Proc. Nat. Acad. Sci. USA Vol. 70, No. 1, pp. 71-75, January 1973

Alteration of Ribosomal S4 by Mutation Linked to Kasugamycin-Resistance in Escherichia coli (ribosomal /ribosomal RNA/reconstitution/phosphocellulose chromatography) ROBERT A. ZIMMERMANN*, YOSHIKO IKEYAt, AND P. FREDERICK SPARLINGt§ * D~partement de Biologie Mol6culaire, Universit6 de Gen6ve, 30, Quai de l'Ecole-de-Medecine, CH-1211, Geneva 4, Switzerland; and t Departments of Medicine and Bacteriology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514 Communicated by Cyrus Levinthal, October 20, 1972

ABSTRACT An alteration in the chromatographic We report experiments that confirm many of these findings. mobility of 30S ribosomal protein S4 from two kasugamy- In contrast to Helser et al. (7, 8), however, we have found that cin-resistant (ksgA) strains of E. coli was observed. The locus determining this alteration frequently cotrans- two ksgA mutants possess an altered 30S subunit protein as duced with ksgA but could be separated from it by trans- well as undermethylated 16S RNA. Both of the mutants were duction and conjugation. Since the structural gene for obtained by nitrosoguanidine mutagenesis and are probably protein S4 is thought to lie some 30 min from ksgA on the double mutants. The altered protein has been identified as E. coli chromosome, the product of the newly-identified or structural gene may modify protein S4. We propose to designate this S4 (9) p4a (10). Several reports have described gene ramB. changes in protein S4 (11-14) resulting from mutations in the Reconstitution of 30S subunits from RNA and protein cluster of ribosomal protein genes near strA (12-15). The gene components of ksgA ramB and ksgA+ ramB+ strains dem- for alteration of protein S4 that we describe is shown to be onstrated that ribosomal resistance to kasugamycin was close to, but separable from, ksgA, and is therefore at a con- due to altered 16S RNA and not to altered protein S4. The altered 16S RNA was undermethylated, and a methyl- siderable distance fromn strA on the E. coli chromosome. The ating enzyme that acts on 16S RNA from ksgA strains implications of this finding are discussed. was present in ksgA+ but not in ksgA strains. MATERIALS AND METHODS Resistance to the , kasugamycin Bacterial Strains. Bacterial strains are listed in Table 1. results from alteration of the 30S ribosomal subunit in The kasugamycin-resistant strains in which altered protein Escherichia coli (1). The gene for ribosomal kasugamycin S4 was found were selected on tryptone-yeast extract plates resistance (ksgA) is closely linked to leu and maps near containing 1 mg of kasugamycin/ml after nitrosoguanidine 0.5 min on the E. coli chromosome (1-3). By contrast, loci mutagenesis (16) to the extent of 10% survival of exposed determining ribosomal resistance to the , cells. Spontaneous kasugamycin-resistant mutants were streptomycin and spectinomycin, have been placed in the selected on the same plates containing 250 lsg of kasugamycin cluster of 30S and 50S ribosomal protein genes near strA at per ml. Kasugamycin was a gift of Bristol Laboratories. 64 min (see ref. 4), and in both cases, antibiotic sensitivity or Strains were designated ksgA if the locus for kasugamycin resistance is mediated by specific ribosomal proteins (5, 6). resistance cotransduced 90% with pdxA, and if cell-free The unusual map position of the ksgA locus suggested a novel extracts were resistant to kasugamycin (2). Strains were mechanism for kasugamycin resistance (1). designated ksgB if they contained a locus for kasugamycin In a recent report, Helser, Davies, and Dahlberg demon- resistance that was located between 30-35 min on the E. strated that resistance to kasugamycin is attributable to an coli chromosome, and if cell-free extracts were sensitive to alteration of the 16S RNA component of the 30S subunit (7). kasugamycin (2). Transductions were performed with phage