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.     

Suppression of spectinomycin resistance in a mutant of Escherichia coli K-12.

A mutant of Escherichia coli has been isolated which exhibits partial suppression of spectinomycin resistance. The site of mutation is in the streptomycin (strA) region and is closely linked to the spcA gene. However, this gene, which we propose to call mod, is phenotypically distinguishable from both the neomycin-kanamycin (nek) and the ribosomal ambiguity gene (ram). The relative gene order is mod spcA strA. In a cell-free protein synthesizing system, altered ribosomes appear to be responsible for the suppression of spectinomycin resistance caused by mod.     

A spectinomycin dependent mutant of Escherichia coli.

Summary A mutant of Escherichia coli B has been isolated which shows a novel phenotype of spectinomycin dependence. The mutant, termed RD, needs spectinomycin to grow at temperatures of 37° or below; it is unable to grow at 42° in either the presence or absence of spectinomycin. Secondary mutants which grow well in the absence of spectinomycin can be isolated spontaneously at a frequency of about 10^-6. Two-dimensional gel electrophoresis of ribosomal proteins from 25 of these revertants showed that two revertants had an alteration in S4; one other showed an alteration in L5, and one showed an apparent absence of L1. Mutant RD itself had an altered less basic S5, which was maintained in all the revertants that were checked.Genetic analysis indicated that RD was a double mutant: one mutation, which alone conferred a spectinomycin resistant phenotype on the strain, was located in the strA region of the E. coli chromosome and was represented by the mutation in S5. The other mutation, which conferred the dependence on spectinomycin, mapped close to the rif locus.     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

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.     

Mutations conferring lincomycin, spectinomycin, and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (Ll7A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSpl) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3) were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A C change at codon 87 of rps 12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.     

Analysis of ribosomal proteins in streptomycin resistant and dependent mutants isolated from streptomycin independent Escherichia coli strains

Summary Mutants resistant to (Str-R) or dependent on streptomycin (Str-D) were isolated from several streptomycin independent (Str-I) strains of Escherichia coli. From 90 of these mutants ribosomes were isolated and the ribosomal proteins analyzed by two-dimensional polyacrylamide gel electrophoresis. The results which are summarized in Tables 1-4 led to the following conclusions:a) The phenotype (Str-R or Str-D) of the mutants isolated from the Str-I strains strongly depends on the parental strain. b) No other ribosomal proteins than S4, S5 and S12 seem to be altered by mutations leading to dependence on, independence from or resistance to streptomycin. c) The S4 proteins of the analyzed mutants belong to three groups. The ratio between the groups depends more on the origin of the mutants than on their phenotype. d) Eight new types of altered S4 proteins were detected. It is very likely that many, if not all, of the altered S4 proteins originated by frame shift mutations. e) Some of the mutants differ from the wild type by alterations in three ribosomal proteins (S4, S5 and S12). The alteration in one protein, S4, apparently compensates for that in another protein, S5, in such a way that the original phenotype is expressed. These mutants are therefore an excellent tool for studies at the molecular level on the interaction of ribosomal components within the particle.     

A simple procedure for the isolation of streptomycin resistant plants in Solanaceae

A system has been developed for rapid selection of streptomycin resistant mutants, as adventitious shoots arising from explants of several Solanaceous species. Efficient mutagenesis was achieved by incubating shoot culture-derived leaf strips with 1 or 5 mM nitroso-methylurea, for 90 or 120 min. In Nicotiana tabacum and Lycopersicon peruvianum these treatments resulted in white or variegated adventitious shoots from up to 3.5% of explants placed on medium promoting shoot regeneration. Chlorophyll deficiencies were only observed very rarely in Solanum nigrum. Streptomycin resistant shoots were obtained from leaf explants placed on medium containing 500 mg l^-1 streptomycin sulphate, under which conditions explants are bleached and adventitious shoot development suppressed. Green adventitious s shoots appeared at a frequency dependent both on the mutagenic treatment and on the species. The best response was with S. nigrum where >70% of the explants produced streptomycin resistant shoots, most of which retained their resistance on subsequent testing. Maternal inheritance of streptomycin resistance has been confirmed for several N. tabacum and S. nigrum mutants, and there is also evidence for paternal transmission in the latter species. The procedure has been successfully extended to other species, including N. sylvestris and N. plumbaginifolia, and also to obtain spectinomycin resistant mutants.Communicated by R. Hagemann     

Antibiotic-resistant mutants of Bacillus subtilis conditional for sporulation.

Among spontaneously occurring antibiotic-resistant mutants of Bacillus subtilis 168 we have identified a sub-class that is conditionally sporulative. Mutants in this sub-class are resistant to antibiotic during vegetative growth but are sensitive during sporulation. Mutants conditionally-resistant to erythromycin, kanamycin, spectinomycin, and streptomycin have been isolated and characterized by phase contrast microscopy and with respect to their ability to synthesize heat-resistant endospores or the sporulation-associated enzyme alkaline phosphatase. The results suggest that several entirely different genetic lesions may result in this single phenotype. This group includes mutants whose properties suggest that both th 30S and 50S ribosomal subunits may be altered concomitant with early spore specific metabolism. The blockage imposed by antibiotic may be at or near Stage 2 of sporulation.     

Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants.

Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered. Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process. Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants. In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells. We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein. 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin. The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively. This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops.     

Nuclear Mutation Increases Streptomycin and Spectinomycin Sensitivity in Chlamydomonas

A spontaneously arising nuclear mutation, ss-1, has been identified in Chlamydomonas reinhardtii that decreases both streptomycin and spectinomycin resistance levels about 10-fold after its introduction into all wild-type, streptomycin-resistant and spectinomycin-resistant strains examined. The mutations for resistance map to nuclear and uniparentally inherited (chloroplast) loci. In contrast, no modification of erythromycin resistance was detected after introducing ss-1 into wild-type strains or into strains carrying nuclear or uniparentally inherited erythromycin-resistance mutations. We suggest that ss-1 affects the small subunit of the chloroplast ribosome because others have shown that streptomycin and spectinomycin resistance in C. reinhardtii are associated with this subunit, whereas erythromycin resistance is associated with the large subunit. ss-1 shows no linkage with the nuclear locus for streptomycin resistance.     

Pleiotropic effects of ribosomal protein s4 studied in Escherichia coli mutants

Summary A temperature sensitive mutant of Escherichia coli was found to have two mutational alterations of its ribosomes: one of these was a streptomycin dependent mutation and the other was a suppressor alteration of S4, with a marked structural change. The altered form of S4 was studied in a strain that was constructed so that this alteration was the only one effecting the structure of the ribosome. Here, it was shown that the mutant form of S4 cause a temperature sensitive defect in the assembly of 30S subunits in vivo which is reflected in the inability of this mutant to properly process ribosomal RNA at the restrictive temperatures. An analysis of both transductants and revertants of this mutant show that the suppression of the streptomycin dependence phenotype, temperature sensitivity, and a defect in RNA processing all have their origin in a single mutational event effecting the structural gene for S4.     

Genetic studies of the ribosomal proteins in Escherichia. coli. II. Altered 30s ribosomal protein component specific to spectinomycin resistant mutants

Summary Spectinomycin resistant (spc ^r) mutants were obtained by treating the cells of E. coli K12, W3637 with nitrosoguanidine. The compositions of ribosomal proteins were analyzed for six out of eleven such spc ^r-mutants with chromatography on a carboxymethyl cellulose (CMC) column. The 30s ribosomal subunit from all of the spc ^r-mutants was found to contain the altered 30-4 protein component, while no difference was detected in 50s ribosomal proteins between spc ^r and spc ^s bacteria.Abbreviations used CMC carboxymethyl cellulose - str streptomycin - spc spectinomycin     

Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.     

Mutations conferring lincomycin,spectinomycin, and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (Ll7A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSpl) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3′) were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A → C change at codon 87 of rps 12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.     

Coresistance to Neomycin and Kanamycin by Mutations in an Escherichia coli Locus that Affects Ribosomes

Mutant strains resistant to neomycin or to kanamycin sulfate were isolated from Escherichia coli K-12. Nine mutants were analyzed; all were resistant to both antibiotics (about 150 and 100 mug/ml, respectively), and were designated nek. In the mutant strains, the ribosomes are changed from those of the parental strain; for when they were used in assays for polypeptide formation directed by polyadenylic acid or polycytidylic acid, coding fidelity in presence of the drugs was increased and inhibition of synthesis by the drugs was lessened. Mating experiments and transduction tests showed that all of the nine nek mutants are either closely linked or allelic, and the nek locus is closely linked to two genes-str (streptomycin) and spc (spectinomycin)-known to affect the 30S ribosome. The two nek mutants tested were recessive to the sensitive, wild-type allele. When the nek mutants were compared to the parental strain, pleiotropic effects of the nek mutations were observed. Resistance to low levels of streptomycin and spectinomycin was increased, whereas resistance to chloramphenicol was decreased. Also, the mutants were less able to adapt to high concentrations of lincomycin, and could no longer show phenotypic suppression of an arginine requirement by neomycin or kanamycin. Such pleiotropic effects are suggested to be the rule for mutations in genes that participate in the biosynthesis of a cellular organelle.     

Ribosomal protein S12 as a site for streptomycin resistance in Nicotiana chloroplasts

Summary A streptomycin resistant Nicotiana plastome mutant, X/str ^R6, was subjected to molecular analysis. In this mutant, a single nucleotide transition, C » T, in the chloroplast gene for ribosomal protein S12 alters codon 90 from proline to serine while the nucleotide sequence of the chloroplast 16 S rRNA gene is identical to that of the wild type. Mutant X/str ^R6 thus differs from several previously reported streptomycin resistant chloroplast mutants which are altered in the gene for 16 S rRNA.     

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.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.