The ribosomal components responsible for kasugamycin dependence, and its suppression, in a mutant of Escherichia coli

Summary The phenotype of a kasugamycin dependent mutant, MV17, was found to be the product of a kasugamycin resistance mutation in ksgA, together with a dependentizing mutation in rplW, the gene for large ribosomal subunit protein L23. Revertants from dependence on this small subunit targeted antibiotic were found to have mutational alterations in ribosomal proteins L23, L1, L11, and S9. The mutations causing alterations in L1 and L23 were shown to be responsible for the reversion and that altering L11 to be involved in the reversion.     

Two Genetic Loci for Resistance to Kasugamycin in Escherichia coli

There are two loci for resistance to the antibiotic kasugamycin (Ksg) in Escherichia coli. Mutations at ksgA resulted in 30S ribosomal subunit resistance to Ksg. The map location of ksgA was near minute 0.5: ksgA was 95% cotransducible with pdxA, and the apparent gene order was thr... ksgA... pdxA. Studies in stable ksgA/ksgA⁺ merodiploids showed that sensitivity was dominant over resistance. Mutations at a second gene (ksgB), located between minutes 25 and 39, resulted in phenotypic Ksg^R indistinguishable from ksgA mutations, but ribosomes from ksgB strains were sensitive to the drug in vitro. Spontaneous and induced mutations to Ksg^R were usually of the ksgA (ribosomal) type.     

A Third Kasugamycin Resistance Locus, ksgC, Affecting Ribosomal Protein S2 in Escherichia coli K-12

A third kasugamycin-resistant mutant affecting ribosomal protein S2 has been isolated from Escherichia coli K-12. Mating and transduction revealed that this newly recognized kasugamycin resistance locus, designated as ksgC, is located at 0.1 to 0.2 min from purE.     

Differential inhibition of coliphage MS2 protein synthesis by ribosome-directed antibiotics

Of thirteen ribosome-directed antibiotics surveyed for their inhibitory effect on viral protein synthesis in Escherichia coli infected with coliphage MS2, three antibiotics, kasugamycin, tetracycline and chloramphenicol, were found to exert differential inhibition, with synthesis of maturation protein being more sensitive than coat protein synthesis. Differential effects of kasugamycin and tetracycline were also observed in vitro. Such differential inhibition might reflect the presence of cistron-speciflc ribosomes or the induction of functional ribosomal heterogeneity by these antibiotic ligands.     

Conditional lethal mutants of Bacillus subtilis dependent on kasugamycin for growth

Summary Mutants of Bacillus subtilis dependent on the antibiotic kasugamycin have been isolated and characterised. The mutant phenotype was the result of a kasugamycin resistance mutation mapping near leu, together with a mutation conferring dependence which mapped elsewhere on the chromosome. In some cases, the latter mutation caused spectinomycin dependence in a spectinomycin resistant strain. Four mutants had detectable alterations in ribosomal proteins, which were not, however, responsible for the phenotype. These alterations were in proteins BS3, BS7, BS9, and BL15. Some mutants had defects in ribosomal subunit assembly, or altered cell morphology associated with the mutant phenotype.     

The Phenotype of Many Independently Isolated +1 Frameshift Suppressor Mutants Supports a Pivotal Role of the P-Site in Reading Frame Maintenance

The main features of translation are similar in all organisms on this planet and one important feature of it is the way the ribosome maintain the reading frame. We have earlier characterized several bacterial mutants defective in tRNA maturation and found that some of them correct a +1 frameshift mutation; i.e. such mutants possess an error in reading frame maintenance. Based on the analysis of the frameshifting phenotype of such mutants we proposed a pivotal role of the ribosomal grip of the peptidyl-tRNA to maintain the correct reading frame. To test the model in an unbiased way we first isolated many (467) independent mutants able to correct a +1 frameshift mutation and thereafter tested whether or not their frameshifting phenotypes were consistent with the model. These 467+1 frameshift suppressor mutants had alterations in 16 different loci of which 15 induced a defective tRNA by hypo- or hypermodifications or altering its primary sequence. All these alterations of tRNAs induce a frameshift error in the P-site to correct a +1 frameshift mutation consistent with the proposed model. Modifications next to and 3′ of the anticodon (position 37), like 1-methylguanosine, are important for proper reading frame maintenance due to their interactions with components of the ribosomal P-site. Interestingly, two mutants had a defect in a locus (rpsI), which encodes ribosomal protein S9. The C-terminal of this protein contacts position 32–34 of the peptidyl-tRNA and is thus part of the P-site environment. The two rpsI mutants had a C-terminal truncated ribosomal protein S9 that destroys its interaction with the peptidyl-tRNA resulting in +1 shift in the reading frame. The isolation and characterization of the S9 mutants gave strong support of our model that the ribosomal grip of the peptidyl-tRNA is pivotal for the reading frame maintenance.     

