strA Locus Research Articles

Preparation of ultrafine monosized gold particles

UV-induced mutability to prototrophy depended on the alleles of the strA locus. The yield of UV-induced suppressor mutations was decreased ten-fold in a Strr strain compared with an isogenic Strs strain. This decrease was due to the inability of the majority of suppressors to be expressed phenotypically in the Strr strain in the course of selection. The addition of streptomycin to the selective media raised the number of selected suppressor revertants, by making such expression possible. The probable relation of streptomycin resistance and UV-induced suppressors is discussed.

Bacillus subtilis mutants dependent on streptomycin

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.

Ribosome slowed by mutation to streptomycin resistance.

ALTHOUGH the basic mechanisms of protein synthesis are now rather well understood, particularly in Escherichia coli, it is less clear how the high accuracy of this process is achieved. (The error rate in protein synthesis in E. coli seems to be about one in 104 amino acid misincorporations (Edelman and Gallant, unpublished results, and our own unpublished data)). A major site determining accuracy in protein synthesis is the ribosome, and in addition to the ribosome-mediated effects on accuracy (in vivo and in vitro) of various metal ions, aminoglycoside antibiotics and organic solvents, there are ribosomal mutations which increase or decrease accuracy2. Certain mutations of the 30S ribosomal protein SI2 conferring streptomycin resistance (the strA locus) enhance accuracy3–5, whereas certain mutations in the S4 protein (the ram locus) are known to decrease accuracy6. The strA mutations are restrictive (reducing) in their effect on both missense and nonsense suppression in vivo and on miscoding in vitro3, the ram mutation, on the other hand, has the opposite effect in both cases6. It is possible that it is the kinetics of polypeptide synthesis at the ribosome that determines accuracy and that the kinetics in turn are affected by the strA and ram mutations. Because the same transfer RNA (tRNA) discrimination kinetics that may determine accuracy may also contribute to the elongation speed, we have investigated the effect of mutation to streptomycin resistance on the speed of polypeptide elongation.

Growth of bacteriophages MS2 and T7 on streptomycin-resistant mutants of Escherichia coli.

Streptomycin-resistant mutants of an Hfr strain of Escherichia coli K were examined for their ability to support the growth of male-specific ribonucleic acid phage MS2 and female-specific deoxyribonucleic acid phage T7. Normally, the Hfr strain allows propagation of MS2 and is lysed by it (efficiency of plating equal to 1), whereas the same strain restricts propagation of T7 and is not lysed by it (efficiency of plating smaller than 10-7). Twenty-four isolates out of 26 independently obtained streptomycin-resistant mutants are partially or completely derestricted for propagation of T7; efficiency of plating of T7 in such strains ranges from 10-3-1. Depending on their response to plating of MS2 and T7, the streptomycin-resistant mutants can be divided into four classes. The mutants in all four classes continue to be "male" in conjugation with F- strains. Genetic analysis is presented to show that restriction of MS2, derestriction of T7, and resistance to streptomycin are the pleiotropic effects of a single mutation at the strA locus.

Genetic position and amino acid replacements of several mutations in ribosomal protein S5 from Escherichia coli.

