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.     

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.     

Is efficiency of suppressor tRNAs controlled at the level of ribosomal proofreading in vivo?

Ribosomal rpsD mutations did not stimulate nonsense suppressor tRNAs in a general manner according to their increased ribosomal ambiguity and decreased proofreading efficiency. Streptomycin, which stimulates error production by blocking proofreading in vitro, did not increase efficiency of suppressor tRNAs in strains with normal or streptomycin-resistant (rpsL) ribosomes. It did so only in combination with one rpsL mutation which is associated with streptomycin pseudodependence.     

A new class of mutations altering the response of the ribosome to streptomycin

Summary Mutants were isolated from high-level streptomycin dependent strains of Escherichia coli B, which do not spontaneously revert to antibiotic independence. In these mutants the requirement for streptomycin was much reduced, but not abolished. The relieving of the antibiotic dependence was caused by qui (for quasi-independent) mutations. These were analogous to the ramA (rpsD) mutations which relieve the streptomycin requirement of other classes of streptomycin dependent mutants, but strains harboring qui mutations exhibited novel streptomycin phenotypes in conjunction with all rpsL (strA) alleles. RamA mutations increase ribosomal misreading; qui mutations either did not significantly alter misreading, or else reduced it.This work was done in partial fulfilment of the requirements for the Ph. D. degree in the Division of Medical Sciences of Harvard University     

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.     

Mutant ribosomes can generate dominant kirromycin resistance.

Mutations in the two genes for EF-Tu in Salmonella typhimurium and Escherichia coli, tufA and tufB, can confer resistance to the antibiotic kirromycin. Kirromycin resistance is a recessive phenotype expressed when both tuf genes are mutant. We describe a new kirromycin-resistant phenotype dominant to the effect of wild-type EF-Tu. Strains carrying a single kirromycin-resistant tuf mutation and an error-restrictive, streptomycin-resistant rpsL mutation are resistant to high levels of kirromycin, even when the other tuf gene is wild type. This phenotype is dependent on error-restrictive mutations and is not expressed with nonrestrictive streptomycin-resistant mutations. Kirromycin resistance is also expressed at a low level in the absence of any mutant EF-Tu. These novel phenotypes exist as a result of differences in the interactions of mutant and wild-type EF-Tu with the mutant ribosomes. The restrictive ribosomes have a relatively poor interaction with wild-type EF-Tu and are thus more easily saturated with mutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomes are inherently kirromycin resistant and support a significantly faster EF-Tu cycle time in the presence of the antibiotic than do wild-type ribosomes. A second phenotype associated with combinations of rpsL and error-prone tuf mutations is a reduction in the level of resistance to streptomycin.     

Compensatory evolution reveals functional interactions between ribosomal proteins S12, L14 and L19

Certain mutations in S12, a ribosomal protein involved in translation elongation rate and translation accuracy, confer resistance to the aminoglycoside streptomycin. Previously we showed in Salmonella typhimurium that the fitness cost, i.e. reduced growth rate, due to the amino acid substitution K42N in S12 could be compensated by at least 35 different mutations located in the ribosomal proteins S4, S5 and L19. Here, we have characterized in vivo the fitness, translation speed and translation accuracy of four different L19 mutants. When separated from the resistance mutation located in S12, the three different compensatory amino acid substitutions in L19 at position 40 (Q40H, Q40L and Q40R) caused a decrease in fitness while the G104A change had no effect on bacterial growth. The rate of protein synthesis was unaffected or increased by the mutations at position 40 and the level of read-through of a UGA nonsense codon was increased in vivo, indicating a loss of translational accuracy. The mutations in L19 increased sensitivity to aminoglycosides active at the A-site, further indicating a perturbation of the decoding step. These phenotypes are similar to those of the classical S4 and S5 ram (ribosomal ambiguity) mutants. By evolving low-fitness L19 mutants by serial passage, we showed that the fitness cost conferred by the L19 mutations could be compensated by additional mutations in the ribosomal protein L19 itself, in S12 and in L14, a protein located close to L19. Our results reveal a novel functional role for the 50 S ribosomal protein L19 during protein synthesis, supporting published structural data suggesting that the interaction of L14 and L19 with 16 S rRNA could influence function of the 30 S subunit. Moreover, our study demonstrates how compensatory fitness-evolution can be used to discover new molecular functions of ribosomal proteins.     

