Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase

Summary The wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.Abbreviations bp base pairs - EDTA ethylenediaminetetraacetic acid - kb kilobases' - TES n-tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid     

A temperature sensitive mutant of Saccharomyces cerevisiae defective in pre-rRNA processing.

A recessive temperature sensitive mutant has been isolated that is defective in ribosomal RNA processing. By Northern analysis, this mutant was found to accumulate three novel rRNA species: 23S', 18S' and 7S', each of which contains sequences from the spacer region between 25S and 18S rRNA. 35S pre-rRNA accumulates, while the level of the 20S and 27S rRNA processing intermediates is depressed. Pulse-chase analysis demonstrates that the processing of 35S pre-rRNA is slowed. The defect in the mutant appears to be at the first processing step, which generates 20S and 27S rRNA. 7S' RNA is a form of 5.8S RNA whose 5' end is extended by 149 nucleotides to a position just 5 nucleotides downstream of the normal cleavage site that produces 20S and 27S rRNA. 7S' RNA can assemble into 60S ribosomal subunits, but such subunits are relatively ineffective in joining polyribosomes. A single lesion is responsible for the pre-rRNA processing defect and the temperature sensitivity. The affected gene is designated RRP2.     

Temperature sensitive mutations affecting RNA synthesis in Escherichia coli

Summary A streptomycin method has been used for the isolation of mutants with RNA synthesis inhibited at elevated temperature. The method is based on the observation that streptomycin kills bacteria with normal RNA synthesis and does not affect the cells with inhibited synthesis of RNA. This selection method increases the yield of temperature sensitive mutants by a factor 10–20, the amount of mutants with disturbed RNA synthesis is increased 3–5 fold as compared with the method of replicas.Several types of mutants were found among the temperature sensitive strains: those possessing temperature sensitivity of one, two or three types of cellular macromolecules DNA, RNA and protein. The screening among the mutants with affected RNA synthesis revealed a strain ts-19 showing low RNA polymerase activity in cell extracts and partially purified RNA polymerase preparations. The presented evidence suggests that ts-19 mutation affects the structural gene of one of the RNA polymerase subunits.The mapping of the corresponding locus indicated that it was located between the str and thy loci in E. coli K 12 chromosome at a distance of about 20 recombination units from the first locus.     

Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

Summary The bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.     

Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). I. Initial characterization

Three mutant strains of Escherichia coli were independently isolated based upon their inability to propagate bacteriophage λ. The strain most extensively studied, NS-1, has a pleiotropic temperature sensitive alteration that affects cell growth, stable RNA synthesis and λ propagation. Labeling experiments and colorimetric determinations of total RNA carried out in this strain demonstrate that within the first five minutes after raising the temperature to 44.5 °C the rate of total RNA accumulation is reduced to a level that is about 15% that of the control, while protein and DNA synthesis continue at nearly normal rates for at least 30 min. This effect is either due to a very rapid degradation of stable RNA species or a reduced synthesis of RNA. Although the accumulation of all stable RNA species (23, 16 and 4 S RNAs) is reduced co-ordinately to levels ranging from 12 to 16% that of the control, the synthesis of messenger RNA is affected to a lesser degree, if at all. The defect in RNA accumulation can be partially reversed by the addition of chloramphenicol at the moment of temperature shift.In addition to phage λ these strains are unable to propagate RNA phage R17 and lambdoid phages φ80, 21 and 434 at elevated temperatures. The growth of phages T4, T7, P1 and P2 is normal.A genetic analysis of strain NS-1 indicates that all of its temperature sensitive properties depend on a mutation, designated groE-1, which co-transduces with a mel (melibiose) marker. However, the expression of the RNA synthesis defect requires, in addition, a second mutation which does not co-transduce with mel.     

Transcription of bacteriophage Mu. I. Hybridization analysis of RNA made in vitro

Summary A mutation in the cyR1 gene of the fungus Podospora anserina confers resistance to cycloheximide and leads to an alteration of the 60S ribosomal protein L21 (Bégueret et al. 1977). Nine revertants of this mutant were isolated and the properties of these strains were analyzed. It was found that one revertant strain contains a new mutant form of L21. It is proposed that the cyR1 gene is the structural gene for protein L21 and that the alteration of this protein is responsible for the resistance to cycloheximide in vivo.     

