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.     

Escherichia coli kasugamycin dependence arising from mutation at the rpsI locus

Escherichia coli mutants with alterations in the electrophoretic mobility of ribosomal protein S9 were used to locate rpsI, the gene for this protein, on the linkage map. rpsI was located at about 70 min, roughly halfway between argG and fabE. It was very close to the gene for ribosomal protein L13, rplM. Another mutation at the rpsI locus gave rise to a phenotype of kasugamycin dependence and resistance. In this mutant, dependence on antibiotic came from kasugamycin being necessary to slow the rate of protein synthesis.     

Isolation and characterization of temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

Summary The ribosomal proteins of temperature-sensitive mutants of Escherichia coli isolated independently after mutagenesis with nitrosoguanidine were analyzed by two-dimensional gel electrophoresis. Out of 400 mutants analyzed, 60 mutants (15%) showed alterations in a total of 22 different ribosomal proteins. The proteins altered in these mutants are S2, S4, S6, S7, S8, S10, S15, S16, S18, L1, L3, L6, L10, L11, L14, L15, L17, L18, L19, L22, L23 and L24. A large number of them (25 mutants) have mutations in protein S4 of the small subunit, while four mutants showed alterations in protein L6 of the large subunit. The importance of these mutants for structural and functional analyses of ribosomes is discussed.     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Proteins occurring at, or near, the subunit interface of E. coli ribosomes

Summary The identification of ribosomal proteins that occur at, or near, the subunit interface of the 30S and 50S subunits in the E. coli 70S ribosome was attempted by studying the effect of antibodies on the Mg⁺⁺ dependent dissociation-association equilibrium of 70S ribosomes. Dissociated ribosomes were mixed with monovalent fragments of IgG antibodies (Fab's) specific for each ribosomal protein and then reassociated into intact 70S particles. Various degrees of inhibition of this reassociation were observed for proteins S9, S11, S12, S14, S20, L1, L6, L14, L15, L19, L20, L23, L26 and L27. A small amount of aggregation of 50S subunits was caused by IgG's specific for the proteins S9, S11, S12, S14 and S20 and purified 50S subunits. It was inferred that the presence of small amounts of these proteins on 50S subunits was compatible with their presence at the subunit interface. Finally, the capacity of proteins S11 and S12 to bind to 23S RNA was demonstrated.Paper No. 84 on Ribosomal Proteins. Preceding paper is by Rahmsdorf et al., Molec. gen. Genet. 127, 259–271 (1973).     

Localization of the structural gene for ribosomal protein L19 (rplS) in Escherichia coli

Summary A temperature-sensitive mutant derived from an E. coli K12 strain, PA3092, was found to have an alteration in the ribosomal protein L19 (Isono et al., 1977). This mutant is a double mutant with a temperature-sensitivity mutation and a mutation leading to the structural alteration of L19 protein. Crosses with various Hfr strains and transductions with P1kc have revealed that the latter mutation maps at 56.4 min, between pheA and alaS. From the fact that two other mutations causing different types of alterations in L19 protein also map at this locus, the gene affected by these mutations was concluded to be the structural gene for the ribosomal protein L19 (rplS).     

The cytoplasmic ribosomes of Chlamydomonas reinhardtii: characterization of antibiotic sensitivity and cycloheximide-resistant mutants

Summary In vitro protein synthesis was used to characterize the antibiotic sensitivity of cytoplasmic ribosomes from wild-type and antibiotic-resistant strains of Chlamydomonas reinhardtii. Cytoplasmic ribosomes from two cycloheximide-resistant mutants, act-1 and act-2, were resistant to the antibiotic in vitro. The alteration effected by the act-1 mutation, which was dominant in diploids, was localized to the large subunit of the cytoplasmic ribosomes, but no ribosomal protein alterations were detected using two-dimensional gel electrophoresis. The act-2 mutation, which was semidominant in diploids, was frequently associated with a charge alteration in the large subunit ribosomal protein (r-protein) cyL38 that segregated independently from the antibiotic-resistant phenotype in crosses.     