Accuracy modulating mutations of the ribosomal protein S4-S5 interface do not necessarily destabilize the rps4-rps5 protein–protein interaction

During the process of translation, an aminoacyl tRNA is selected in the A site of the decoding center of the small subunit based on the correct codon–anticodon base pairing. Though selection is usually accurate, mutations in the ribosomal RNA and proteins and the presence of some antibiotics like streptomycin alter translational accuracy. Recent crystallographic structures of the ribosome suggest that cognate tRNAs induce a “closed conformation” of the small subunit that stabilizes the codon–anticodon interactions at the A site. During formation of the closed conformation, the protein interface between rpS4 and rpS5 is broken while new contacts form with rpS12. Mutations in rpS12 confer streptomycin resistance or dependence and show a hyperaccurate phenotype. Mutations reversing streptomycin dependence affect rpS4 and rpS5. The canonical rpS4 and rpS5 streptomycin independent mutations increase translational errors and were called ribosomal ambiguity mutations (ram). The mutations in these proteins are proposed to affect formation of the closed complex by breaking the rpS4-rpS5 interface, which reduces the cost of domain closure and thus increases translational errors. We used a yeast two-hybrid system to study the interactions between the small subunit ribosomal proteins rpS4 and rpS5 and to test the effect of ram mutations on the stability of the interface. We found no correlation between ram phenotype and disruption of the interface.     

Escherichia coli cells bearing a ribosomal ambiguity mutation in rpsD have a mutator phenotype that correlates with increased mistranslation

Escherichia coli cells bearing certain mutations in rpsD (coding for the 30S ribosomal protein S4) show a ribosomal ambiguity (Ram) phenotype characterized by increased translational error rates. Here we show that spontaneous mutagenesis increases in Ram cells bearing the rpsD14 allele, suggesting that the recently described translational stress-induced mutagenesis pathway is activated in Ram cells.     

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.     

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.     

Genetic characteristics and protective properties of neamin-resistant mutant Salmonella typhimurium and S. dublin of the Nea(r) Str(s) and Nea(r) Str(r) 500 classes

The genetic analysis of attenuated mutants, class Nea(r) Str(s), with the use of bacteriophage P 22 has shown that mutation rendering the mutants resistant to neamine is localized in gene nea A. In experiments with the intraperitoneal infection of mice, the appearance of this mutation in S. typhimurium and S. dublin virulent strains has been found to lead to the decrease of virulence in 100% of clones. On the basis of the data obtained in this investigation, region str-spc in S. typhimurium and S. dublin has been mapped. In contrast to mutation spc A, mutations nea A and str A have been shown to inhibit the action of amber suppressor. The investigation has confirmed the regularity, previously established for Shigella flexneri, concerning the relationship between the influence of mutations, occurring in the genes which determine resistance to neamine and streptomycin and control the synthesis of ribosomal proteins S4, S5, S12 and S17, on the virulence of S. typhimurium and S. dublin and the effect of these mutations on the accuracy of the translation of genetic information in the biosynthesis of protein: mutation spc A has been found to produce no changes in the virulence of salmonellae, while mutations nea A and str A cause its loss. Salmonella strains carrying mutations nea A and nea B have shown pronounced protective properties in experiments on mice.     

Evidence for a unique first position codon-anticodon mismatch in vivo

The Ser68(AGC) codon of the beta-lactamase gene was changed to the glycine codons GGA and GGC. With glycine at position 68, beta-lactamase is inactive because it does not have a nucleophilic side-chain to function in the reaction mechanism. The mutant SG68(GGA) allele had no detectable beta-lactamase activity; however, the mutant SG68(GGC) did produce a small amount of activity. Both mutant alleles produce comparable amounts of beta-lactamase protein in a maxi-cell system. To identify why these two "same-sense" beta-lactamase mutants differ phenotypically, we introduced the alleles into Escherichia coli strains with mutations that affect translational fidelity. The rpsD mutation, which decreases fidelity, significantly increased activity with the SG68(GGC) allele, while the rpsL mutation, which increases translational fidelity, had little effect on the beta-lactamase activity. The rpsD and rpsL alleles had no effect on the SG68(GGA) allele. From the allele specificity of the activity produced by the bla mutants, and from the differential effect of translational fidelity on the activity of the SG68(GGC) allele, we infer that tRNA(GCU)Ser, the AGU/C reading tRNA(Ser), mistranslates SG68(GGC) at a frequency of about 0.1%, and subsequently produces active beta-lactamase. This is the first observation of an A/G wobble with a wild-type tRNA at the first position of the codon-anticodon interaction.     

A single base substitution in 16S ribosomal RNA suppresses streptomycin dependence and increases the frequency of translational errors

A C to U substitution at position 1469 of 16S ribosomal RNA (rRNA) from Escherichia coli suppresses streptomycin dependence and causes increased translational error frequencies. Strains containing the rpsL252 or StrM287 streptomycin-dependent alleles are able to grow in the absence of streptomycin when transformed with plasmids containing the U1469 mutation in 16S rRNA. Ribosomes containing wild-type proteins and U1469 mutant 16S rRNA misincorporate leucine in vitro at elevated levels, comparable to that of some typical S4 ram ribosomes. These results provide additional support for the participation of 16S rRNA in maintaining translational accuracy.     

Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)

Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying DeltarsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the DeltarsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the DeltarsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the DeltarsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.     

UGA suppressor that maps within a cluster of ribosomal protein genes.