W-reactivation of phage lambda in recF, recL, uvrA, and uvrB mutants of E. coli K-12.

Summary Assay conditions are described which permit detection of cryptic temperature sensitive RNA polymerases in vitro. RNA polymerase was prepared from fifteen different temperature sensitive mutants of Salmonella typhimurium chosen at random from a larger group isolated by localized mutagenesis and uridine suicide techniques. The dependence of enzyme activity on temperature, ionic strength and pH was studied in vitro. Assays at higher ionic strength (0.23 M) and temperature (50°C) distinguish three classes of mutants (Table 2). Activity of seven mutant RNA polymerases (called Class 1) under these conditions was 1% to 5% that of the parental RNA polymerase. Five mutant RNA polymerases (called Class 2) had 18% to 64% of the parental activity and three were not distinguishable from the parental enzyme under these conditions. Mixing experiments showed that the defect in Class 1 mutant enzymes is a property of the enzymes and not due to a diffusible inhibitor. In one case the lesion was shown to reside in the core enzyme. Class 1 mutant RNA polymerases were shown to be irreversibly inactivated during the assay at higher temperature and ionic strength. This suggests that the Class 1 enzymes may be more thermolabile than the wild type enzyme or may fail to be protected from thermal denaturation by formation of a ternary complex with template and product. We conclude that the method used to isolate these mutants (Young et al., 1976) and the assay described here (Table 2) are efficient ways to isolate and detect temperature sensitive RNA polymerase mutants of Salmonella typhimurium.     

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

Summary Revertants 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.Paper No. 60 on Ribosomal Proteins. Preceding paper is by B. Wittmann-Liebold, Hoppe-Seyler's Z. physiol. Chemie, in press.     

A cold-sensitive cytoplasmic mutation of Saccharomyces cerevisiae affecting assembly of the mitochondrial 50S ribosomal subunit.

Summary A cytoplasmic mutant of Saccharomyces cerevisiae (E23-1) has been isolated that is resistant to erythromycin and cold sensitive for growth on nonfermentable carbon sources at 18°. Genetic analysis has shown that both of these properties probably result from a single mutation at the rib2 locus which maps close to or within the gene for the 21S rRNA of the mitochondrial 50S ribosomal subunit. Electrophoresis of total RNA extracted from purified mitochondria demonstrated that the 21S and 14S rRNA species from both mutant and wild-type cells were present in roughly equimolar quantities regardless of growth temperature. The mutant is therefore not defective in the synthesis of the 21S rRNA. Sucrose gradient analysis of the mitochondrial ribosomes in Mg²⁺-containing buffers revealed that approximate values for the ratio of 50S to 37S subunits were 1:1 for wild-type cells grown at either 18° or 32°, 0.5:1 for the mutant grown at 32° and 0.2:1 for the mutant grown at 18°. The subunit ratios were approximately 1:1 when Ca²⁺-containing buffers were used, however, In alls cases, 50S particles from the mutant grown at 18° lacked or contained markedly reduced amounts of two distinctive protein components that were present in the mutant at 32° and in the wild-type at both temperatures. In addition, no intact 21S RNA could be recovered from the mitochondrial ribosomes of the mutant grown at the restrictive temperature, even in the presence of Ca²⁺. These findings indicate that mitochondrial 50S ribosomal subunits produced by the mutant at 18° are structurally defective and raise the possibility that the defect results from an alteration in the gene for 21S rRNA.A preliminary report of this work was presented at the meeting on The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, August 18–22, 1977     

Mitochondrially encoded resistance to paromomycin in Saccharomyces cerevisiae: Reinvestigation of a controversy

