H. G. Wittmann

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

SummaryRibosomes 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.

Correlation of 30S ribosomal proteins ofEscherichia coli isolated in different laboratories

SummaryRibosomal proteins isolated from 30S subunits ofE. coli in four laboratories have been correlated by using two-dimensional gel electrophoresis, immunological techniques, amino acid compositions and molecular weights. The results are given in the Table. A common nomenclature for naming 30 S ribosomal proteins and their genetic loci is proposed.

Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence

SummaryRevertants from streptomycin dependence to independence were isolated as single step mutants from six different streptomycin dependent strains. The ribosomal proteins from 100 such mutants were analyzed by two-dimensional polyacrylamide gel electrophoresis and some of them were also examined by immunological techniques. Altered proteins were found in 40 mutants, 24 in protein S4 and 16 in protein S5. No change in any other protein was detected.Altered S5 proteins migrated into five different positions on the polyacrylamide plate and it can be concluded that the mutant proteins differ from the wild type probably by single amino acid replacements. The altered S4 proteins migrated into 17 different positions on the plate. Extensive changes of length, both shorter and longer than wild type S4 protein, are postulated for many of the mutant S4 proteins.Analysis of the ribosomal proteins of four ram mutants revealed altered S4 protein in two of them. The alterations in these mutant proteins are probably very similar to those found in streptomycin independent mutants.Among the revertants there was no apparent correlation between the protein alteration and the particular response to streptomycin.These studies suggest a strong interaction between protein S12, which confers streptomycin dependence, and protein S4 or S5, which can suppress this dependence.

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

SummaryRibosomes 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.

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

SummarySpontaneous 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 μg/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 μg 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.

Coat proteins of strains of two RNA viruses: Comparison of their amino acid sequences

SummaryThe amino acid sequences of four strains of tobacco mosaic virus isolated in different parts of the world are compared. The differences between the strains are discussed with respect to special proteinchemical features (such as beginning of the chain, deletion of amino acids, number of different amino acids, sizes and distribution of regions with invariable amino acids) and with respect to the possibility of deducing the most probable nucleotide sequence for the coat protein cistron of tobacco mosaic virus.The complete amino acid sequences of the two RNA bacteriophage strains fr and f2 are compared. According to their coat proteins three groups of phages can be formed: 1) MS 2, f2 M 12 and R 17, 2) fr and 3) Qβ.

Altered S5 and S20 ribosomal proteins in revertants of an alanyl-tRNA synthetase mutant of Escherichia coli

Summary120 phenotypic revertants of a temperature-sensitive alanyl-tRNA synthetase (alaS) mutant of Escherichia coli were isolated and screened for the ribosomal suppressor phenotype reported recently. About 20% of the mutants showed altered ribosomal sedimentation patterns which indicated a defect in ribosome assembly. The mutants were analysed by two-dimensional polyacrylamide gel electrophoresis and immunological methods for changes in ribosomal proteins.Alterations of ribosomal proteins could be identified in three mutants. One of them (0–1) had an alteration in protein S5. The mutated protein was immunologically different not only from wild type S5 but also from the altered S5 of spectinomycin resistant mutants and from the altered S5 of mutants suppressing streptomycin dependence. Two other mutants (39-1 and 64-2) had an altered S20 protein. In one of them (64-2) protein S20 was present in a reduced amount compared to the amount of S20 in wild type ribosomes.For the mutant (0–1) with the altered protein S5 a clear correlation could be demonstrated between the presence of the altered ribosomal protein and the suppression of temperature-sensitive growth of the alaS mutant.

Immunological and electrophoretical comparison of ribosomal proteins from eight species belonging to Enterobacteriaceae

SummaryThe ribosomal proteins of seven different Enterobacteriaceae were compared with those of E. coli by two-dimensional gel electrophoresis and by immunological methods. The ribosomal proteins of all Enterobacteriaceae were found to be very similar in molecular weight and in their electrophoretic properties. However, more dissimilarities could be detected by immunological methods thus indicating that few, if any, of the ribosomal proteins among the tested Enterobacteriaceae are identical. Ribosomes from all Enterobacteriaceae possess a protein which is electrophoretically identical with protein S7 from strain B (and not strain K) of E. coli.

Genetic analysis of an alteration of ribosomal protein S20 in revertants of an alanyl-tRNA-synthetase mutant of Escherichia coli

SummaryThe genetic location has been determined of two mutations which suppress the temperature-sensitive phenotype of an alanyl-tRNA-synthetase mutant of Escherichia coli and which are correlated with alterations of the ribosomal protein S20. Both mutations map at the same chromosomal site; the gene order relative to other markers of the Escherichia coli map is thr-sup-pyrA-araC-leu.Replacement of the suppressor allele by the wild-type allele via P1 transduction results in the appearance of the wild-type S20 protein; concomitantly suppression of temperature-sensitivity is released.Strains of Escherichia coli were contructed which are partially diploid for the region of the chromosome containing the suppressor allele. Investigation of these strains revealed that the wild-type suppressor is dominant as judged by the activity to suppress the alaS mutation since the partial diploids are no longer able to suppress the alaS-3 mutation. Investigation of the ribosomal protein pattern of these partial diploids by means of two-dimensional polyacrylamide gel electrophoresis did not reveal two distinct spots characteristic for the normal and the altered forms of S20; rather, an elongated spot was observed trailing from the wild-type S20 position towards the anode.

Ribosomes after infection with bacteriophage T4 and T7.

SummaryThe synthesis of E. coli ribosomal proteins ceases after infection with bacteriophages T4 or T7 as does the synthesis of most other host proteins. The shut-off does not affect all ribosomal proteins to the same extent. After T7 infection no new proteins were detected in NH4Cl-washed ribosomal particles. Bacteriophage T4, however, induces 3–4 new protein bands demonstrated by one-dimensional gel electrophoresis. The appearance of these bands is prevented by the addition of rifampicin at the time of infection but not when rifampicin is added one minute after infection. The NH4Cl-washed ribosomal particles present at the time of T7 or T4 infection do not show any structural changes by sedimentation, subunit dissociation, or protein analysis on two-dimensional polyacrylamide gels. However, by labeling the T7 infected cells with 32P-phosphate, it is seen that the ribosomes become phosphorylated. The 32P-label comigrates with ribosomal proteins. This phosphorylating activity depends on a T7 gene. The T7 protein phosphokinase utilizes ribosomes as phosphate acceptor in vitro. The T7 ribosomes (NH4Cl-washed) still function in vitro as do ribosomal particles from uninfected cells.