Increased kasugamycin sensitivity in Escherichia coli caused by the presence of an inducible erythromycin resistance (erm) gene of Streptococcus pyogenes

Summary An inducible erythromycin resistance gene (erm) of Streptococcus pyogenes was introduced into Escherichia coli by transformation with a plasmid. The recipient E. coli cells were either kasugamycin sensitive (wildtype) or kasugamycin resistant (ksgA). The MIC values of erythromycin increased from 150 g/ml to>3000 g/ml for E. coli. An extract of transformed cells, particularly a high-salt ribosomal wash, contained an enzyme that was able to methylate 23S rRNA from untransformed cells in vitro; however, 23S rRNA from transformed cells was not a substrate for methylation by such an extract. 165 rRNA and 30S ribosomal subunits of either the wild type or a kasugamycin resistant (ksgA) mutant were not methylated in vitro. Transformation of E. coli by the erm-containing plasmid led to a reduction of the MIC values for kasugamycin. This happened in wild-type as well as in ksgA cells. However, in vitro experiments with purified ksgA encoded methylase demonstrated that also in erm transformed E. coli, the ksgA encoded enzyme was active in wild-type, but not in ksgA cells. It was also shown by in vitro experiments that ribosomes from erm ksgA cells have become sensitive to kasugamycin. Our experiments show that in vivo methylation of 23S rRNA, presumably of the adenosine at position 2058, leads to enhanced resistance to erythromycin and to reduced resistance to kasugamycin. This, together with previous data, argues for a close proximity of the two sites on the ribosome that are substrates for adenosine dimenthylation.Abbreviations MLS macrolide, lincosamide, streptogramin B     

An intron in a ribosomal protein gene from Tetrahymena

We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two-dimensional polyacrylamide gels of the protein synthesized by translation in vitro of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene of a ciliate to be described at the nucleotide sequence level. The intron obeys the GT/AG rule for splice junctions of nuclear mRNA introns from higher eukaryotes but lacks the pyrimidine stretch usually found in the immediate vicinity of the 3' splice junction. The structure of the intron and the fact that it is found together with the well described self-splicing rRNA intron is discussed in relation to the evolution of RNA splicing.     

Cloning and sequencing of ribosomal protein L27a and a gene similar to human GS1 in Drosophila

Two closely linked genes were identified and characterized in the 24F region on the left arm of chromosome 2 in Drosophila. One cDNA predicts a protein of 231 amino acids, with a molecular mass of 25.7 kDa. The predicted amino-acid sequence of this protein is 47.2% identical to that of the previously reported human GS1 protein, which is encoded by a gene that is of interest because it is one of the few X-linked genes that escapes X-inactivation. We have accordingly named our gene GS1like (GS1l). The second cDNA begins 383 bp proximal to the first. This cDNA encodes a protein of a predicted 149 amino acids and a molecular mass of 17.0 kDa. This protein represents a homolog of ribosomal protein L27a; thus, we have named the gene RpL27a. This gene might be responsible for the Minute mutation located at 24F. An rpL27a gene was previously localized to 87F/88A; thus, this gene might be present in two locations in Drosophila.     

Ribosomal assembly deficiency in an Escherichia coli thermosensitive mutant having an altered L24 ribosomal protein.