Specifically, the 16S RNA from resistant mutants lacks Plkc by standard methods (17). methylation of two adjacent adenine residues that are both dimethylated in the RNA of sensitive strains. The same Chromatography of Ribosomal Proteins. Growth of cells, authors later found that ksgA+, but not ksgA, bacteria con- preparation of ribosomal proteins, and chromatographic tain an enzyme that dimethylates the relevant adenine resi- analysis were performed as described by Zimmerman et al. dues of "resistant" RNA and that 30S particles reconstituted (11) with two exceptions: cells were labeled with either from such RNA after in vitro methylation are sensitive to [3H]- or ['4C]amino-acid mixtures (New England Nuclear the drug (8). Moreover, the enzyme acted only on under- Corp.) and high-capacity phosphocellulose (Mann, 1.1 methylated 16S RNA from kasugamycin-resistant mutants meq/g) was used for chromatography. and not on mature 16S RNA from sensitive strains, 23S RNA, Preparation of Ribosomal Components for Reconstitution. or protein. It was concluded that the product of the ksgA gene Cells were grown to mid log-phase in tryptone-yeast extract is a methylating enzyme that modifies 16S RNA at some broth and harvested after pouring on ice. were time during maturation of the 30S subunit. prepared as described (18), except that no DNase was used. Ribosomes were not washed in high-salt (0.5 M NH4Cl) buffers. 30S and 50S subunits were prepared by suspension of § To whom reprint requests should be sent. pelleted 70S ribosomes in standard buffer [10 mM Tris HCI 71 Downloaded by guest on September 29, 2021 72 Biochemistry: Zimmerman et al. Proc. Nat. Acad. Sci. USA 70 (1978) TABLE 1. Strains of E. coli K12 used TABLE 2. Reconstitution of SOS ribosomes from total protein and 16S RNA Strain Source Genotype Reconstituted 30S JC411 W. K. Mass* F-; argG met leu his strA ribosomes Incorporation cpm (% JC12 W. K. Mass* HFr; purC met control) FS131 Nitrosoguanidine As JC12 but ramBI ksgAl9 16S Total ug of kasugamycin per ml mutagenesis RNA protein 0 50 100 FS157 Nitrosoguanidine As JC12 but ramB2 ksgA23 ksgB1 mutagenesie S S 607 367 (61) 289 (48) FS215 Recombinant from As JC411 but his+ ramB2 ksgA23 S R 503 251 (50) 179 (35) FS157 x JC411 R S 324 281 (87) 220 (68) FS216 Recombinant from As JC411 but his+ ramB2 ksgBl R R 357 288 (81) 290 (73) FS157 x JC411 FS232 Transductant of As JC411 but leu+ ramBI ksgAl9 30S ribosomal subunits were reconstituted as described in JC411 from Methods and assayed for activity in poly(U,G)-dependent FS131 [P4C]valine incorporation in vitro. Reaction mixture (100 pl FS233 Transductant of As JC411 but leu+ ramB2 ksgA23 total) contained 0.7-0.8 A26 units each of reconstituted 30S par- JC411 from ticles and 50S subunits, 10ul of S-100 fraction, 10 pg of poly(U,G) FS157 (1:1, Miles Laboratories), 8 mM Mg acetate, 0.01 mM [14C]- Q13 W. Gilbert via met tyr pnp-13 valine (50 pCi/pmol, New England Nuclear Corp.), and other M. Nomurat components as described (18). S indicates component derived from FS240 Spontaneous As Q13 but ksgA3O strain JC411 (ksgA+ ramB+); R indicates component derived from mutant strain FS232 (ksgAI9 ramBi). FS241 Nitrosoguanidine As Q13 but leu mutagenesis FS242 Transductant of As FS241 but leu+ ksgA23 gation on a 5-20% linear sucrose gradient in TAS buffer for FS241 from 9.5 hr at 21,000 rpm in an SW27 rotor. Ribosomes and sub- FS157 units were stored at -70° in standard buffer containing 10 mM MgC12. * Dept. of Microbiology, New York University School of Med- The 16S RNA and total 30S ribosomal protein fractions icine, New York, N.Y. were prepared by phenol extraction and ureag-LiCl treatment, t Institute for Enzyme Research, Univ. of Wisconsin, Madison, respectively (20). Reconstitution of 30S subunits from Wisc. 16S RNA and total 30S proteins was performed according to Traub and Nomura (20). The RNA precipitate resulting from (pH 7.6)-30 mM NH4C1-6 mM 2-mercaptoethanol] con- treatment of 30S subunits with urea-LiCl was also used and taining 0.3 mMI MgCl2, followed by dialysis against TAS was as active as that prepared by phenol extraction. buffer [20 mM Tris HCl (pH 7.6)-100 mM NH4Cl-1 mM Fingerprint Analysis. [14C]Methyl-labeled oligonucleotides MgCl2-0.5 mM EDTA-4 mM 2-mercaptoethanol] for 3-4 hr from 16S RNA were prepared and fingerprinted (21). at 0° (19). Subunits were separated on discontinuous 0.2-1.0 M sucrose gradients (19) by centrifugation in an SW27 rotor for 20 Methykation. 