The relative genetic position of the following four mutations of ribosomal protein S5 has been determined: spc-13, a mutation to spectinomycin resistance; stri N421 and strid1023, mutations suppressing dependence on streptomycin and sup0-1, a mutation suppressing partially the temperature-sensitive phenotype of an alanyl-tRNA synthetase mutation. The transduction experiments performed indicate that the spc-13 site is located in the S5 cistron proximal to the strA locus, that sup0-1 maps proximal to the aroE gene and that the striN421 and strid1023 loci are located between these two mutational sites. Proteinchemical analysis of the amino acid replacement in protein S5 of strain N421 (carrying the striN421 allele) has shown that an arginine residue is replaced by leucine which results in the appearance of a trypsin intensitive bond between the tryptic peptides T2 and T16. The same alteration has been previously found by Itoh and Wittmann (1973) in the S5 protein of strain d1023. Determination of the alteration of ribosomal protein S5 of strain 0-1 (sup0-1 allele) revealed that the C-terminal tryptic peptide is altered. It differs from that of the wild-type protein by the lack of five amino acids and the appearance of a C-terminal glycine residue instead of a lysine residue. This change can be explained by the deletion of eleven nucleotides in the S5 cistron of strain 0-1. The recent determination of the primary structure of ribosomal protein S5 (Wittmann-Liebold and Greuer, 1975) allows the ordering of the S5 alterations employed: The order is spc-13-strid1023 (striN421)-sup0-1 with the spc-13 amino acid replacement being located at the NH2-terminal portion of the S5 sequence and the alteration of strain 0-1 at the COOH-terminal end. The proteinchemical results are therefore in full agreement with the genetic data and unambiguously allow the conclusion that the S5 cistron is transcribed counterclock-wise on the Escherichia coli chromosome.

The dependence of UV-induced reversions to prototrophy on the streptomycin resistance allele in Escherichia coli

UV-induced mutability to prototrophy depended on the alleles of the strA locus. The yield of UV-induced suppressor mutations was decreased ten-fold in a Str r strain compared with an isogenic Str s strain. This decrease was due to the inability of the majority of suppressors to be expressed phenotypically in the Str r strain in the course of selection. The addition of streptomycin to the selective media raised the number of selected suppressor revertants, by making such expression possible. The probable relation of streptomycin resistance and UV-induced suppressors is discussed.

Conditional dihydrostreptomycin resistance in Bacillus subtilis.

Mutants resistant to dihydrostreptomycin were isolated and genetically analyzed in Bacillus subtilis. Two new classes of mutants distinct from the ribosomal strA locus were found. One class, strB, was located between metC3 and ura-1 on the chromosome. The second class, strC, mapped in the spore gene region close to the spoA locus. Both mutant classes were resistant to dihydrostreptomycin during growth but sensitive to the antibiotic during sporulation. Resuspension sporulation experiments with a strB mutant showed that sensitivity to the antibiotic was acquired early in the sporulation process. The germination and outgrowth of strB spores was sensitive to the antibiotic until growth commenced, whereupon the culture was resistant. Thus the mutants are sensitive to dihydrostreptomycin during both sporulation and germination but resistant during the growth phase.

A mutation suppressing streptomycin dependence: II. An altered protein on the 30 s ribosomal subunit

Escherichia coli, strain K106, is a spontaneous revertant from a streptomyc-independent strain in which the requirement for streptomycin for growth is suppressed by a mutation outside the strA locus. Previous studies have shown that the mutant cells have a slower growth rate than the streptomycin-sensitive wild type and have a defect in the assembly of the 50 s ribosomal subunit; the monosomes from the mutant have an altered response to streptomycin when assayed for the ability to support in vitro polypeptide synthesis primed with polyuridylic acid. In this report we show that the relative sensitivity of the mutant ribosomes to streptomycin is a property of the 30 s subunit. In addition, when natural messenger is used in a cell-free system, combinations containing either 30 s or 50 s subunits from the mutant have substantially reduced functional capacity. Chromatography on phosphocellulose of the ribosomal proteins of the mutant and the wild type shows no differences in 50 s proteins; however, K106 30 s subunits contain one protein which elutes from the column at a position different from the analogous protein of either the wild-type or the streptomycin-dependent strains. This protein is not the protein altered in streptomycin-resistant or dependent ribosomes. The relation of the altered ribosomal protein to the complex mutant phenotype is discussed.

A slow-growing, streptomycin resistant mutant of Escherichia coli affected in protein synthesis and ribosomal assembly.

The Escherichia coli K12 strain derivative MX 109, carries the strA938 mutant allele. It was isolated as a spontaneous high-level streptomycin resistant mutant, having the additional characteristic of growing slowly at a wide range of temperatures. Thus far, these two phenotypic characteristics have not been genetically segregated and, probably, are the result of a single mutational event at the strA locus.