Localized P1 mutagenesis was used to screen for conditionally lethal mutations in ribosomal protein genes. One such mutation, 2859mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and cotransduces at 98% with rpsE (S5). The 2869mis mutation leads to thermosensitivity and impaired assembly in vivo of 50 S ribosomal particles at 42 °C. The strain carrying the mutation has an altered L24 ribosomal protein which at 42 °C shows weaker affinity for 23 S RNA than the wild-type protein. The mutational alteration involves a replacement of glycine by aspartic acid in protein L24 from the mutant. We conclude therefore that the 2859mis mutation affects the structural gene for protein L24 (rplX).     

Expression vectors for quatitating in vivo translational ambiguity: Their potential use to analyse frameshifting at the HIV gag-pol junction

Translational errors are necessary so as to allow gene expression in various organisms. In retroviruses, synthesis of pol gene products necessitates either readthrough of a stop codon or frameshifting. Here we present an experimental system that permits quantification of translational errors in vivo. It consists of a family of expression vectors carrying different mutated versions of the luc gene as reporter. Mutations include both an in-frame stop codon and 1-base-pair deletions that require readthough or frameshift, respectively, to give rise to an active product. This system is sensitive enough to detect background errors in mammalian cells. In addition, one of the vectors contains two unique cloning sites that make it possible to insert any sequence of interest. This latter vector was used to analyse the effect of a DNA fragment, proposed to be the target of high level slippage at the gag-pol junction of HIV. The effect of paromomycin and kasugamycin, two antibiotics known to influence translational ambiguity, was also tested in cultured cells. The results indicate that paromomycin diversely affects readthrough and frameshifting, while kasugamycin had no effect.This family of vectors can be used to analyse the influence of structural and external factors on translational ambiguity in both mammalian cells and bacteria.     

Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana

The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5′ untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.     

The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus has been completely determined. The sequence data were mainly obtained by manual sequencing of peptides derived from digestion with trypsin, Staphylococcus aureas protease and pepsin. A few overlaps of tryptic peptides were established by DNA sequence analysis of a chromosomal fragment containing the rpsL gene coding for ribosomal protein S12. The protein contains 138 amino acid residues and has an M_r of 15208. Comparison of this sequence with the sequences of the ribosomal S12 proteins from E. coli as well as from Euglena, tobacco and liverwort chloroplasts shows that 75% of the amino acid residues are identical within the S12 proteins of all four species. Therefore, S12 is the most strongly conserved ribosomal protein known so far.     

Increased translational fidelity caused by the antibiotic kasugamycin and ribosomal ambiguity in mutants harbouring the ksgA gene

The aminoglycoside kasugamycin, which has previously been shown to inhibit initiation of protein biosynthesis in vitro, also affects translational accuracy in vitro. This is deduced from the observation that the drug decreases the incorporation of histidine relative to alanine into the coat protein of phage MS2, the gene of which is devoid of histidine codons. The read-through of the MS2 coat cistron, due to frameshifts in vitro, is also suppressed by the antibiotic. In contrast, streptomycin enhances histidine incorporation and read-through in this system. The effects of kasugamycin take place at concentrations that do not inhibit coat protein biosynthesis. Kasugamycin-resistant mutants (ksgA) lacking dimethylation of two adjacent adenosines in 16 S ribosomal RNA, show an increased leakiness of nonsense and frameshift mutants (in the absence of antibiotic). They are therefore phenotypically similar to previously described ribosomal ambiguity mutants (ram).     

Cloning of the Ribosomal Protein L41 Gene of Phaffia rhodozyma and Its Use as a Drug Resistance Marker for Transformation

The ribosomal protein L41 gene of Phaffia rhodozyma was cloned and used as a dominant selectable marker for cycloheximide resistance in the transformation of P. rhodozyma. Electrotransformation with a plasmid containing a ribosomal DNA fragment as a targeting signal typically yielded 800 to 1,200 transformants/μg of DNA with an integrated copy number of about seven per haploid genome.     

The Gene for Ribosomal Protein L13, rplM, Is Located Near argR, at About 70 Minutes on the Escherichia coli Chromosomal Linkage Map

Mutants of Escherichia coli with alterations in the electrophoretic mobility of ribosomal protein L13 were used to locate rplM, the gene for this protein, on the chromosomal linkage map. rplM was situated between gltB and argR, at about 70 min.