Analysis of lincomycin resistance mutations in Escherichia coli.

Summary High level lincomycin resistant strains of Escherichia coli were isolated and screened for altered ribosomal proteins and functions. Amongst 58 strains investigated by electrophoresis one had an altered ribosomal protein S7, another one a mutated L14 and two showed altered L15 proteins.A correlation between these alterations and lincomycin resistant growth could not be demonstrated by genetic analysis for any of the mutants. In vitro, however, extracts from the two L15 mutants were less sensitive to inhibition by the drug. A gene locus (lin ^R) responsible for the lincomycin resistance phenotype was mapped at min 30 of the Escherichia coli chromosome near tyrR; it seems to be identical to the previously described linB locus (Apirion, 1967); however, in contrast to these reports it does not seem to alter any ribosomal function.     

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 strain of Escherichia coli which gives rise to mutations in a large number of ribosomal proteins

Summary A strain of E. coli K12 has been isolated which gives rise to mutations in a large number of ribosomal proteins. Mutant VT, which was derived from A19, shows a novel type of streptomycin dependence and has an altered ribosomal protein S8. Streptomycin-independent isolates from mutant VT contain a great variety of changed proteins on two-dimensional polyacrylamide gels. 120 revertants screened in this way have changes in thirteen 30S proteins and fifteen 50S proteins. Several mutants were found in which additional proteins are present on the ribosome. Further, there is one instance of a ribosomal protein (L1) being absent, and one of apparent doubling of a ribosomal protein (L7/12). The unique properties of mutant VT probably are the result of the altered S8.     

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.     

Ribosomal protein modification in Escherichia coli

Summary Among mutants of E. coli selected for temperaturesensitive growth, four were found to possess alterations in ribosomal proteins L7/L12. Of these, three apparently lack protein L7, the acetylated form of protein L12. Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E. coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min. Hence, the gene affected in these mutants was termed rimL. Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein. It was found that the acetylase activity specific for protein L12 was negligible, when assayed in vitro, in the high-speed supernatant prepared from mutant cells. These results indicated that the three mutants contain mutations in the gene rimL that codes for an acetylating enzyme specific for ribosomal protein L12.Previous paper in this series is Isono and Isono (1980)     

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.     

Differential subcellular localization of ribosomal protein L7 paralogs in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In Saccharomyces cerevisiae, ribosomal protein L7, one of the ∼46 ribosomal proteins of the 60S subunit, is encoded by paralogous RPL7A and RPL7B genes. The amino acid sequence identity between Rpl7a and Rpl7b is 97 percent; they differ by only 5 amino acid residues. Interestingly, despite the high sequence homology, Rpl7b is detected in both the cytoplasm and the nucleolus, whereas Rpl7a is detected exclusively in the cytoplasm. A site-directed mutagenesis experiment revealed that the change in the amino acid sequence of Rpl7b does not influence its sub-cellular localization. In addition, introns of RPL7A and RPL7B did not affect the subcellular localization of Rpl7a and Rpl7b. Remarkably, Rpl7b was detected exclusively in the cytoplasm in rpl7a knockout mutant, and overexpression of Rpl7a resulted in its accumulation in the nucleolus, indicating that the subcellular localization of Rpl7a and Rpl7b is influenced by the intracellular level of Rpl7a. Rpl7b showed a wide range of localization patterns, from exclusively cytoplasmic to exclusively nucleolar, in knock-out mutants for some rRNA-processing factors, nuclear pore proteins, and large ribosomal subunit assembly factors. Rpl7a, however, was detected exclusively in the cytoplasm in these mutants. Taken together, these results suggest that although Rpl7a and Rpl7b are paralogous and functionally replaceable with each other, their precise physiological roles may not be identical.     

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.     

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.     

Ribosomal proteins of Streptomyces aureofaciens producing tetracycline

Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens. Proteins of small subunits were resolved into 21 spots. Larger ribosomal subunits contained 35 proteins. The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations. Antibodies developed against 50 S proteins of S. aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species. Results of the experiments indicate that about one half of the 50 S proteins of S. aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E. coli. Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S. aureofaciens can be assembled to E. coli P₀ cores lacking proteins L7/L12. Reconstitution of the P₀ cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis.     

Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins

Summary A battery of immunological tests were used to investigate mutants which had been determined as lacking one or two ribosomal proteins on the basis of two-dimensional polyacrylamide gels. Proteins which were confirmed as missing from the ribosome in one or more mutants were large subunit proteins L1, L15, L19, L24, L27, L28, L30 and L33 and small subunit proteins S1, S9, S17 and S20. Cross-reacting material (CRM) was also absent from the post-ribosomal supernatant except in the case of protein S1. Since mutants lacking protein L11 have been previously described, any one of 13 of the 52 ribosomal proteins can be missing. None of these 13 proteins, except S1, can therefore have an indispensable role in ribosome function or assembly. In several mutants in which a protein was not missing but altered, it was present as several moieties of differing charge and size.     