We have reinvestigated the nature of mitochondrially inherited resistance to paromomycin in Saccharomyces cerevisiae. Resistance to this antibiotic can arise by a nucleotide alteration in the gene coding for 15 S ribosomal RNA at a recognition site for the restriction endonuclease ThaI (CGCG), as has been observed by Li (M. Li, K. Lyon, N. Martin and A. Tzagoloff (1981). “Abstracts, Cold Spring Harbor Meeting on Mitochondrial Genes,” p. 56. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.). We have confirmed this finding and document here also a new type of paromomycin resistance that is unrelated to this ThaI restriction site. Certain petite mutants derived from different locations of the mtDNA of S. cerevisiae KL14-4A can elicit resistance to paromomycin when crossed with a wild-type sensitive strain. These petite mutants lack detectable sequence homology with the 15 S ribosomal RNA gene and they have no extensive sequence homology with each other. We have constructed paromomycin-resistant diploids by crossing such KL14-4A petite mutants with a sensitive wild-type strain. The diploids that receive the paromomycin-resistant allele from a petite mutant retaining the 15 S ribosomal RNA gene no longer contained the ThaI site. However, diploids that become resistant after a cross with petite mutants retaining fragments from other mtDNA regions than the 15 S ribosomal RNA, still contain the ThaI site. This shows that paromomycin resistance can occur in the presence of the ThaI site. After sporulation, suitable paromomycin-resistant haploids were crossed with each other and sensitive recombinant diploids were found, indicating the existence of more than one form of paromomycin resistance. Possible explanations for this novel type of paromomycin resistance and the unorthodox way in which it arises, are presented.     

Chemical and functional characterization of an altered form of ribosomal protein S4 derived from a strain of E. coli defective in auto-regulation of the alpha operon.

We have isolated a mutant form of Escherichia coli ribosomal protein S4. This mutant is temperature sensitive and apparently fails to autogenously regulate the gene products of the alpha operon, which consists of the genes for proteins S13, S11, S4, L17, and the alpha subunit of RNA polymerase (1). We have shown that this mutation results in the production of an S4 protein with a molecular weight approximately 4,000 daltons less than the wild-type protein. Our chemical analyses demonstrate that the mutant protein is missing its C-terminal section consisting of residues 170-203. However, our studies to determine the capacity of this mutant protein to bind 16S RNA show that this protein is unimpaired in RNA binding function. This observation suggests that the functional domain of protein S4 responsible for translational regulation of the S4 gene products requires more of the protein than the 16S RNA binding domain.     

Analysis of rpsD mutations in Escherichia coli. I. Comparison of mutants with various alterations in ribosomal protein S4.

Summary Streptomycin-independent revertants were selected from streptomycin-dependent mutants. Twenty-five out of 150 such revertants were temperature sensitive. Ribosomal proteins from 18 temperature-sensitive and 10 temperature-insensitive revertants were analysed by SDS-polyacrylamide gel electrophoresis. Seventeen of the former but none of the latter category showed an alteration of protein S4. The mutated rpsD allele of 6 temperature-sensitive revertants was transduced into a rpsL ⁺ strain. In all cases an increased suppressibility of T4 amber phages was observed. Such suppressibility was not observed in the original rpsD, rpsL strains. All 18 temperature-sensitive mutants were disturbed in the processing of 17s to 16s RNA at non-permissive temperature and the accumulated 17s RNA was degraded. Temperature-insensitive rpsD revertants could be isolated, which had gained a second alteration in S4. Such revertants, which had lost the temperature-sensitive property, were also unable to suppress growth of T4 amber phages.It is concluded that temperature-sensitive growth, inability to process 17s RNA and to assemble 30S ribosomes at non-permissive temperature as well as increased translational ambiguity are highly correlated properties in rpsD mutants.     

A temperature-sensitive mutation in asparaginyl-tRNA synthetase causes cell-cycle arrest in early S phase

The Chinese hamster temperature-sensitive cell-cycle mutant ts24 was analyzed biochemically in order to determine the nature of this lesion. The inability of these cells to proceed through S phase at the restrictive temperature could be complemented by the addition of asparagine to the growth medium, and enzymological analysis showed that this line contains a temperature-sensitive asparaginyl-tRNA synthetase. Normal asparaginyl-tRNA synthetase activity was restored in cells transfected with cloned genomic DNA that overcomes the mutational defect. In corroboration with these results it was shown that a different temperature-sensitive asparaginyl-tRNA synthetase mutant isolated in another laboratory was blocked in S phase in a manner similar to that of ts24. While the mechanism by which asparaginyl-tRNA synthetase affects cell-cycle progression has not been elucidated, it can be shown that it is not mediated through alteration in overall levels of protein synthesis.     

