6ffler, G., Daya, L., Rak, K.H., Garrett, R.A.: Ribosomal proteins. XXVL The number of specific protein binding sites on 16S and 23S and 23S RNA of Escherichia coli. J. molec. Biol. 62, 411414 (1971) Takata, R. : Genetic studies of the ribosomal proteins in Escherichia coll. IX. Mapping of the ribosomal proteins, $2 and $20, by intergeneric mating experiments between Serratia marcescens and Escherichia coli K12. Molec. gen. Genet. 146, 233-238 (1976) Wittmann, H.G., St6ffler, G., Hindennach, I., Kurland, C.G., Randall-Hazelbauer, L., Birge, E.A., Nomura, M., Kaltschmidt, E., Mizushima, S., Traut, R.R., Bickle, T.A.: Correlation of 30S ribosomal proteins of Escherichia coli isolated in different laboratories. Molec. gen. Genet. 111, 327-333 (1971)

Communicated by E. Bautz

Received October 2, 1976