23S core particles from 30S subunits (22) were hr at 27,000 rpm. 30S and 50S subunits were precipitated with methylated by enzymes prepared from 8-100 fractions and 0.6 volume of ethanol (19), and further purified by centrifu- from the 1.0 M NH4Cl supernatant of washed 70S ribosomes (8); S-adenosyl-['H ]methionine was purchased from New England Nuclear Corp. RESULTS .1. :.: i i 1500 1. AI Altered Ribosomal Protein S4 in Certain Kasugamycin- Resistant Strains. In our early efforts to identify the ribosomal I I00o .I looo l component altered in kasugamycin-resistant strains of :11 E. coli, 'H- and "C-labeled 30S subunit proteins from JC411 0 0 j.., I. (ksgA+) and FS215 (a ksgA23 recombinant derived by con- 500 jugation of JC411 with FS157) were compared by cochroma- tography on phosphocellulose columns. Fig. 1 demonstrates that one 30S protein from the mutant eluted at a significantly o -O 50 100 150 200 250 300 lower salt concentration than its counterpart from the FRACTION NUMBER wild-type strain, whereas the elution positions of all other 30S FIG. 1. Elution profile of 30S total proteins from 3H-labeled proteins from the two strains coincided. No chromatographic JC411 and 14C-labeled FS215. About 10 mg of proteins, including differences were observed in a parallel analysis of 50S subunit equal contributions from the 30S subunits of each strain, was proteins. applied to a phosphocellulose column in initial elution buffer locus (0.05 M NaH2PO4, titrated to pH 6.5 with methylamine-6 M To further examine the relationship between the ksgA urea-4 mM 2-mercaptoethanol) and eluted with a linear 0-0.5 M and the altered protein, two independently isolated, kasuga- NaCl gradient in the same buffer (35). Procedures have been mycin-resistant mutations were introduced into JC411 described (11). 3H-labeled JC411 ( ); "4C-labeled FS215 (ksgA +) from FS131 and FS157 by cotransduction with (- -). Arrows indicate displaced peaks. leu. Chromatographic comparison of the parental 30S proteins Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) New Gene for Altered Ribosomal Protein S4 73

b ! 'A. 'gX a *:ai,, i.t<,r S7 S3 S4 54 S9+S18 FS232 JC411 *.^W ,.:, ;' TSEy' .::*

y 2000 V 1500 S3 _ .,''.. S 4 _ S 7* - * .@i .d

1500 * x S 9 II a. 1000 0l C.)l C.) I C) 1000 F s 8*--+ 500 500 -

160 170 180 190 200 210 TOTAL 30S 161 170 178 88 202 FRACTION NUMBER PROTEINS FRACTION NUMBER FIG. 2. (a) Detail of elution profile of 30S total proteins from IH-labeled JC411 and 'IC-labeled FS232. Preparation and chromatog- raphy of sample was performed as described in Fig. 1 and ref. 11. 3H-labeled JC411 (- -); '4C-labeled FS232 ( - -). (b) Polyacrylamide gels of peaks from chromatogram depicted in a. Gel of total 30S proteins is shown at left; gels from individual column fractions are shown at right. Gels contained 7.5% acrylamide and 0.8% methylene bis-acrylamide and were run according to Moore et al. (36). Protein S4 is unambiguously identified in this system since it migrates as a separate band. Similar analyses of FS215 and FS233 yielded identical results. Bands marked by asterisk (*) also contain other proteins that elute at different positions in the chromatogram. A faint band due to pro- tein S11 is visible in tube 188 at the same level as protein S9; this band is not present in tube 178. Protein Sil most likely accounts for the small 14C-peak between tubes 185 and 188.