The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis

The isolation and properties of a single site temperature sensitive protease mutant of Bacillus subtilis are described. Numerous criteria suggest that the mutation resides in the structural gene coding for a basic serine protease. The mutation has been mapped between aroD and lys-1 on the Bacillus subtilis chromosome. This protease exists as an intracellular and extracellular enzyme. The mutant cells are temperature sensitive for sporulation, antibiotic production, and the sporulation-specific alteration in DNA-dependent RNA polymerase β subunit. Several types of evidence indicate a direct involvement of this enzyme in a limited proteolytic cleavage of vegetative RNA polymerase β subunit, which produces the lower molecular weight β subunit found in sporulating cells. The derangement in this process is sufficient to account for the stoppage of sporulation at stage 0 when the mutant cells are grown at the non-permissive temperature.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.     

Control of translational repression by protein-protein interactions.

The coat protein of the RNA bacteriophage MS2 is a translational repressor and interacts with a specific RNA stem-loop to inhibit translation of the viral replicase gene. As part of an effort to dissect genetically its RNA binding function, mutations were identified in the coat protein sequence that suppress mutational defects in the translational operator. Each of the mutants displayed a super-repressor phenotype, repressing translation from the wild-type and a variety of mutant operators better than did the wild-type coat protein. At least one mutant probably binds RNA more tightly than wild-type. The other mutants, however, were defective for assembly of virus-like particles, and self-associated predominantly as dimers. It is proposed that this assembly defect accounts for their super-repressor characteristics, since failure to assemble into virus-like particles elevates the effective concentration of repressor dimers. This hypothesis is supported by the observation that deletion of thirteen amino acids known to be important for assembly of dimers into capsids also resulted in the same assembly defect and in super-repressor activity. A second class of assembly defects is also described. Deletion of two amino acids from the C-terminus of coat protein resulted in failure to form capsids, most of the coat protein having the apparent molecular weight expected of trimers. This mutant (dl-8) was completely defective for repressor activity, probably because of an inability to form dimers. These results point out the inter-dependence of the structural and regulatory functions of coat protein.     

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.     

Alteration of ribosomal protein S17 by mutation linked to neamine resistance in Escherichia coli: II. Localization of the amino acid replacement in protein S17 from a neaA mutant

A mutant of Escherichia coli strain K12S, nea^R301, resistant to the antibiotic neamine was found to have an altered 30 S ribosomal protein S17. The modification involves a change in the electrophoretic mobility of this protein. S17 proteins wore purified from the mutant and the parental strain, respectively, and the amino acid compositions of all tryptic peptides were compared. The results show that the mutational alteration involves a replacement of histidine by proline in peptide T8 from mutant nea^R301. The amino acid replacement is located at position 30 of the S17 protein chain. We conclude, therefore, that the mutation nea^R301 affects the structural gene for protein S17 (rps Q).     

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.     

A mutation in the secretion pathway of the yeast Yarrowia lipolytica that displays synthetic lethality in combination with a mutation affecting the signal recognition particle.

In an attempt to identify proteins involved in the translocation step of protein secretion, a genetic screen was carried out in the yeast Yarrowia lipolytica. A conditional lethal mutant which has a defect in the 7S RNA of the signal recognition particle was mutagenized and screened for second-site mutations that specifically exacerbate its temperature sensitivity. This approach had previously allowed the characterization of an endoplasmic reticulum component, Sls1p, involved in protein translocation. A second mutation, sls2-1, was isolated that causes synthetic lethality when combined with the 7S RNA mutation. On its own, the sls2-1 mutation confers a temperature-sensitive growth phenotype. The secretory phenotype of the sls2 mutant consists in abnormal secretion of several polypeptides, and thus differs from the defect in secretory protein synthesis associated with the 7S RNA and sls1-1 mutations. Two new Y. lipolytica genes were identified which can relieve the growth defect of sls2-1 cells: SLS2 itself and SSL2, a multicopy suppressor of the temperature sensitivity of the sls2 mutant. The SLS2 gene encodes a polypeptide that can potentially be farnesylated and phosphorylated, and shares some homology with an S. cerevisiae protein of unknown function. Ssl2p resembles calmodulin-dependent serine/threonine protein kinases. These two proteins may interact to regulate protein sorting. Received: 9 June 1998 / Accepted: 10 February 1999