Nature of phenotypic masking exhibited by drug-dependent streptomycin a mutants of Escherichia coli

A class of drug-dependent mutants that occur at the strA locus in Escherichia coli requires any one of several factors for growth: streptomycin, paromomycin or ethanol. Streptomycin and paromomycin in combination, however, kill these mutants at the same doses and with the same kinetics as they kill the wild type when used individually. Each drug can sensitize the culture to the other by fixation to non-metabolizing cells; four generations of growth are required to completely dilute out the induced sensitivity. This property persists in a class of revertants to drug independence. Dependence on, or resistance to, either streptomycin or paromomycin can thus be phenotypically masked in cells of these strains which have been previously grown in the presence of the other antibiotic. The amount of misreading by ribosomes extracted from strains exhibiting phenotypic masking is small when induced by either streptomycin or paromomycin, but is quite large in the presence of both. Ribosomes from cells grown in one of the drugs are induced by the other to the same high level of misreading as by the drug combination. This phenomenon can be attributed to the binding of drugs by ribosomes during growth in the presence of antibiotics. The association is very stable and two ammonium sulfate precipitations are required to dissociate the drug from the ribosome. These observations demonstrate (1) that exposure of a culture to streptomycin or paromomycin results in firm binding of the antibiotics to the ribosomes; (2) that in vivo the complex between drug and ribosome disappears only through dilution of the bacterial mass by growth; (3) that drug binding results in misreading of the genetic code, at least when the ribosomes are programmed with synthetic messenger RNA; (4) that killing by streptomycin and paromomycin in vivo is related to high levels of misreading in vitro in a strictly parallel way; and (5) that mutation at the strA gene, which codes the structure of a ribosomal protein, affects bacterial response to several antibiotics and not to streptomycin alone.

A ribosomal ambiguity mutation

The existence of a genetic determinant ram ‡ ‡ Abbreviations used: for the designation of genetic markers the proposals of Demerec, Adelberg Clark & Hartman (1966) have been followed and abbreviations correspond generally to those of Taylor & Trotter (1967) with the addition of apt for the speotinomycin gene. Other abbreviations are the following: CSD, conditionally streptomycin dependent (Gorini &Kataja, 1984u); Arg-CSD, streptomycin dependent under condition of absence of arginine; Sm. streptomycin; Sp, speotinomyoin; Phe, phenylalanine; De, isoleucine; Ser, serine; His, histidine; Pro, proline; aromix, mixture of growth factors required by aroE mutants which are blocked in the dehydrosbikimate reductase (Pittard & Wallace, 1966); poly-I?, polyuridine; poly-C, polycytidine. for a 30 s ribosomal component controlling translation ambiguity has been demonstrated. It is located in the strA (streptomycin sensitivity) region and is 99% cotransducible with the spc (spectinomycin sensitivity) gene. In vitro, ram mutant ribosomes misread extensively and by interchanging the subunits of ram and ram + ribosomes it is shown that misreading is a property of the 30 s subunit. In vivo, the ram mutation confers on the cell the ability to suppress all three nonsense codons. Streptomycin induces a similar generalized ambiguity phenotypically both in vitro and in vivo. The effects of ram and of streptomycin are additive. Ambiguity caused by ram, by streptomycin and by their combination is antagonized in vivo and in vitro by additional mutations at the strA locus. Thus the strA40 (resistant competent) and strA1 (resistant incompetent) alleles increasingly restrict ribosomal (as well as tRNA) suppression and return to normal the reduced growth rate produced by ram mutation in a wild-type strA + strain. Growth is inhibited completely in a ram1, strA + strain by sublethal doses of streptomycin and in ram1, strA40 strains by higher doses, while strain ram1, strA1 is indifferent to streptomycin. The bactericidal action of the drug is unrelated to these effects.