Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

Analysis of ribosomal proteins in streptomycin resistant and dependent mutants isolated from streptomycin independent Escherichia coli strains

Summary Mutants resistant to (Str-R) or dependent on streptomycin (Str-D) were isolated from several streptomycin independent (Str-I) strains of Escherichia coli. From 90 of these mutants ribosomes were isolated and the ribosomal proteins analyzed by two-dimensional polyacrylamide gel electrophoresis. The results which are summarized in Tables 1-4 led to the following conclusions:a) The phenotype (Str-R or Str-D) of the mutants isolated from the Str-I strains strongly depends on the parental strain. b) No other ribosomal proteins than S4, S5 and S12 seem to be altered by mutations leading to dependence on, independence from or resistance to streptomycin. c) The S4 proteins of the analyzed mutants belong to three groups. The ratio between the groups depends more on the origin of the mutants than on their phenotype. d) Eight new types of altered S4 proteins were detected. It is very likely that many, if not all, of the altered S4 proteins originated by frame shift mutations. e) Some of the mutants differ from the wild type by alterations in three ribosomal proteins (S4, S5 and S12). The alteration in one protein, S4, apparently compensates for that in another protein, S5, in such a way that the original phenotype is expressed. These mutants are therefore an excellent tool for studies at the molecular level on the interaction of ribosomal components within the particle.     

Properties of the interaction of ribosomal protein S4 and 16S RNA in Escherichia coli revertants from Streptomycin dependence to independence

Summary The binding properties of altered S4 proteins from E. coli revertants from streptomycin dependence to independence were investigated. Three of the proteins with the same length as the wild type protein, from mutants N424, N428 and N430, exhibited unchanged binding and conformational properties. However, three proteins with an altered length, from mutants N422, N425 and N433, bound more weakly to the 16S RNA, and their conformations were different from that of the wild type S4 protein. In the presence of the other 16S RNA binding proteins, no stimulation of the binding of the latter three proteins could be detected.Paper No. 40 on Ribosomal Proteins. Preceding paper is in press in Molec. gen. Genetics (1972).     

A strain of Escherichia coli which gives rise to mutations in a large number of ribosomal proteins

Summary A strain of E. coli K12 has been isolated which gives rise to mutations in a large number of ribosomal proteins. Mutant VT, which was derived from A19, shows a novel type of streptomycin dependence and has an altered ribosomal protein S8. Streptomycin-independent isolates from mutant VT contain a great variety of changed proteins on two-dimensional polyacrylamide gels. 120 revertants screened in this way have changes in thirteen 30S proteins and fifteen 50S proteins. Several mutants were found in which additional proteins are present on the ribosome. Further, there is one instance of a ribosomal protein (L1) being absent, and one of apparent doubling of a ribosomal protein (L7/12). The unique properties of mutant VT probably are the result of the altered S8.     

Ribosomal proteins

Summary Ribosomal proteins fromE. coli mutant N421 which is a spontaneous revertant from streptomycin dependence to independence have been compared to those of the wild type by two-dimensional polyacrylamide gel electrophoresis and by four immunochemical methods. The only detectable difference is a change in protein S5. This finding suggests that reversion from streptomycin dependence to independence can be caused not only by a mutation in protein S4, as described earlier, but also in protein S5.     

Bacillus subtilis mutants dependent on streptomycin

Summary Six streptomycin-dependent mutants of Bacillus subtilis, two of which were asporogenous, were isolated. All six mutants, SD1, SD2, SD6, SD7, SD9 and SD10, contained a single mutation causing streptomycin dependence and asporogeny, but four of these mutants (SD6, SD7, SD9, SD10) contained a second mutation which phenotypically suppressed the asporogenous character of the streptomycin dependence mutation. All six mutants grew more slowly than the wild type strain BR151, but those defective in sporulation grew the slowest. The streptomycin dependence mutations of SD9 and SD10B (a sporeplus transformant from SD10 carrying both the dependence mutation and the phenotypic suppressor) lie near or possibly within the strA locus. Ribosomes from SD9, SD10A (a spore-minus transformant from SD10 carrying only the dependence mutation), and SD10B were stimulated in vitro by concentrations of streptomycin that inhibit the activity of wild type strain BR151 ribosomes. The level of misreading as measured by poly(U)-directed isoleucine incorporation was greatly enhanced by streptomycin in wild type strain BR151 ribosomes, but misreading of mutant SD9, SD10A, and SD10B ribosomes, irrespective of the sporulation phenotype, was little affected by streptomycin. There were no apparent differences in the patterns obtained by two-dimensional polyacrylamide gel electrophoresis of the 70S ribosomal proteins of the mutants SD9, SD10A, SD10B, and wild type strain BS151.     

Alteration of ribosomal protein L6 in mutants of Escherichia coli resistant to gentamicin.

