Summary
Mutants able to grow in the presence of 1.2 mg/ml streptomycin were isolated from Escherichia coli WP2 after exposure to ultraviolet light (UV) or in the absence of any treatment (spontaneous), and from a umuC derivative after exposure to UV and delayed photoreversal. These mutants, characterized as streptomycin resistant (Smr) or dependent (Sud), carry mutations in the rpsL gene. This gene was amplified using the polymerase chain reaction and sequenced. Mutations induced by UV were largely (76%) of the Smr phenotype, all of which were changes at an A: T base pair at codons 42 or 87. Mutations induced by UV plus delayed photoreversal in the non-UV-mutable umuC122 derivative of WP2 were exclusively of the Smd phenotype and all occurred at G: C base pairs at codons 41, 90 or 91. These results are consistent with current understanding of the mechanism of mutagenesis by UV and delayed photoreversal. A broader spectrum of mutations was seen in the spontaneous series including three-base deletions leading to amino acid loss (2 of codon 93, 1 of codon 87). Of particular note was the number of intragenic second site mutations in the spontaneous series, most if not all of which appeared to be silent with respect to streptomycin phenotype. It is necessary to postulate a high rate of formation of such mutations at some stage during the experiment. One possibility is that spontaneous mutation may often occur in bursts when an error correction mechanism (eg., proofreading, mismatch correction) is temporarily inactive. In all, 12 different mutations conferring the ability to grow in the presence of streptomycin were identified, five Smr and seven Smd, including two three-base deletions. The mutations fall into two small regions of the gene, one spanning codons 40–43, the other 87–93. Both Smr and Smd mutations were found in each region. The sequence of the wild-type rpsL gene of E. coli WP2 (a B/r strain) was identical to that of E. coli K12, in contrast to the situation with a number of other genes. The sequence conservation may be a consequence of codon usage bias in this highly expressed gene.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
van Acken U (1975) Proteinchemical studies on ribosomal proteins S4 and S12 from ram (ribosomal ambiguity) mutants of Escherichia coli. Mol Gen Genet 140:61–68
Allen PN, Noller HF (1989) Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 168 ribosomal RNA. J Mol Biol 208:457–468
Andersson DI, Kurland CG (1983) Ram ribosomes are defective proofreaders. Mol Gen Genet 191:378–381
Böck A, Petzet A, Piepersberg W (1979) Ribosomal ambiguity (ram) mutations facilitate dihydrostreptomycin binding to ribosomes. FEBS Lett 104:317–321
Bockrath R (1989) Streptomycin-resistant and dependent mutants of E. coli: possible indicators of two important types of DNA alteration by UV mutagenesis. Mutagenesis 4:78–79
Bockrath RC, Ruiz-Rubio M, Bridges BA (1987) Specificity of mutation by ultraviolet light and delayed photoreversal in umuC defective Escherichia coli K-12: a targeting intermediate at pyrimidine dimers. J Bacteriol 169:1410–1416
Bohman K, Ruusala T, Jelenc PC, Kurland CG (1984) Kinetic impairment of restrictive streptomycin-resistant ribosomes. Mol Gen Genet 198:90–99
Bridges BA, Mottershead RP (1971) RecA+ dependent mutagenesis occurring before DNA replication in UV and gamma-irradiated Escherichia coli. Mutat Res 13:1–8
Bridges BA, Mottershead RP (1978) Mutagenic DNA repair in Escherichia coli. VIII. Involvement of DNA polymerase III in constitutive and inducible DNA repair after ultraviolet and gamma irradiation. Mol Gen Genet 162:35–41
Bridges BA, Sharif F (1986) Mutagenic DNA repair in Escherichia coli. XII. Ultraviolet mutagenesis in excision proficient umuC and lex(Ind−) bacteria as revealed by delayed photoreversal. Mutagenesis 1:111–117
Bridges BA, Southworth M (1982) Constitutive error-prone repair at sites of excision repair in Escherichia coli: a re-examination. Biochimie 64:655–659