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     

Functional interdependence among ribosomal elements as revealed by genetic analysis

Summary Escherichia coli strains with preexisting ribosomal mutations were used in order to isolate further ribosomal mutations. The ribosomal mutations used were resistance to erythromycin, spectinomycin, streptomycin or kasugamycin. These mutations cause alteration of specific ribosomal elements, L4, S5, S12 proteins and 16S rRNA respectively. Mutations have been introduced into strains carrying one, two or three of these mutations. Strains with all possible combinations of these four mutations were constructed. The phenotypes of all isolated mutants were tested, and frequently the strains lost one or more of their pre-existing resistances.Thus, functional interactions were revealed among proteins, as well as RNA and proteins within the 30 S ribosomal subunit and as well as between the 30 S and the 50 S ribosomal subunits.     

Requirement for C-terminal Extension to the RNA Binding Domain for Efficient RNA Binding by Ribosomal Protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

Establishment of Arabidopsis thaliana ribosomal protein RPL23A-1 as a functional homologue of Saccharomyces cerevisiae ribosomal protein L25

Arabidopsis thaliana ribosomal protein (r-protein) RPL23A-1 shows 54% amino acid sequence identity to the Saccharomyces cerevisiae equivalent r-protein, L25. AtRPL23A-1 also shows high amino acid sequence identity to members of the L23/L25 r-protein family in other species. R-protein L25 in S. cerevisiae has been identified as a primary rRNA-binding protein that directly binds to a specific site on yeast 26S rRNA. It is translocated to the nucleolus where it binds to 26S rRNA during early large ribosome subunit assembly; this binding is thought to play an important role in ribosome assembly. The S. cerevisiae mutant strain YCR61 expresses L25 when grown on galactose, but not glucose, medium. Transformation of YCR61 with a shuttle vector containing the AtRPL23A-1 cDNA allowed transformed colonies to grow in and on glucose selection medium. R-protein AtRPL23A-1 can complement the L25 mutation, demonstrating the functional equivalence of the two r-proteins and introducing AtRPL23A-1 as the first plant member of the L23/L25 r-protein family.     

Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Summary Specitinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc1–11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1–11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.     

RNA-protein interactions in the ribosome: VIII. Co-operative interactions in the 50 S subunit of Escherichia coli

Six 50 S ribosomal subunit proteins, each unable to interact independently with the 23 S RNA, were shown to associate specifically with ribonucleoprotein complexes consisting of intact 23 S RNA, or fragments derived from it, and one or more RNA-binding proteins. In particular, L21 and L22 depend for attachment upon L20 and L24, respectively; L5, L10 and L11 interact individually with complexes containing L2 and L16; and one or both proteins of the $\text{L}\text{17}\text{L}\text{27}$ mixture are stimulated to bind in the presence of L1, L3, L6, L13 and L23. Moreover, L14 alone was found to interact with a fragment from the 3′ end of the 23 S RNA, even though it cannot bind to 23 S RNA. By correlating the data reported here with the findings of others, it has been possible to formulate a partial in vitro assembly map of the Escherichia coli 50 S subunit encompassing both the 5 S and 23 S RNAs as well as 21 of the 34 subunit proteins.     

Cold-sensitive growth of a mutant of Escherichia coli with an altered ribosomal protein S8: analysis of revertants.

