Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium

Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE . Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium . To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium . We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL , being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL . This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity ( ram ) phenotype.     

A mutation in the small (alpha) subunit of glycyl-tRNA synthetase affects amino acid activation and subunit association parameters

A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity. For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme. Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS). While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered. One severely affected glyS mutant (N302) was studied in more detail. For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA. There also was a severely reduced adenylate synthesis activity (greater than 100-fold). In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme. The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity. The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.     

Media dependence of translational mutant phenotype

Abstract We have measured the growth rates of some ribosomal mutants of Escherichia coli in different growth media. The mutants are a streptomycin resistant (SmR) mutation in rpsL; a partially streptomycin dependent (SmP) mutation in rpsL; a ribosome ambiguity mutant (ram) in rpsD; a ram mutant in rpsE as well as a mutant defective in tRNA modification, mia A. The data show that the growth rates of all mutants are less inhibited in poor media than they are in rich ones. The translation rates and nonsense suppression levels for each mutant are not significantly different in rich and poor media, which shows that the ribosomal mutant phenotypes are maintained under different growth conditions. These results suggest that the degree of growth inhibition for mutants with altered translation machinery is dependent on the growth conditions. In addition, the data suggest that bacteria are able to physiologically compensate for the loss of growth efficiency in such mutants, particularly, under poor growth conditions.     

A novel mutation in ribosomal protein S4 that affects the function of a mutated RF1

Release factors (RF) 1 and 2 trigger the hydrolysis of the peptide from the peptidyl-tRNA during translation termination. RF1 binds to the ribosome in response to the stop codons UAG and UAA, whereas RF2 recognizes UAA and UGA. RF1 and RF2 have been shown to bind to several ribosomal proteins. To study this interaction in vivo, prfA1, a mutant form of RF1 has been used. A strain with the prfA1 mutation is temperature sensitive (Ts) for growth at 42 degrees C and shows an increased misreading of UAG and UAA. In this work we show that a point mutation in ribosomal protein S4 can, on the one hand, make the RF1 mutant strain Ts(+); on the other hand, this mutation increases the misreading of UAG, but not UAA, caused by prfA1. The S4 mutant allele, rpsD101, is a missense mutation (Tyr51 to Asp), which makes the cell cold sensitive. The behaviour of rpsD101 was compared to the well-studied S4 alleles rpsD12, rpsD14, and rpsD16. These three mutations all confer both a Ts (44 degrees C) phenotype and show a ribosomal ambiguity phenotype, which rpsD101 does not. The three alleles were sequenced and shown to be truncations of the S4 protein. None of the three mutations could compensate for the Ts phenotype caused by the prfA1 mutation. Hence, rpsD101 differs in all studied characteristics from the three above mentioned S4 mutants. Because rpsD101 can compensate for the Ts phenotype caused by prfA1 but enhances the misreading of UAG and not UAA, we suggest that S4 influences the interaction of RF1 with the decoding center of the ribosome and that the Ts phenotype is not a consequence of increased readthrough.     

Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells.

Translational stress-induced mutagenesis (TSM) refers to the mutator phenotype observed in Escherichia coli cells expressing a mutant allele (mutA or mutC) of the glycine tRNA gene glyV (or glyW). Because of an anticodon mutation, expression of the mutA allele results in low levels of Asp-->Gly mistranslation. The mutA phenotype does not require lexA-regulated SOS mutagenesis functions, and appears to be suppressed in cells defective for RecABC-dependent homologous recombination functions. To test the hypothesis that the TSM response is mediated by non-specific mistranslation rather than specific Asp-->Gly misreading, we asked if streptomycin (Str), an aminoglycoside antibiotic known to promote mistranslation, can provoke a mutator phenotype. We report that Str induces a strong mutator phenotype in cells bearing certain alleles of rpsL, the gene encoding S12, an essential component of the ribosomal 30 S subunit. The phenotype is strikingly similar to that observed in mutA cells in its mutational specificity, as well as in its requirement for RecABC-mediated homologous recombination functions. Expression of Str-inducible mutator phenotype correlates with mistranslation efficiency in response to Str. Thus, mistranslation in general is able to induce the TSM response. The Str-inducible mutator phenotype described here defines a new functional class of rpsL alleles, and raises interesting questions on the mechanism of action of Str, and on bacterial response to antibiotic stress.     