with those from the two transductants, FS232 (ksgAl9) and ksgA, although precise determination of its map position has FS233 (ksgA23), resulted in elution patterns indistinguishable been impeded by lack of a convenient method for scoring the from that of Fig. 1; a detail of one of these chromatograms is mutant phenotype in vivo. Nonetheless, the separability of the presented in Fig. 2a. Thus, the locus that determines the two genes is strongly suggested by the following observations. altered protein must be close to or identical with ICsgA, since (a) Strain FS216 (ksgA + ksgBI) has been shown to possess an it was present in two independent transductants selected for altered S4 similar in its chromatographic properties to that of leu+ and scored for kasugamycin resistance. FS215, and is therefore ramB. Since FS216 was constructed by The altered 30S protein in strains FS215, FS232, and FS233 conjugation of FS157 (ksgA23 ksgB1 ramB2) with JC411 was identified by polyacrylamide gel electrophoresis of (ksgA + ksgB+ ramB+), the presence of the altered protein in material from individual column fractions, as illustrated in FS216 can be most easily explained by postulation of a cross- Fig. 2b for FS232. The analysis demonstrates that samples over between ksgA and ramB. (b) Transduction of the ksgA23 from the displaced mutant peak (tube 178) and from the wild- allele into FS241, a leu- derivative of Q13, by the same type peak (tube 188) both contained a single component that methods used to construct FS232 and FS233 (selection for migrated to a position characteristic of 30S protein S4. At the leu+, followed by scoring for kasugamycin resistance) re- pH used for electrophoresis, there was no difference in the sulted in the ksgA23 strain FS242 whose 30S subunits did not mobilities of the chromatographically altered and unaltered possess a chromatographically altered protein S4. Thus, proteins. although ramB and ksgA are sufficiently close to be cotrans- There is considerable evidence that the structural gene for duced, as in the construction of FS233, they are not oblig- protein S4 is located in the ribosomal protein gene cluster atorily cotransduced. (c) No chromatographic differences near 64 min on the E. coli chromosome map (11-15). Since were observed upon comparison of 30S proteins from Q13 and the original mutant of the S4 structural gene was designated FS240, a spontaneous kasugamycin-resistant derivative of ram (15), we propose that the new locus for altered S4 near Q13. 0.5 min be designated ramB, in contrast to the original locus Kasugamycin Resistance Attributable to Abnormal 16S RNA. near 64 min, which is redesignated ramA. The evidence that ramB is genetically distinct from ksgA Separability of ksgA from the Locus for Altered S4. The locus suggests that ramB is not directly related to the mechanism for altered protein S4 (ramB) is apparently distinct from of ribosomal resistance to kasugamycin. Accordingly, 30S Downloaded by guest on September 29, 2021 74 Biochemistry: Zimmerman et al. Proc. Nat. Acad. Sci. USA 70 (1973) ribosomal subunits were reconstituted from 30S total proteins struction of an isogenic set of strains carrying one or both of and 16S RNA of JC411 (ksgA + ramB+) and FS232 the same ramB and ksgA alleles, which has not yet been (ksgA19 ramBi) and tested for their susceptibility to the possible due to the cumbersome necessity of scoring for ramB antibiotic in an in vitro polypeptide-synthesizing system. As by column chromatography. It is possible that Coincidence of shown in Table 2, reconstituted subunits were resistant to kegA and ramB mutations occurred by chance in our strains kasugamycin only when 168 RNA was derived from a because it is known that after exposure to nitrosoguanidine, kasugamycin-resistant strain; total proteins from the same multiple mutations are frequently found within a 2-min map strain, which included altered S4, did not produce kasuga- distance of the mutation for which initial selection was made mycin-resistant subunits unless combined with homologous (27). RNA. Cotransduction of a gene (ramB) for altered protein S4 Ribosomal subunits were also reconstituted with com- with ksgA, which is located near 0.5 min on the E. coli ponents from the kegA + strains Q13 or FS241 and their k8gA linkage map, would appear to be paradoxical, since the same derivatives, FS240 (ksgASO) or FS242 (ksgA23), all of which protein is altered by ramA (11) and by certain mutations re- are ramB+. In all cases, it was found that the presence of 16S versing streptomycin dependence (12-14), all of which RNA from a kasugamycin-resistant strain was required to map near 64 min. Insofar as is known, protein S4 does not form particles resistant to the drug in vitro (data not shown). consist of more than one polypeptide chain (28). Moreover, Moreover, the levels of resistance observed with proteins although examples of gene duplication in bacteria are known from the ramB+ strains were not significantly different from (29), it is unlikely that both ramA and ramB are structural those obtained with