Genetic fine structure of the pyrE region containing the genes for ribosomal proteins L28 and L33 in Escherichia coli

Summary Temperature-sensitive mutants harbouring alterations in ribosomal proteins L28 and L33 have been isolated and used in mapping the genes coding for the two proteins. It was found that they mapped very close to each other and near pyrE at 80.7 min on the E. coli genetic map. The genes affected by the mutations have been concluded to be the structural genes for proteins L28 (rpmB) and L33 (rpmG) by constructing merodiploids heterozygous for pyrE and for the two ribosomal proteins. Various transduction studies with P1kc phages indicate the gene order in this region to be (rpmB, rpmG)-pyrE-spoT-gltC.     

Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast

A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized.It was found that the enzyme utilizes GTP and ATP as phosphoryl donors. Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 × 10^-6M and 5.5 × 10^-5M, respectively.Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the [-³²P] nucleotides used. It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro. The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E. coli ribosomes. The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.     

A spontaneous mutant of Escherichia coli with protein L24 lacking from the ribosome

Summary A spontaneous mutant that lacked ribosomal protein L24 was isolated and its derivatives investigated. The lesion responsible was close to, or in, rplX, the gene for protein L24. It led to a severe reduction in the amount of the large ribosomal subunit, even under permissive growth conditions. The mutation also led to a very slow growth rate and a temperature sensitive phenotype of carrier strains. Temperature indifferent secondary mutants frequently showed recovery of protein L24, but the protein was usually in a form larger than wildtype. Other secondary mutants had acquired an external suppressor that resulted in the simultaneous alteration of several other ribosomal proteins as well as the fractional presence of protein L24. Secondary mutants had normal amounts of the large ribosomal subunit, but it sedimented more slowly than normal.     

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [³⁵ S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.     

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.     

Selection in Bacillus subtilis giving rise to strains with mutational alterations in a variety of ribosomal proteins

Summary To facilitate mapping of ribosomal protein genes in Bacillus subtilis, a selection was devised which gave rise to strains with alterations in any one of a variety or ribosomal proteins. Alterations in eighteen ribosomal proteins were identified when eighty mutants were analysed. In addition, one strain showed a major assembly defect in the large ribosomal subunit resulting in the presence of a particle sedimenting at about 40S. Eighteen large subunit proteins were present on this particle in normal amounts, while twelve proteins were much reduced in amount or undetectible.     

Comparison of ribosomal proteins of chloroplast from spinach and of E. coli

Summary A comparison of ribosomal proteins from Escherichia coli and from chloroplasts of Spinach was made using two separate methods: electrophoretic migration and immunochemical cross-reaction between blotted E. coli ribosomal proteins and chloroplast ribosomal subunits antisera. It is shown that L2 from E. coli (E-12) and L4 from chloroplasts (CS-L4) comigrated and that E-L4 immunologically cross-reacted with the isolated CS-L4 antibody. Co-migration was observed for three additional couples of 50S ribosomal proteins. It is also shown that at least one 30S E. coli ribosomal protein immuno-cross reacted with a 30S chloroplast antiserum and that three couples of 30S ribosomal proteins comigrated.     

The ribosomal stalk is required for ribosome binding,depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga‐like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α‐sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the α‐sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild‐type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the ΔP1 and ΔP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild‐type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.     

Decrease in ribosomal proteins 1, 2/3, L4, and L7 in Drosophila melanogaster in the absence of X rDNA

Summary The rRNA genes (rDNA) in Drosophila melanogaster are found in two clusters, one on the X and one on the Y chromosome. We have compared the ribosomal protein composition of wild-type Oregon-R flies containing both X-linked and Y-linked rDNA with that of flies containing only the Y-linked rDNA by two-dimensional polyacrylamide gel electrophoresis. Four basic proteins (1, 2/3, L4, and L7) normally present in wild-type flies were either electrophoretically not detectable (1, 2/3, and L4) or marginally detectable (L7) in flies with only Y-linked rDNA. No additional proteins were observed in these flies. However, immunodiffusion assays using specific antibodies raised against purified protein L4 confirmed that L4 was present but in relatively lower amounts in these Y-linked rDNA flies. An investigation was carried out to determine whether these electrophoretically undetectable proteins were more readily lost during ribosome preparation and hence were not readily detectable in the 80S particles by gel electrophoresis or whether they had been modified. Thus the proteins in the post-ribosomal cell supernatant and the high salt sucrose gradient were analyzed by two-dimensional gel electrophoresis and immunochemical assays with antibodies raised against protein L4 and total 80S ribosomal proteins. The experimental evidence indicates that there is a small amount of protein L4 and probably proteins 1, 2/3, and L7 in flies with only Y-linked rDNA but significantly less of these proteins than in wild-type flies.