Expression of mutant alanine tRNAs increases spontaneous mutagenesis in Escherichia coli

The expression of mutA, an allele of the glycine tRNA gene glyV, can confer a novel mutator phenotype that correlates with its ability to promote Asp-->Gly mistranslation. Both activities are mediated by a single base change within the anticodon such that the mutant tRNA can decode aspartate codons (GAC/U) instead of the normal glycine codons (GCC/U). Here, we investigate whether specific Asp-->Gly mistranslation is required for the unexpected mutator phenotype. To address this question, we created and expressed 18 individual alleles of alaV, the gene encoding an alanine tRNA, in which the alanine anticodon was replaced with those specifying other amino acids such that the mutant (alaVX) tRNAs are expected to potentiate X-->Ala mistranslation, where X is one of the other amino acids. Almost all alaVX alleles proved to be mutators in an assay that measured the frequency of rifampicin-resistant mutants, with one allele (alaVGlu) being a stronger mutator than mutA. The alaVGlu mutator phenotype resembles that of mutA in mutational specificity (predominantly transversions), as well as SOS independence, but in a puzzling twist differs from mutA in that it does not require a functional recA gene. Our results suggest that general mistranslation (as opposed to Asp-->Gly alone) can induce a mutator phenotype. Furthermore, these findings predict that a large number of conditions that increase translational errors, such as genetic defects in the translational apparatus, as well as environmental and physiological stimuli (such as amino acid starvation or exposure to antibiotics) are likely to activate a mutator response. Thus, both genetic and epigenetic mechanisms can accelerate the acquisition of mutations.     

Induction of the heat shock regulon in response to increased mistranslation requires oxidative modification of the malformed proteins

The Escherichia coli rpsD12 allele, which reduces translational fidelity and elevates expression of heat shock protein (Hsp) genes, only enhanced Hsp gene expression in the presence of oxygen. Similarly, the rpsL141 allele, which reduces mistranslation and Hsp gene expression, failed to affect the Hsp regulon in cells grown anaerobically. Increased production of Hsps in response to starvation is associated with increased mistranslation and was demonstrated to likewise require the presence of oxygen. Thus, mistranslation triggered by starvation or mutations in the accuracy centre of the ribosome appear to elevate Hsp gene expression via an oxidative modification of mistranslated proteins. In contrast, Hsp gene induction during temperature upshifts was independent of oxygen availability. The data further suggest that it is the oxidative modification of mistranslated DnaK substrates rather than oxidation of DnaK itself that triggers Hsp gene expression upon starvation.     

Isolation and characterization of a new globomycin-resistant dnaE mutant of Escherichia coli.

We isolated a globomycin-resistant, temperature-sensitive mutant of Escherichia coli K-12 strain AB1157. The mutation mapped in dnaE, the structural gene for the alpha-subunit of DNA polymerase III. The in vivo processing of lipid-modified prolipoprotein was more resistant to globomycin in the mutant strain 307 than in its parent. The prolipoprotein signal peptidase activity was also increased twofold in the mutant, and there was a threefold increase in the activity of isoleucyl-tRNA synthetase. The results suggest that a mutation in dnaE may affect the expression of the ileS-lsp operon in E. coli. In addition, strain 307 showed a reduced level of streptomycin resistance compared with its parental strain AB1157 (rpsL31). Strain 307 was killed by streptomycin at a concentration of 200 micrograms/ml, which did not affect the rate of bulk protein synthesis in this mutant. A second mutation which was involved in the reduced streptomycin resistance in strain 307 was identified and found to be closely linked to or within the rpsD (ramA, ribosomal ambiguity) gene. Both dnaE and rpsD were required for the reduced streptomycin resistance in strain 307.     

Bacillus subtilis mutants with alterations in ribosomal protein S4.

Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis. The mutations causing the S4 alterations, designated rpsD1 and rpsD2, were located between the argGH and aroG genes, at 263 degrees on the B. subtilis chromosome, distant from the major ribosomal protein gene cluster at 12 degrees. The mutant rpsD alleles were isolated by hybridization using a wild-type rpsD probe, and their DNA sequences were determined. The two mutants contained alterations at the same position within the S4-coding sequence, in a region containing a 12-bp tandem duplication; the rpsD1 allele corresponded to an additional copy of this repeated segment, resulting in the insertion of four amino acids, whereas the rpsD2 allele corresponded to deletion of one copy of this segment, resulting in the loss of four amino acids. The effects of these mutations, alone and in combination with streptomycin resistance mutations, on growth, sporulation, and streptomycin resistance were analyzed.     

Primary structure of an unusual glycine tRNA UGA suppressor.

We have determined the nucleotide sequences of two UGA-suppressing glycine transfer RNAs. The suppressor tRNAs were previously shown to translate both UGA and UGG and to have arisen as a consequence of mutation in glyT, the gene for the GGA/G-reading glycine tRNA of Escherichia coli. In each mutant tRNA, the primary sequence change was the substitution of adenine for cytosine in the 3' position of the anticodon. In addition, a portion of mutant glyT tRNA molecules contained N6-(delta 2-isopentenyl)-2-thiomethyl adenine adjacent to the 3' end of the anticodon (nucleotide 37). The presence or absence of this hypermodification may be a determinant in some of the biological properties of the mutant tRNA.     