Summary Spontaneous and ethylmethane-sulfonate induced mutants of Escherichia coli resistant to gentamicin sulfate were isolated and investigated for alterations in the ribosomal protein pattern. It was found by two-dimensional polyacrylamide gel electrophoresis that three independently isolated strains did not show any spot for ribosomal protein L6. On cochromatography of radioactively labelled mutant and wild-type ribosomal proteins on carboxymethyl-cellulose columns a shift of the elution position of protein L6 was observed, the new elution positions being characteristic for the individual mutants analyzed which indicates that they possess different alterations in the L6 primary structure.Genetic analysis showed that the gentamicin resistant strains contain at least two mutations. One of them correlates with the altered L6 protein and causes an increased minimal inhibitory concentration of the drug by about 5 to 10-fold. The other mutation is not yet biochemically characterized. Its presence is connected with an about 10 to 20-fold increase in the resistance. Both mutations, when put together, confer resistance to 50 to 100 g/ml of the antibiotic in a low salt rich medium and to 1 mg/ml in a defined medium with a high concentration of phosphate. Cross-resistance analysis demonstrated that the three gentamicin-resistant (double-mutant) strains with the altered L6 protein are resistant to 50–100 g per ml of all other aminoglycoside antibioties tested. This forms a sharp contrast to the streptomycin resistance mutations present in strA1, strA40 or strA60 mutants which do not confer markedly increased levels of resistance to most of the other aminoglycosides.     

Assembly analysis of ribosomes from a mutant lacking the assembly-initiator protein L24: lack of L24 induces temperature sensitivity

Summary Previously, we have shown that the ribosomal protein L24 is one of two assembly-initiator proteins. L24 is essential for early steps of the assembly of the 50S ribosomal subunit but it is not involved in both the late assembly and the ribosomal functions. Surprisingly, an E. coli mutant (TA109-130) exists which lacks L24. This apparent paradox is analyzed and resolved in this paper. The phenotypic features of the mutant lacking L24, are a temperature sensitivity (growth severely reduced beyond 34° C), a very low growth rate already at permissive temperatures (at least six-fold slower than wild type) and an underproduction of 50S subunits (molar ratio of 30S to 50S about 1:0.5). The S value of the mutant large subunits is 47S, and they are normally active in poly(Phe) synthesis. The total protein of the mutant large subunits show negligible activity in the total reconstitution assay using the standard two-step procedure. Number analysis of the assembly-initiator proteins revealed that only one initiator protein is effective, as expected. The activity is restored upon addition of wild-type L24. However, when the temperature of the first step is lowered from 44° to 36° C, reconstitution of active particles occurs with a 50% efficiency in the absence of L24. The recovery of activity is accompanied by the appearance of again two initiator proteins, when the mutant TP50 lacking L24 is used in the reconstitution assay at the permissive temperature of 36° C during the first step. These findings indicate that at least another protein or, alternatively, two other proteins take over the function of the assembly initiation at the lower temperature. Although the extent of the formation of active particles becomes independent of L24 below 36° C, the rate of formation is still strongly affected even at permissive temperatures. The presence of L24 reduces the activation energy of the rate-limiting step of the early assembly, i.e., the activation energy of RI ₅₀ ^* (1) formation is 43±4 kcal/mol in the presence and 83±9 kcal in the absence of L24. The results presented provide an explanation of the phenotypic features of the mutant solely due to the assembly effects caused by the lack of L24.     

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin.Biochemical analysis shows that the mutants each have two modified 30S ribosomal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).     

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.     

Streptomyces relC mutants with an altered ribosomal protein ST-L11 and genetic analysis of a Streptomyces griseus relC mutant

Several relaxed (rel) mutants have been obtained from Streptomyces species by selecting colonies resistant to thiopeptin, an analogue of thiostrepton. Using two-dimensional gel electrophoresis, I compared the ribosomal proteins from rel and rel+ pairs of S. antibioticus, S. lavendulae, S. griseoflavus, and S. griseus. It was found that all of the Streptomyces rel mutants thus examined had an altered or missing ribosomal protein, designated tentatively ST-L11. These rel mutants therefore could be classified as relC mutants and were highly sensitive to erythromycin or high temperature. A relC mutant of S. griseus was defective in streptomycin production, but phenotypic reversion of this defect to normal productivity was found at high incidence among progeny of the relC mutant. This phenotypic reversion did not accompany a reappearance of ribosomal protein ST-L11, and furthermore the ability of accumulating ppGpp still remained at a low level, thus suggesting existence of a mutation (named sup) which suppresses the streptomycin deficiency phenotype exhibited by the relC mutant. Genetic analysis revealed that there is a correlation between the rel mutation and the inability to produce streptomycin or aerial mycelia. The sup mutation was found to lie at a chromosomal locus distinct from that of the relC mutation. It was therefore concluded that the dependence of streptomycin production on the normal function of the relC gene could be entirely bypassed by a mutation at the suppressor locus (sup). The suppressing effect of the sup mutation on the relC mutation was blocked when the afs mutation (defective in A-factor synthesis) was introduced into a relC sup double mutant. It is proposed that the sup gene or its product can be direct or indirect target for ppGpp.     

