Amino acid replacement in the protein S5 from a spectinomycin resistant mutant of Bacillus subtilis

Summary Ribosomal protein S5 was isolated from wild type Bacillus subtilis ATCC 6633 and from a spectinomycin resistant mutant (BSPC 111) derived from spectinomycin sensitive to resistance is accomtrypsin and all the tryptic peptides were isolated by column- and paper-chromatography. By comparative amino acid analyses of the peptides, it was demonstrated that the S5 from the mutant differs from the wild type S5 by a replacement of one amino acid, namely lysine by isoleucine in the peptide T9. The results are compared with E. coli spectinomycin resistant mutants.     

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.     

Phenotypic and genetic characterization of multidrug-resistant <Emphasis Type="Italic">Salmonella</Emphasis> Infantis strains isolated from broiler chicken meats in Turkey

Twenty Salmonella Infantis strains resistant against kanamycin, tetracycline, neomycin, spectinomycin, sulphonamide, nalidixic acid and trimethoprim were selected for this study out of 103 Salmonella strains isolated from broiler samples collected from several markets in the Bolu and Ankara regions of Turkey. The resistance genes aadA1, aphA1, sul1, tet(A), dfrA5/dfrA14 and gyrA were determined for these multidrug-resistant S. Infantis strains. S. Infantis strains contained a mega plasmid with the molecular size of 206 kb. The strains were divided into three groups according to the pulsed field gel electrophoresis patterns of XbaI digested chromosomal DNA. A Ser83→Tyr83 point mutation was found in the gyrA gene of all quinolone-resistant isolates. Filter mating experiments showed that 206 kb plasmid transferred nalidixic acid resistance associated with class I integrons.     

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.     

Isolation of Spectinomycin Resistance Mutations in the 16S rRNA of Salmonella enterica serovar Typhimurium and Expression in Escherichia coli and Salmonella

Two single-base mutations in 16S rRNA conferring high-level resistance to spectinomycin were isolated on a plasmid-borne copy of the rrnD operon from Salmonella enterica serovar Typhimurium. Neither of the mutations (C1066U and C1192U) had appreciable effects on cell growth, but each had differential effects on resistance to spectinomycin and fusidic acid. Both mutations also conferred resistance to spectinomycin in Escherichia coli strains containing deletions of all seven chromosomal rrn operons and expressing plasmid-encoded Salmonella rRNA exclusively. In contrast, when expressed in E. coli strains containing intact chromosomal rrn operons, the strains were sensitive to spectinomycin. However, chromosomal mutations arose that allowed expression of the rRNA-dependent spectinomycin resistance phenotype. It is proposed that in heterogeneous rRNA populations, the native E. coli rRNA out-competes the heterologous Salmonella rRNA for binding to ribosomal proteins, translation factors, or ribosome assembly, thus limiting entry of the antibiotic-resistant 30S subunits into the functioning ribosome pool. Received: 28 September 2001 / Accepted: 26 March 2002     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

Further similarities between chloroplast and bacterial ribosomes

Summary Protein synthesis by chloroplasts isolated under aseptic conditions from Phaseolus vulgaris leaves is inhibited by the bacterial antibiotics spectinomycin, lincomycin, and erythromycin; that by chloroplasts from Nicotiana tabacum leaves is inhibited by spectinomycin and lincomycin but not by erythromycin. Protein synthesis by cytoplasmic ribosomes from plants and animals is not inhibited by these compounds, nor is amino acid activation by the soluble fraction from bean chloroplasts. These results suggest that chloroplast ribosomes possess sites which bind several unrelated bacterial antibiotics and support the idea that chloroplasts originated from prokaryotic cells. These antibiotics may be useful in studying the process of chloroplast formation in intact cells.     

Biochemical and molecular characterization of a rhizobitoxine-producing <Emphasis Type="Italic">Bradyrhizobium</Emphasis> from pigeon pea plants

Out of a total of 8 bacterial strains isolated from the root nodules of pigeon pea plants grown in arid region, five were identified as rhizobia based on biochemical test and confirmed by 16S rDNA sequencing. PCR based screening for the rtxA gene (involved in biosynthesis of rhizobitoxine) revealed that the gene was present in one strain identified biochemically and genetically as belonging to species Bradyrhizobium (BS KT-24). The strain was resistant to phosphomycin, nalidixic acid, kanamycin, gentamicin and neomycin but sensitive towards streptomycin and spectinomycin. Bioinformatic-tool-guided phylogenetic analysis of rtxA gene revealed its distinctiveness from other known rtxA genes (present in B. japonicum, B. elkanii and Xanthomonas oryzae). The rhizobitoxine producing strain BS KT-24 is considered to exhibit better survival and nodulation protection besides competitiveness for pigeon pea and other legumes grown under abiotic stress and, thus, be a candidate in practical aspect of rhizobitoxine production by rhizobium and its application as rhizobial inoculants.     