De Wilde M, Gabezon T, Villarroel R, Herzog A, Bollen A (1975) Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli. I. Properties of ribosomal mutants whose resistance to neamine is the cumulative effect of two distinct mutations. Mol Gen Genet 142:19–33
Funatsu G, Wittmann HG (1972) Ribosomal proteins XXXIII: Location of amino-acid replacements in protein S12 isolated from Escherichia coli mutants resistant to streptomycin. J Mol Biol 68:547–550
Funatsu G, Yaguchi M, Wittmann-Liebold B (1977) Primary structure of protein S12 from the small Escherichia coli ribosomal subunit. FEBS Lett 73:12–17
Galas DJ, Branscomb EW (1976) Ribosome slowed by mutation to streptomycin resistance. Nature 262:617–619
Gorini L (1974) Streptomycin and misreading of the genetic code. In: Nomura M, Tissieres A, Lengyel P (eds) Ribosomes. Cold Spring Harbor Laboratory Press, New York, pp 791–803
Harwood J, Tachibana A, Meuth M (1991) Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line. Mol Cell Biol 11:3163–3170
Hasenbank R, Guthrie C, Stöffler G, Wittmann HG, Rosen L, Apirion D (1973) Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence. Mol Gen Genet 127:1–18
Hultman T, Stahl S, Hornes E, Uhlen M (1989) Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res 17:4937–4946
Ikemura T (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: A proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol 151:389–409
Itoh T, Wittmann HG (1973) Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence. Mol Gen Genet 127:19–32
Li BH, Larsen S, Pratt V, Bockrath R (1991) Diverse backmutations at an ochre defect in the tyrA gene sequence of E. coli B/r. Mutat Res 246:139–149
Moazed D, Noller HF (1986) Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell 47:985–994
Moazed D, Noller HF (1987) Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 327:389–394
Montandon PE, Wagner R, Stutz E (1986) E. coli ribosomes with a C912 to U base change in the 16S rRNA are streptomycin resistant. EMBO J 5:3705–3708
Nishioka H, Doudney CO (1969) Different modes of loss of photoreversibility of mutation and lethal damage in ultraviolet light resistant and sensitive bacteria. Mutat Res 8:215–228
Piepersberg W, Noseda V, Böck A (1979) Bacterial ribosomes with two ambiguity mutations: Effects on translational fidelity, on the response to aminoglycosides and on the rate of protein synthesis: Mol Gen Genet 171:23–34
Post LE, Nomura M (1980) DNA sequence from the str operon of Escherichia coli. J Biol Chem 255:4660–4666
Post LE, Arfsten AE, Reusser F, Nomura M (1978) DNA sequences of promoter regions for the str and spc ribosomal protein operons in E. coli. Cell 15:215–229
Ruiz-Rubio M, Bridges BA (1987) Mutagenic DNA repair in Escherichia coli XIV. Influence of two DNA polymerase III imitator alleles on spontaneous and UV mutagenesis. Mol Gen Genet 208:542–548
Ruusala T, Kurland CG (1984) Streptomycin preferentially perturbs ribosomal proofreading. Mol Gen Genet 198:100–104
Schaaper RM, Danforth BN, Glickman BW (1986) Mechanisms of spontaneous mutagenesis: An analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene. J Mol Biol 189:273–284
Sharp PM, Li W (1987) The rate of synonymous substitution in enterobacterial genes is inversely related to codon usage bias. Mol Biol Evol 4:222–230
Stern S, Powers T, Changchien L, Noller HF (1988) Interaction of ribosomal protein S5, S6, S11, S12, S18 and S21 with 16S rRNA. J Mol Biol 201:683–695
Yaguchi M, Wittmann HG, Cabezon T, De Wilde M, Villarroel M, Herzog A Bollen A (1975) Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli II. Localization of amino acid replacements in proteins S5 and S12 altered in double mutants resistant to neamine. Mol Gen Genet 142:35–43
Author information
Authors and Affiliations
Additional information
Communicated by R. Devoret
Rights and permissions
About this article
Cite this article
Timms, A., Steingrimsdottir, H., Lehmann, A. et al. Mutant sequences in the rpsL gene of Escherichia coli B/r: Mechanistic implications for spontaneous and ultraviolet light mutagenesis. Molec. Gen. Genet. 232, 89–96 (1992). https://doi.org/10.1007/BF00299141
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00299141