Genetic studies of the ribosomal proteins in Escherichia. coli. II. Altered 30s ribosomal protein component specific to spectinomycin resistant mutants

Summary Spectinomycin resistant (spc ^r) mutants were obtained by treating the cells of E. coli K12, W3637 with nitrosoguanidine. The compositions of ribosomal proteins were analyzed for six out of eleven such spc ^r-mutants with chromatography on a carboxymethyl cellulose (CMC) column. The 30s ribosomal subunit from all of the spc ^r-mutants was found to contain the altered 30-4 protein component, while no difference was detected in 50s ribosomal proteins between spc ^r and spc ^s bacteria.Abbreviations used CMC carboxymethyl cellulose - str streptomycin - spc spectinomycin     

Genetic studies of the ribosomal proteins in Escherichia coli. 7. Mapping of several ribosomal protein components by transduction experiments between different strains of Escherichia coli

Summary Two 50s (50-10 and 50-12) and two 30s (30-4 and 30-7) ribosomal proteins could be distinguished between Shigella dysenteriae Sh/s and Escherichia coli K-12 JC411 with CMC column chromatography. On the other hand, E. coli K-12 AT2472 was shown to have a 30s ribosomal protein, 30-6(AT), which is specific to this strain and distinguishable from 30-6 of other E. coli K-12 strains. Transduction experiments by phage Plkc between Sh. dysenteriae Sh/s and E. coli ATSPCO1, a spectinomycin resistant mutant derived from AT2472 in which the 30-4 protein is altered, indicated that the genes specifying the above five ribosomal protein components are located in the streptomycin region on the E. coli chromosome.The gene order for three 50s (50-8, 50-10 and 50-12) and three 30s [str (30-?), 30-4 and 30-6] ribosomal proteins on the chromosome was determined by transduction technique between Sh. dysenteriae Sh/s and E. coli ATSPC01, between E. coli ATSPC01 and E. coli ER05 (an erythromycin resistant strain in which the 50-8 protein is altered), and between Sh. dysenteriae Sh/s and E. coli ERSPC14 (str ^s spc ^r ery ^r), respectively. It was found that these protein genes are arranged on the chromosome in the order of str (30-?)-30-4-30-6-50-8-50-10-50-12.     

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     

Genetic studies of the ribosomal proteins in Escherichia coli. 3. Compositions of ribosomal proteins in various strains of Escherichia coli

Summary The comparative chromatographic investigations into the ribosomal proteins of various strains of E. coli have demonstrated that most of the strains including three strains of E. coli subsp. communior had ribosomes with the same protein compositions (C-type). The ribosomes from strain B are different from the C-type ribosomes in having the specific 30-4 (B) component in place of 30-4 (B-type), while those from strains K 12 may be distinguished from the type-C ribosomes by the presence of the specific 30-7 (K) component in place of 30-7 (K-type) or, in addition to 30-7 (K), the presence of 30-9 (W3637) in place of 30-9 (K-3637 type). Two strains, IAM 1132 and IAM 1182, have R-type ribosomes, in which at least six 50s proteins and four 30s protein components are distinct from the corresponding protein components in the C-type ribosomes.     

Genetic studies of the ribosomal proteins in Escherichia coli. I. Mutants and strains having 30s ribosomal subunit with altered protein components

Summary Two 30s ribosomal protein components, 30-4_K and 30-7_K, from E. coli K12 strain were clearly distinguished on the CMC column chromatogram from the corresponding protein components, 30-4_B and 30-7_B, from B strain. The 30-7_K component was shown to correspond to the K-character.A mutant strain of K12, W3637, had an altered 30s ribosomal protein component, 30-9_W3637.The characters of 30-4_K, 30-7_K, 30-9_W3637 and str ^r were found to be cotransduced from W3637 to B strain by Plke phage in 16 out of 20 str ^r transductants. The 30-9_W3637 and 30-4_K components were separated from str ^r in 4 str ^r transductants. These results indicate that (1) neither 30-4 nor 30-9 is the protein whose mutational change leads to str ^r, and (2) the genes specifying the 30s ribosomal proteins, 30-4, 30-7, 30-9 and str are linked on the chromosome.Abbreviations used CMC carboxymethyl cellulose - str streptomycin     

Genetic studies of the ribosomal proteins in Escherichia coli. 8. Mapping of ribosomal protein components by intergeneric mating experiments between Serratia marcescens and Escherichia coli

Summary Ribosomal protein compositions of Serratia marcescens and Escherichia coli K12 were analyzed by using carboxymethyl cellulose column chromatography. Nine 50S and nine 30S ribosomal proteins of E. coli K12 could be distinguished from those of S. marcescens on the chromatogram.Episomes of E. coli K12, which cover the streptomycin(str) region of the chromosome, were transferred to S. marcescens. Chromatographic analyses were made on the ribosomal proteins extracted from these hybrid strains. At least nine 30S and six 50S ribosomal proteins of E. coli-type could be detected in the ribosomes of the hybrid strains in addition to the ribosomal proteins of S. marcescens.     

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.