protein mixtures containing altered S4. genes for S4. If they were, one would expect ramA+ ramB Our findings are thus consistent with the work of Helser et al. strains such as FS215 and FS232 to yield two approximately (7), who first demonstrated that kasugamycin resistance was equal peaks of normal and mutant S4. However, analysis of due to altered 16S RNA. the normalized chromatograms presented in Figs. I and 2a In addition, fingerprint analysis of RNase T, digests of reveals that about 85% of the '4C-radioactivity in the S4 ["4C]methyl-labeled 16S RNA from JC411 and FS232 showed region (fractions 175-194) is associated with the mutant S4 that the oligonucleotide AACCUG from FS232 RNA lacks peak; most of the remaining 15%, which is nearly coincident the four methyl groups normally present in mature 16S mol- with, but slightly displaced from, the normal S4 peak, ecules (21). We have also demonstrated that 23S core particles probably consists of protein S11 (see Fig. 2b). Nonetheless, derived from the 30S subunits of ksgA strains are excellent since the presence of a small amount of normal S4 in the 30S substrates for a methylating enzyme present in extracts of subunits of ramA + ramB strains cannot be completely ksgA + but not of ksgA strains; the methylase is present in the excluded, the existence of two structural genes for this S-100 fraction, but is found in relatively higher concentration protein remains a possibility. To explain the chromatographic in extracts from salt-washed ribosomes (8). Core particles made results in such an event, one would have to postulate either from the 30S subunits of ksgA + strains are not substrates for preferential synthesis of mutant S4 or preferential incorpora- the methylating enzyme. These findings are in accord with tion of mutant S4 into the ribosomes. those already reported (7, 8) and are, therefore, not presented A more likely explanation is that the primary structure of in detail here. protein S4 is encoded only by the ramA (15), or rpxD (12), locus in the strA region, consistent with the mounting evidence DISCUSSION that the ribosomal protein genes clustered near strA at 64 min We have shown that mutations in a gene (ramB) close to are structural genes (30), and that the ramB gene product k8gA leads to a change in the chromatographic mobility of is an enzyme that modifies protein S4. It is intriguing in this 30S protein S4, while mutations in ksgA result in under- regard to note that the C-terminal amino-acid sequences of methylation of a specific nucleotide sequence in the 16S RNA several mutant forms of protein S4 have been found to vary (7). A possible explanation for the unexpected finding of substantially in length (31). This observation could be inter- alteration of 30S protein S4 in each of two tested nitroso- preted to mean that protein S4 is cleaved from a longer guanidine-induced mutants (FS131, FS157) selected for high- precursor molecule; if so, the ramB gene product would be a level resistance to kasugamycin is suggested by the fact that potential candidate for the putative cleavage enzyme. Al- protein S4 is one of the five or six 30S ribosomal proteins that ternatively, ramB might alter protein S4 by a group-transfer interact directly with 16S RNA during subunit assembly reaction; several enzymes that perform secondary modifica- (23-25). If interaction of altered 16S RNA with mutant pro- tions of ribosomal proteins by methylation, phosphorylation, tein S4 produces a structure more resistant to and acylation have already been described (32-34). Finally, kasugamycin than that resulting from altered 16S RNA the presence of the ramB gene near ksgA would raise the alone, double mutations would be favored. Although this possibility that there is a grouping of ribosome-modifying conjecture cannot be excluded, it is unlikely since (a) ksgA genes near 0.5 min on the bacterial chromosome. ramB strains are not significantly more resistant to kasuga- The functional significance of protein S4 alteration re- mycin in vivo or in vitro than other k8gA ramB+ strains, sulting from mutation at the ramB locus remains to be (b) altered and unaltered S4 protein have approximately the clarified. It is reasonable to predict that translational fidelity same affinity for 16S RNA (P. Spierer and R. A. Zimmermann, should be affected in ramB strains, since the alteration of unpublished), and (c) methylation of 16S RNA is thought to protein S4 in ramA mutants has a striking influence on occur subsequent to the binding of protein S4 during subunit accuracy of (11, 15). Preliminary experiments have assembly (26). There may be other selective advantages in fact shown that the 70S ribosomes of strains FS232 and (apart from resistance to kasugamycin) to kegA mutants, FS233 (both ramB ksgA) differ notably from those of strain which are also ramB. Testing this hypothesis requires con- JC411 (ramB+ ksgA+) in their ability to misread poly(U) Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) New Gene for Altered Ribosomal Protein S4 75

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