Biochemical and genetic studies on two different types of erythromycin resistant mutants of Escherichia coli with altered ribosomal proteins

Summary Ribosomes from nine E. coli mutants with high level resistance to the antibiotic erythromycin were isolated and their proteins were compared with those of the parental strains by two-dimensional polyacrylamide gel electrophoresis, by carboxymethylcellulose column chromatography and by immunological techniques. Two 50S proteins were found to be altered in the mutants: either L 4 or L 22.Ribosomes with an altered L4 protein bound erythromycin rather poorly and the formation of N-acetylphenylalanyl puromycin was drastically decreased. On the other handribosomes with an altered L22 protein bound erythromycin as efficiently as wild type ribosomes and their puromycin reaction was at least as good as that of wild type ribosomes.Transduction experiments showed that the mutations affecting both proteins, L4 and L22, are located very close to the str and spc genes, nearer to the spc than to str gene.Paper No. 61 on Ribosomal Proteins. Preceding paper is by Hasenbank et al., Molec. gen. Genet., 127, 1–18 (1973).Communicated by E. Bautz     

Pleiotropic effects resulting from mutations in genes for ribosomal proteins: analysis of revertants from streptomycin dependence

In order to study the functions of the individual ribosomal proteins and their interaction, a group of revertants from streptomycin dependence to independence was analyzed. Reversion from dependence resulted from a number of different mutational events, all resulting in altered ribosome function. The mutants selected for study exhibited extensive pleiotropy—in addition to the elimination of the requirement for streptomycin for growth, the strains differed from the dependent parent and each other in growth rate, level of streptomycin resistance, effect of antibiotics on viability, rate of subunit assembly in vivo, affinity of isolated ribosomes for streptomycin and functionality of ribosomes in various cell-free assays.There appear to be strong correlations between the level of resistance to streptomycin in growing cells and the ability of the isolated ribosomes to bind streptomycin, the effect of antibiotic on cell-free protein synthesis programmed with natural message (but not poly(U)) and the degree of translational fidelity. There seems to be no relation between level of antibiotic resistance and the overall growth rate, the presence of a defect in ribosome assembly or the ribosomal protein altered by the mutation. Mutations in genes for 30 S proteins S4 and S5 can result in the same phenotype, while different changes in S4 in otherwise isogenic strains result in widely varying phenotypes.The wide variety of effects resulting from single mutational events suggests that each of these changes in a ribosomal protein changes the conformation of the ribosome or its ability to undergo configurational changes.     

An improved method for two-dimensional gel-electrophoresis: Analysis of mutationally altered ribosomal proteins of Escherichia coli

Summary An improved method for the two-dimensional electrophoretic analysis of ribosomal proteins on acrylamide gel slabs has been developed by combining the procedures for the first dimension of Mets and Bogorad (1974) and for the second dimension of Kaltschmidt and Wittmann (1970) and by introducing several modification. Ribosomal proteins of various Escherichia coli mutants have been analyzed by the new method. Advantages are that (1) it requires only small amounts of protein (100–200 g 70S ribosomal proteins), (2) reproducibility is very high, and (3) it makes it easier to identify mutational alterations in proteins S10, L4, L10, and L21 which hardly migrate out of the sample gel with our previous electrophoresis procedure. Furthermore, the new method can be nicely adapted to analysis of the ribosomal proteins from other organisms, such as Bacilli or yeast.     

Ribosomal protein S12 as a site for streptomycin resistance in Nicotiana chloroplasts

Summary A streptomycin resistant Nicotiana plastome mutant, X/str ^R6, was subjected to molecular analysis. In this mutant, a single nucleotide transition, C » T, in the chloroplast gene for ribosomal protein S12 alters codon 90 from proline to serine while the nucleotide sequence of the chloroplast 16 S rRNA gene is identical to that of the wild type. Mutant X/str ^R6 thus differs from several previously reported streptomycin resistant chloroplast mutants which are altered in the gene for 16 S rRNA.     

Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA

We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS-unreactive. In this model, the balance between these two states would be influenced by proteins S4 and S12. Mutations in S12 generally cause a shift toward the unreactive conformer, and in the case of SmD and SmP ribosomes, this shift can be suppressed phenotypically by streptomycin, ram mutations in protein S4 cause a shift toward the reactive conformer, but only when tRNA is bound. This suggests that the opposing effects of these two classes of mutations influence the proof-reading process by somewhat different mechanisms.     

Improved electrophoretic and immunochemical techniques for the identification and characterization of mutant proteins, applied to ribosomal protein S8 in Escherichia coli mutants.

Summary The ribosomal proteins of 11 mutants which are sensitive to starvation at elevated temperature and of 36 transductants derived from them were studied with several electrophoretic, immunochemical and proteinchemical methods. The following results were obtained: (1) Ribosomal protein S8 is altered in three of these mutants. (2) The amino acid exchange in protein S8 of mutant N4128 is GluLys in position 59 of the protein chain. (3) Temperature sensitivity and inability to recover from starvation at elevated temperatures are caused by the same mutational event which is, however, unrelated to the alteration in protein S8.Several electrophoretic and immunological procedures were applied during the characterization of these mutants. A modified immunoelectrophoresis on cellulose acetate gels was developed, and proved to be the most applicable procedure for the detection of mutationally altered ribosomal proteins. This procedure may gain general importance for detecting mutational alterations in other proteins.     

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.