Summary 26 cold-resistant revertants of a cold-sensitiveEscherichia coli mutant with an altered ribosomal protein S8 were analyzed for their ribosomal protein pattern by two-dimensional polyacrylamide gel electrophoresis. It was found that 16 of them had acquired the apparent wild-type form of protein S8, one exhibits a more strongly altered S8 than the original mutant and two revertants regained the wild-type form of S8 and, in addition, possess alterations in protein L30. The ribosomes of the residual revertants showed no detectable difference from those of the parental S8 mutant.The mutation leading to the more strongly altered S8 was genetically not separable from the primary S8 mutation; this indicates that both mutations are very close to each other or at the same site. The structural gene for ribosomal protein L30 was mapped relative to two other ribosomal protein genes (for proteins S5 and S8) by the aid of one of the L30 mutants: The relative order obtained is:aroE....rpmD(L30)....rpsE(S5)....rpsH(S8)....THe L30 mutation impairs growth and ribosomal assembly at 20°C and is therefore the first example of a mutant with a defined 50S alteration that has (partial) cold-sensitive ribosome assembly. A double mutant was constructed which possesses both the S8 and the L30 mutations. It was found that the L30 mutation had a slight antagonistic effect on the growth inhibition caused by the S8 mutation. Thus the L30 mutants might have possibly arisen from the original S8 mutants first as S8/L30 double mutants which was followed by the loss of the original S8 lesion.     

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli.

Summary The conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).     

Mutations affecting the structural genes and the genes coding for modifying enzymes for ribosomal proteins in Escherichia coli.

Summary Temperature-sensitive mutants of an Escherichia coli K-12 strain PA3092 have been isolated following mutagenesis with nitrosoguanidine, and their ribosomal proteins analyzed by two-dimensional gel electrophoresis This method was found to be very efficient in obtaining mutants with various structural alterations in ribosomal proteins. Thus a total of some 160 mutants with alterations in 41 different ribosomal proteins have so far been isolated. By characterizing these mutants, we could isolate, not only those mutants with alterations in the structural genes for various ribosomal proteins, but also those with impairments in the modification of proteins S5, S18 and L12. Furthermore, a mutant has been obtained which apparently lacks the protein S20 (L26) with a concomitant reduction to a great extent of the polypeptide synthetic activity of the small subunit. The usefulness of these mutants in establishing the genetic architecture of the genes coding for the ribosomal proteins and their modifiers is discussed.     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

The phenotypic suppression of a mutation in the gene rplX for ribosomal protein L24 by mutations affecting the lon gene product for protease LA in Escherichia coli K12

Summary A suppressor mutation of a temperature-sensitive mutant of ribosomal protein L24 (rplX19) was mapped close to the lon gene by genetic analysis and was shown to affect protease LA. The degradation and the synthesis rates of individual ribosomal proteins were determined. Proteins L24, L14, L15 and L27 were found to be degraded faster in the original rplX19 mutant than in the rplX19 mutant containing the suppressor mutation. Other ribosomal proteins were either weakly or not at all degraded in both mutants. Temperature-sensitive growth was also suppressed by the overproduction of mutant protein L24 from a plasmid. Our results suggest that (1) either free ribosomal proteins or proteins bound to abortive assembly precursors are highly susceptible to the lon gene product and (2) the mutationally altered protein L24 can still function at the nonpermissive growth temperature of the mutant, if it is present in sufficient amounts.     

Erythromycin inhibits the assembly of the large ribosomal subunit in growing Escherichia coli cells

Erythromycin and other macrolide antibiotics have been examined for their effects on ribosome assembly in growing Escherichia coli cells. Formation of the 50S ribosomal subunit was specifically inhibited by erythromycin and azithromycin. Other related compounds tested, including oleandomycin, clarithromycin, spiramycin, and virginiamycin M₁, did not influence assembly. Erythromycin did not promote the breakdown of ribosomes formed in the absence of the drug. Two erythromycin-resistant mutants with alterations in ribosomal proteins L4 and L22 were also examined for an effect on assembly. Subunit assembly was affected in the mutant containing the L22 alteration only at erythromycin concentrations fourfold greater than those needed to stop assembly in wild-type cells. Ribosomal subunit assembly was only marginally affected at the highest drug concentration tested in the cells that contained the altered L4 protein. These novel results indicate that erythromycin has two effects on translation, preventing elongation of the polypeptide chain and also inhibiting the formation of the large ribosomal subunit.