Methylation of 16S ribosomal RNA and resistance to aminoglycoside antibiotics in clones of Streptomyces lividans carrying DNA from Streptomyces tenjimariensis

Summary A single gene from Streptomyces tenjimariensis, conferring resistance to kanamycin, apramycin and sisomicin, has been cloned in Streptomyces lividans. The mechanism of resistance involves methylation of 16S RNA in the 30S ribosomal subunit.     

Cloning and characterization of two genes from Streptomyces lividans that confer inducible resistance to lincomycin and macrolide antibiotics.

Inducible resistance to lincomycin and macrolides in Streptomyces lividans TK21 results from expression of two linked genes: lrm, encoding a ribosomal RNA methyltransferase that confers high-level resistance to lincomycin with lower levels of resistance to macrolides, and mgt, encoding a glycosyl transferase that specifically inactivates macrolides using UDP-glucose as cofactor. The lrm and mgt genes have been cloned and sequenced. The deduced lrm product is a 26-kDa protein with much similarity to other ribosomal RNA methyltransferases, such as the carB, tlrA and ermE products, whereas the mgt product (predicted to be 42 kDa) resembles a eukaryotic glycosyl transferase. Macrolides that induce the lrm-mgt gene pair are substrates for inactivation by the mgt product, and the lrm product confers ribosomal resistance to such inducers.     

Alteration of 23 S ribosomal RNA and erythromycin-induced resistance to lincomycin and spiramycin in Staphylococcus aureus

Functionally active “hybrid” 50 S ribosomal subunits can be reconstituted using 23 S RNA from Staphylococcus aureus (strain 1206) and 5 S RNA, as well as 50 S ribosomal proteins from Bacillus stearothermophilus. Using this system, resistance of S. aureus 50 S subunits to lincomycin and spiramycin was analyzed. When 23 S RNA from either phenotypically resistant (“induced resistance”) S. aureuscells or derived genetically resistant (“constitutive resistance”) S. aureus cells, were used, the reconstituted 50 S subunits showed the resistant phenotype similar to that seen in native 50 S subunits obtained from resistant cells; only very weak inhibition by the antibiotics was observed in poly (U) - directed polyphenylalanine synthesis involving these 50 S subunits. In contrast, the 50 S particles reconstituted using 23 S RNA from uninduced (sensitive) S. aureus were subject to greater inhibition by the antibiotics in cell-free poly-peptide synthesis. It is concluded that modification of 23 S RNA, presumably the previously observed methylation to form dimethyladenine, is responsible for the resistance to the antibiotics in this strain of S. aureus.     

Biotransformation of milbemycin D to milemycin G by Streptomyces lividans conferring avermectin O-methyltransferase activity

Avermectin O-methyltransferase gene was cloned from a cosmid clone covering the first module of polyketide synthase gene cluster of avermectin biosynthesis. Streptomyces lividans transformed with a DNA fragment containing avermectin O-methyltransferase gene efficiently convert milbemycin D to milbemycin G, indicating that biosynthetic genes of milbemycin and avermectin might complement each other. © Rapid Science Ltd. 1998     

Genetic instability of determinants of resistance to chloramphenicol and kanamycin in Streptomyces lividans 66

The frequency of chloramphenicol-sensitive variants (Cmls) in Streptomyces lividans 66 is very high (0.57%). Correlation between chloramphenicol sensitivity and deamplification of PstI fragment with the length of 4.82 kb (RES1 genetic element) was shown. However, in some Cmls variants there was no RES1 deamplification. It was noted that in the cells of the Cmls variants isolated the levels of kanamycin and neomycin resistance determined by the Kanr determinant in the pSU17 plasmid were different. Expression of Kanr and Neor determinants inserted via pSU17 plasmid into the cells of Cmls variants was studied and three classes of chloramphenicol-sensitive variants were defined. After transformation of pSU17 plasmid into cells of Cmls variants of the class I, expression of Kanr and Neor genes, similar to that in S. lividans 66, was observed. The resistance level in Cmls variants of the class II was intermediate. In the cells of the class III no expression was noted. Cmls strains of classes I and II were unstable and those of the class III with impaired expression of Kanr and Neor genes were formed with high frequency. Cmlr variants formed from Cmls strain of the class III were studied. Two types of Cmlr variants were detected. Variants of the first type were identical to S. lividans 66 by their properties. The frequency of Cmls variants occurring in the cells of the first type was similar to that in S. lividans 66. The second type included pseudo-revertants. They were unstable and generated amplifications of the 5.7 kb fragment with high frequency.     

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.     

Molecular cloning of tylosin-producing Streptomyces fradiae B-45 genes determining increased inducible resistance to tylosin in Streptomyces lividans TK64

DNA of S. fradiae B-45 partially cleaved by Sau3A restrictase was cloned in S. lividans TK64 in the plasmid vector pIJ702. Three recombinant plasmids pVG251, pVG262, and pVG253 with tlr1, tlr2 and tlr3 genes were isolated from the transformed clones of S. lividans TK64 with higher inducible resistance to tylosin as compared to the plasmid-free strain. DNA-DNA blot hybridization was performed between the total DNA cleaved by several restrictases from S. fradiae B-45 and some other strains and the DNA probes containing the tlr genes. It was shown that tlr1 and tlr3 genes were unique in S. fradiae B-45. Sequences homologous to tlr2 gene were present both in DNA of S. fradiae B-45 in 7 copies and in strains of S. antibiotics and S. hygroscopicus producing respectively oleandomycin and turimycin.     

Twin-Arginine Translocation Pathway in Streptomyces lividans

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.     

Secondary methylation of yeast ribosomal precursor RNA.

The timing of methylation of the ribosomal sequences of ribosomal precursor RNA (pre-rRNA) from the yeast Saccharomyces carlsbergensis was investigated by fingerprint analysis of the methylated oligonucleotides derived from the various precursors. From the total of 37 ribose and 6 base-methyl groups found in 26-S rRNA, the two copies of the base-methylated nucleoside m3U as well as the doubly methylated sequence Um-Gm psi are not yet present in 37-S RNA, the predominant common precursor of 26-S and 17-S rRNA. Introduction of these methyl groups into the ribosomal sequences appears to take place at the level of 29-S pre-rRNA, the immediate precursor to 26-S rRNA. From the total of 18 ribose-methylated and 6 base-methylated nucleosides found in 17-S rRNA, the latter group (one copy of m7G, the m62A-m62A- sequence and the hypermodified methylated nucleoside "mX") is completely missing in 37-S pre-rRNA. The methyl group of m7G is introduced into 18-S pre-rRNA, the direct precursor of 17-S rRNA, in the nucleus. The -m62A-m62A- sequence is methylated after transport of the 18-S pre-rRNA to the cytoplasm prior to the final maturation into 17-S rRNA.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.     

Intraplasmid recombination in Streptomyces lividans 66

Summary An Escherichia coli-Streptomyces shuttle plasmid pIF132 containing two direct mel repeats was constructed. While pIF132 replicated relatively stably in E. coli (Rec⁺ or recA), its structure was unstable in S. lividans: recombination between the mel repeats resulted in a smaller plasmid, pIF138. Furthermore, pIF132 formed oligomers extensively in E. coli but not in S. lividans.