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.     

A cDNA clone for a pathogenesis-related protein 1 from barley

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.     

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.     

Spectinomycin resistant gonococci.

A study was conducted examining the properties of 10 clinical isolates of spectinomycin resistant gonococci from patients attending clinics at St Mary''s and St Thomas''s Hospitals, London. All of the isolates produced beta-lactamase and contained plasmids of 2.6, 4.4, and 24.5 megadaltons and required proline for growth. None produced aminoglycoside modifying enzymes. Resistance to spectinomycin was transferred from some of the isolates by transformation but at a much lower frequency than resistance to streptomycin. The isolates from St Mary''s Hospital were detected after therapy with spectinomycin, whereas those from St Thomas''s Hospital were not. Four recent non-beta-lactamase producing gonococci isolated at St Mary''s Hospital and two isolated at St Thomas''s Hospital also were not related to use of spectinomycin.     

Chemical and genetic analysis of 16S ribosomal RNA in Escherichia coli

Summary Comparative chemical analyses of oligonucleotides arising from pancreatic RNase digestions of 16S ribosomal RNAs from Escherichia coli strains K12 and B(H) showed that a decanucleotide fragment, (5Ap,4Gp)Cp, could be detected exclusively in strain K12 but not in strain B(H), in spite of gross similarity of nucleotide distributions between the two strains.The K12-specific oligonucleotide could not be cotransduced with streptomycin and/or spectinomycin resistant markers from K12 to B(H) by phage Plkc, indicating that the genes specifying 16S ribosomal RNA are not closely linked to these markers on the chromosome.     

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.     

Reversion of a streptomycin-dependent strain of Escherichia coli

Summary A streptomycin dependent, spectinomycin resistant mutant ofEscherichia coli was used to select spontaneous phenotypic revertants to non-dependence on streptomycin. The ribosomes from one such revertant, which is inhibited by both streptomycin and spectinomycin, were analyzedin vitro. The altered protein responsible for the suppression of the streptomycin dependent phenotype was identified; this protein is 30S-10. The genetic locus for this mutation is a newly identified locus and it has been positioned close to thestr locus. The identification of the altered component responsible for the suppression of the spectinomycin resistant phenotype may be the same as that for the streptomycin dependent phenotype, but this is unproven.     

Spectinomycin resistance due to a mutation in an rRNA operon of Escherichia coli

A spectinomycin resistance mutation was isolated in an Escherichia coli rRNA operon (rrnH) located on a multicopy plasmid. Cell-free protein-synthesizing extracts made from cells containing the plasmid were partially resistant to spectinomycin. Although spectinomycin is an aminoglycoside antibiotic, the mutation did not confer resistance to any other aminoglycoside antibiotic tested.     

Generation of fertile transplastomic soybean

We describe here the development of a plastid transformation method for soybean, a leguminous plant of major agronomic interest. Chloroplasts from embryogenic tissue of Glycine max have been successfully transformed by bombardment. The transforming DNA carries a spectinomycin resistance gene (aadA) under the control of tobacco plastid regulatory expression elements, flanked by two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon. All generated spectinomycin resistant plants were transplastomic and no remaining wild type plastome copies were detected. No spontaneous mutants were obtained. The transformation efficiency is similar to that of tobacco plastids. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. This is the first report on the generation of fertile transplastomic soybean.     

Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO₃ supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l α-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Nucleotide sequence of a spectinomycin adenyltransferase AAD(9) determinant from Staphylococcus aureus and its relationship to AAD(3") (9)

Summary The nucleotide sequence of the spc determinant of the Staphylococcus aureus transposon Tn554 has been determined. This gene encodes a spectinomycin adenyltransferase, AAD(9), that mediates resistance to spectinomycin but not to streptomycin. The sequence predicts a 260 amino acid protein of molecular weight 28,943. A spectinomycin-sensitive mutant (spc-1) contains a GA transition resulting in substitution of threonine (ACA) for alanine (GCA) at residue 165. The predicted amino acid sequence is 36% homologous to that of a widely distributed, gramnegative streptomycin/spectinomycin adenyltransferase, AAD(3) (9), specified by the aadA determinant (Holingshead and Vapnek 1985).