Suppressors of a UGG missense mutation in Escherichia coli

As part of our investigation of tRNA structure-function relationships, we isolated and preliminarily characterized translational suppressors of the tryptophan codon UGG in a trpA missense mutant of Escherichia coli. the parent strain also contained two other mutant alleles relevant to the suppressor search; these were supD, which codes for a serine-inserting amber suppressor tRNA, and gly V55, the gene for a GGA/G-reading mutationally altered glycine tRNA. On the basis of map location, reversed-phase (RPC-5) column chromatography of glycyl-tRNA, and codon response, several classes have been distinguished so far. The number of suppressors in each class, their codon responses, and their apparent genic identities, respectively, are as follows: class 1--4 suppressors, UGG, supD; class 2--12 suppressors, UGG, glyU; class 3--9 suppressors, UGA and UGG, glyT; class 4--2 suppressors, UGG, glyT; class 5--7 suppressors, UGG, gly V55. Besides these, one suppressor retains supD activity, but so far its map location has not been distinguished from that of supD. Another suppressor clearly does not map near supD or any of the glycine tRNA genes mentioned. These last two suppressors may represent novel missense suppressors such as misacylated tRNA's or mutationally altered aminoacyl-tRNA synthetases, tRNA modification enzymes, or ribosomes. Finally, three other suppressors were obtained from a strain containing glyT56, the gene for an AGA/G-reading form of glyT tRNA. All three occurred at the expense of glyT56 activity and exhibited the the transductional linkage to argH that is characteristic of glyT.     

Streptomycin- and rifampin-resistant mutants of Escherichia coli perturb F exclusion of bacteriophage T7 by affecting synthesis of the F plasmid protein PifA.

Certain alleles of rpsL that confer resistance to the antibiotic streptomycin almost completely relieve F exclusion of bacteriophage T7. Introduction of a specific rpoB allele conferring resistance to rifampin into the rpsL strain restores the ability of the F-containing strain to exclude T7. This variation in the severity of F exclusion is reflected in the levels of the F-encoded inhibitor protein PifA: F'-containing cells that harbor specific rpsL alleles are phenotypically Pif-, but become Pif+ by the further acquisition of a specific rpoB allele. F-containing cells harboring the gyrA43(Ts) mutation also appear phenotypically Pif-, possibly because repression of the pif operon is enhanced by an altered DNA conformation in the gyrase mutant strain.     

Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.     

Autoregulation of yeast pyruvate decarboxylase gene expression requires the enzyme but not its catalytic activity.

In the yeast, Saccharomyces cerevisiae, pyruvate decarboxylase (Pdc) is encoded by the two isogenes PDC1 and PDC5. Deletion of the more strongly expressed PDC1 gene stimulates the promoter activity of both PDC1 and PDC5, a phenomenon called Pdc autoregulation. Hence, pdc1Delta strains have high Pdc specific activity and can grow on glucose medium. In this work we have characterized the mutant alleles pdc1-8 and pdc1-14, which cause strongly diminished Pdc activity and an inability to grow on glucose. Both mutant alleles are expressed as detectable proteins, each of which differs from the wild-type by a single amino acid. The cloned pdc1-8 and pdc1-14 alleles, as well as the in-vitro-generated pdc1-51 (Glu51Ala) allele, repressed expression of PDC5 and diminished Pdc specific activity. Thus, the repressive effect of Pdc1p on PDC5 expression seems to be independent of its catalytic activity. A pdc1-8 mutant was used to isolate spontaneous suppressor mutations, which allowed expression of PDC5. All three mutants characterized had additional mutations within the pdc1-8 allele. Two of these mutations resulted in a premature translational stop conferring phenotypes virtually indistinguishable from those of a pdc1Delta mutation. The third mutation, pdc1-803, led to a deletion of two amino acids adjacent to the pdc1-8 mutation. The alleles pdc1-8 and pdc1-803 were expressed in Escherichia coli and purified to homogeneity. In the crude extract, both proteins had 10% residual activity, which was lost during purification, probably due to dissociation of the cofactor thiamin diphosphate (ThDP). The defect in pdc1-8 (Asp291Asn) and the two amino acids deleted in pdc1-803 (Ser296 and Phe297) are located within a flexible loop in the beta domain. This domain appears to determine the relative orientation of the alpha and gamma domains, which bind ThDP. Alterations in this loop may also affect the conformational change upon substrate binding. The mutation in pdc1-14 (Ser455Phe) is located within the ThDP fold and is likely to affect binding and/or orientation of the cofactor in the protein. We suggest that autoregulation is triggered by a certain conformation of Pdc1p and that the mutations in pdc1-8 and pdc1-14 may lock Pdc1p in vivo in a conformational state which leads to repression of PDC5.