Abstract
Resistance to streptomycin in bacterial cells often results from a mutation in the rpsL gene that encodes the ribosomal protein S12. We found that a particular rpsL mutation (K87E), newly identified in Escherichia coli, causes aberrant protein synthesis activity late in the growth phase. While protein synthesis decreased with age in cells in the wild-type strain, it was sustained at a high level in the mutant, as determined using living cells. This was confirmed using an in vitro protein synthesis system with poly(U) and natural mRNAs (GFP mRNA and CAT mRNA). Other classical rpsL mutations (K42N and K42T) tested did not show such an effect, indicating that this novel characteristic is typical of ribosomes bearing the K87E mutant form of S12, although the K87E mutation conferred the streptomycin resistance and error-restrictive phenotypes also seen with the K42N and K42T mutations. The K87E (but not K42N or K42T) mutant ribosomes exhibited increased stability of the 70S complex in the presence of low concentrations of magnesium. We propose that the aberrant activation of protein synthesis at the late growth phase is caused by the increased stability of the ribosome.
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References
Bilgin N, Claesens F, Pahverk H, Ehrenberg M (1992) Kinetic properties of Escherichia coli ribosomes with altered forms of S12. J Mol Biol 224:1011–1027
Bjorkman J, Samuelsson P, Andersson DI, Hughes D (1999) Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium. Mol Microbiol 31:53–58
Carter AP, Clemons WM, Brodersen DE, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V (2000) Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407:340–348
Gregory ST, Cate JH, Dahlberg AE (2001) Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus. J Mol Biol 309:333–338
Hesketh A, Ochi K (1997) A novel method for improving Streptomyces coelicolor A3(2) for production of actinorhodin by introduction of rpsL (encoding ribosomal protein S12) mutations conferring resistance to streptomycin. J Antibiot 50:532–535
Hosoya Y, Okamoto S, Muramatsu H, Ochi K (1998) Acquisition of certain streptomycin-resistant ( str) mutations enhances antibiotic production in bacteria. Antimicrob Agents Chemother 42:2041–2047
Inaoka T, Kasai K, Ochi K (2001) Construction of an in vivo nonsense readthrough assay system and functional analysis of ribosomal proteins S12, S4, and S5 in Bacillus subtilis. J Bacteriol 183:4958–4963
Kigawa T, Yabuki T, Yoshida Y, Tsutsui M, Ito Y, Shibata T, Yokoyama S (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett 442:15–19
Kurland CG, Jorgensen F, Richter A, Ehrenberg M, Bilgin N, Rojas AM (1990) Through the accuracy window. In: Warner JR (ed) The Ribosome: structure, function, and evolution. ASM Press, Washington, D.C., pp 513–526
Kurland CG, Hughes D, Ehrenberg M (1996) Limitations of translational accuracy. In: Frederick CN (ed) Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, D.C., pp 979–1004
Legault-Demare L, Chambliss GH (1974) Natural messenger ribonucleic acid-directed cell-free protein-synthesizing system of Bacillus subtilis. J Bacteriol 120:1300–1307
Lodmell JS, Dahlberg AE (1997) A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA. Science 277:1262–1267
Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747
Ofverstedt LG, Zhang K, Tapio S, Skoglund U, Isaksson LA (1994) Starvation in vivo for aminoacyl-tRNA increases the spatial separation between the two ribosomal subunits. Cell 79:629–638
Okamoto-Hosoya Y, Hosaka T, Ochi K (2003a) An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2). Microbiology 149:3299–3309
Okamoto-Hosoya Y, Okamoto S, Ochi K (2003b) Development of antibiotic-overproducing strains by site-directed mutagenesis of the rpsL gene in Streptomyces lividans. Appl Environ Microbiol 69:4256–4259
Ramakrishnan V (2002) Ribosome structure and the mechanism of translation. Cell 108:557–572
Rheinberger HJ, Geigenmuller U, Wedde M, Nierhaus KH (1988) Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding. Methods Enzymol 164:658–670
Ruusala T, Kurland CG (1984) Streptomycin preferentially perturbs ribosomal proofreading. Mol Gen Genet 198:100–104
Sells BH, Ennis HL (1970) Polysome stability in relaxed and stringent strains of Escherichia coli during amino acid starvation. J Bacteriol 102:666–671
Shaw WV (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol 43:737–755
Shima J, Hesketh A, Okamoto S, Kawamoto S, Ochi K (1996) Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). J Bacteriol 178:7276–7284
Tamehiro N, Hosaka T, Xu J, Hu H, Otake N, Ochi K (2003) An innovative approach for strain improvement with antibiotic overproducing industrial strain of Streptomyces albus. Appl Environ Microbiol 69:6412–6417
Timms AR, Steingrimsdottir H, Lehmann AR, Bridges BA (1992) Mutant sequences in the rpsL gene of Escherichia coli B/r: mechanistic implications for spontaneous and ultraviolet light mutagenesis. Mol Gen Genet 232:89–96
Toivonen JM, Boocock MR, Jacobs HT (1999) Modelling in Escherichia coli of mutations in mitoribosomal protein S12: novel mutant phenotypes of rpsL. Mol Microbiol 31:1735–1746
Wada A, Yamazaki Y, Fujita N, Ishihama A (1990) Structure and probable genetic location of a “ribosome modulation factor” associated with 100S ribosomes in stationary-phase Escherichia coli cells. Proc Natl Acad Sci USA 87:2657–2661
Wada A, Igarashi K, Yoshimura S, Aimoto S, Ishihama A (1995) Ribosome modulation factor: stationary growth phase-specific inhibitor of ribosome functions from Escherichia coli. Biochem Biophys Res Commun 214:410–417
Wimberly BT, Brodersen DE, Clemons WM, Morgan-Warren RJ, Carter AP, Vonrhein C, Hartsch T, Ramakrishnan V (2000) Structure of the 30S ribosomal subunit. Nature 407:327–339
Xu J, Tozawa Y, Lai C, Hayashi H, Ochi K (2002) A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2). Mol Genet Genomics 268:179–189
Yoshida H, Maki Y, Kato H, Fujisawa H, Izutsu K, Wada C, Wada A (2002) The Ribosome Modulation Factor (RMF) binding site on the 100S ribosome of Escherichia coli. J Biochem 132:983–989
Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JHD, Noller HF (2001) Crystal structure of the ribosome at 5.5 A resolution. Science 292:883–896
Zhang K, Pettersson-Landen L, Fredriksson MG, Ofverstedt LG, Skoglund U, Isaksson LA (1998) Visualization of a large conformation change of ribosomes in Escherichia coli cells starved for tryptophan or treated with kirromycin. Exp Cell Res 238:335–344
Acknowledgements
We are grateful to K. Kasai for providing plasmid DNA (pSREG) used in this study, E. Dabbs for certain E. coli mutants, and to Y. Okamoto-Hosoya and K. Hosokawa for discussions. This work was supported by a grant from the Organized Research Combination System of Ministry of Education, Culture, Sports, Science and Technology
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Hosaka, T., Tamehiro, N., Chumpolkulwong, N. et al. The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in Escherichia coli . Mol Genet Genomics 271, 317–324 (2004). https://doi.org/10.1007/s00438-004-0982-z
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DOI: https://doi.org/10.1007/s00438-004-0982-z