A suppressor of UGA mutations (supU) maps near or within a cluster of ribosomal protein genes at 72 min on the Salmonella typhimurium genetic map. The suppressor is relatively inefficient, and its activity is abolished by rpsL (formerly strA) mutations. The suppressor is dominant to a wild-type supU allele. The map position of this suppressor suggests that it may owe its activity to an alteration of ribosome structure.     

A novel single amino acid change in small subunit ribosomal protein S5 has profound effects on translational fidelity

S5 is a small subunit ribosomal protein (r-protein) linked to the functional center of the 30S ribosomal subunit. In this study we have identified a unique amino acid mutation in Escherichia coli S5 that produces spectinomycin-resistance and cold sensitivity. This mutation significantly alters cell growth, folding of 16S ribosomal RNA, and translational fidelity. While translation initiation is not affected, both +1 and -1 frameshifting and nonsense suppression are greatly enhanced in the mutant strain. Interestingly, this S5 ribosome ambiguity-like mutation is spatially remote from previously identified S5 ribosome ambiguity (ram) mutations. This suggests that the mechanism responsible for ram phenotypes in the novel mutant strain is possibly distinct from those proposed for other known S5 (and S4) ram mutants. This study highlights the importance of S5 in ribosome function and cell physiology, and suggests that translational fidelity can be regulated in multiple ways.     

Antagonistic effects of mutant elongation factor Tu and ribosomal protein S12 on control of translational accuracy, suppression and cellular growth

Kirromycin-resistant mutant forms of elongation factor Tu, which are coded by tufA (Ar) or tufB (Bo) and are associated with an increased rate of translational error formation, have been analysed. In vivo, Ar was found to increase misreading as well as suppression of non-sense codons irrespective of Bo in a strain with wild type ribosomes. It is therefore not necessary to evoke both tufA (Ar) and tufB (Bo) mutations together in order to increase translational error as suggested earlier [1]. When combined with a hyperaccurate ribosomal rpsL (S12) mutation, Ar counteracts the restrictive effects on translational error formation caused by the altered protein S12, thus restoring the levels of missense error in vitro and non-sense error and suppression in vivo to near wild type values. As judged from in vitro experiments this results principally from a lowered selectivity of the Ar ternary complex at the initial discrimination step on the ribosome during translation. In vivo, this compensatory effect on the rpsL mutation on non-sense error formation and suppression is seen irrespective of the nature of tRNA or codon context. Furthermore, the tufA mutation enhances the cellular growth rate of the rpsL mutant, whereas it decreases growth of strains with normal ribosomes. Inactivation of one of the two genes coding for EF-Tu (tufB), while leaving the other gene (tufA) intact, can by itself, increase non-sense error formation and suppression.     

Suppressors of Recb Mutations in Salmonella Typhimurium

Using a screen that directly assesses transductional proficiency, we have isolated suppressors of recB mutations in Salmonella typhimurium. The alleles of sbcB reported here are phenotypically distinct from those isolated in Escherichia coli in that they restore recombination proficiency (Rec(+)), resistance to ultraviolet light (UV(R)), and mitomycin C resistance (MC(R)) in the absence of an accompanying sbcCD mutation. In addition the sbcB alleles reported here are co-dominant to sbcB(+). We have also isolated insertion and deletion mutants of the sbcB locus. These null mutations suppress only the UV(S) phenotype of recB mutants. We have also isolated sbcCD mutations, which map near proC. These sbcCD mutations increase the viability, recombination proficiency and MC(R) of both the transductional recombination suppressors (sbcB1 & sbcB6) and the sbcB null mutations. S. typhimurium recB sbcB1 sbcCD8 strains are 15-fold more recombination proficient than wild-type strains. The increase in transductants in these strains is accompanied by a loss of abortive transductants suggesting that these fragments are accessible to the mutant recombination apparatus. Using tandem duplications, we have constructed sbcB merodiploids and found that, in a recB mutant sbcCD(+) genetic background, the sbcB(+) allele is dominant to sbcB1 for transductional recombination but co-dominant for UV(R) and MC(R). However, in a recB sbcCD8 genetic background, the sbcB1 mutation is co-dominant to sbcB(+) for all phenotypes. Our results lead us to suggest that the SbcB and SbcCD proteins have roles in RecBCD-dependent recombination.     

Functional interactions between mutated forms of ribosomal proteins S4, S5 and S12

Here we show that ram mutations, either in ribosomal protein S4 or S5, decrease the proofreading flows for both cognate and noncognate ternary complexes bound by streptomycin-dependent (SmD) ribosomes. This effect is accompanied by a slight increase in the overall error frequency. More important, however, is the decreased proofreading of the cognate species which is almost reduced to wild-type levels. The data suggest that it may be the reduction of the proofreading of the cognate substrate that is important for suppressing streptomycin dependence. Furthermore, we show that rpsE mutants, selected from streptomycin-dependent strains, behave kinetically very similarly to the previously described rpsD mutants.