A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin.

Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.

Strain- and species-specific distribution of the streptomycin gene cluster and kan-related sequences in Streptomyces griseus.

The streptomycin (SM) gene cluster was investigated for its distribution in streptomycetes by Southern hybridization using nick-translated DNA probes, which were isolated from the SM-6-phosphotransferase (SPH) and amidinotransferase (ADT) regions of the SM gene cluster of Streptomyces griseus SS-1198. Bgl II-digested genomic DNAs from SM-producing strains of S. griseus yielded the same size fragment (7.0 kb) which hybridized to both the SPH and ADT probes as expected from the restriction endonuclease cleavage map of the SM gene cluster. By contrast, no genomic DNA fragments from heterologous Streptomyces strains hybridized to the probes. Thus, only SM-producing strains of S. griseus possess the highly homologous SM gene cluster. Similarly, distribution of DNA sequences homologous to the kanamycin (KM)-resistance determinant (kan) from a KM-resistant regenerant of S. griseus SS-1198 protoplasts was also examined. Using the kan gene fragment as the probe it was revealed that the kan-related sequences are present in all the strains of S. griseus tested, irrespective of the type of antibiotics they produce. However, no hybridization to the kan gene probe (KAN) was observed with DNA digests derived from other Streptomyces species.

Gene cluster for streptomycin biosynthesis in Streptomyces griseus: nucleotide sequence of three genes and analysis of transcriptional activity.

Three streptomycin (SM) production genes from Streptomyces griseus clustered around aphD, the major resistance gene, have been sequenced: strB, coding for an aminocyclitol amidinotransferase, ORF5 (strR), a putative regulatory gene, and ORF1 (strD), possibly coding for a hexose nucleotidylating enzyme. Three promoters and at least five, partially overlapping, transcripts have been identified by S1 mapping and Northern blot experiments. aphD, the resistance gene, is transcribed from two promoters. One of them, located inside the strR gene, seems to be constitutive and the other is switched on later in the growth phase. The late transcripts cover the resistance gene (aphD) and a regulatory gene (strR) which controls the expression of strB.

Cloning of a streptomycin-production gene directing synthesis of N-methyl-L-glucosamine.

A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD 1 used as the recipient in cloning displayed deficiency in biosynthesis of N-methyl-L-glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD 1 was narrowed to a 3.0-kb fragment.

Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance.

An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.

Isolation of streptomycin-nonproducing mutants deficient in biosynthesis of the streptidine moiety or linkage between streptidine 6-phosphate and dihydrostreptose.

Eight streptidine idiotrophic mutants (SD20, SD81, SD141, SD189, SD245, SD261, SD263, and SD274) which required streptidine to produce streptomycin were derived from Streptomyces griseus ATCC 10137 by UV mutagenesis. By both the characterization of intermediates accumulated by the idiotrophs and the assay of enzymes involved in streptidine biosynthesis, the biochemical lesions of the mutants were deduced as follows: SD20 and SD263, transamination; SD81, SD261, and SD274, phosphorylation; SD141, transamidination; SD189, dehydrogenation; SD245, linkage between streptidine 6-phosphate and dihydrostreptose. An accumulation of streptidine 6-phosphate was found in SD245 to impair its aminotransferase activity. This finding suggests that aminotransferase activity might have been negatively controlled by the end product, streptidine 6-phosphate, of the streptidine biosynthetic pathway.

Protein patterns of Streptomyces griseus strains during development.

Protein patterns from mycelial extracts of Streptomyces griseus strains No. 45H and No. 52-1 were studied by one dimensional gradient PAGE with 20 cm run. The results are reproducible, the protein patterns are the same for the same developmental stage in a given strain. There are well-defined characteristic changes of the protein patterns during the life cycle. The proteins of spores show conspicuous differences compared to protein patterns of the old (72 h) mycelia in the same culture. There is no difference between protein patterns of spores from submerged and from solid media. The protein patterns of two closely related Streptomyces griseus strains significantly differ from each other. After pulse labelling with (14C)-labelled protein hydrolyzate for 40 min, the specific activities of the proteins show considerable differences. There are characteristic bands with low specific activity and others with high incorporation. The fluorograms of the 16, 40 and 64 h cultures show that different proteins are being synthesized intensively at different ages of the culture.

Aminoglycoside-modifying enzymes of Staphylococcus aureus; expression in Escherichia coli.

Staphylococcus aureus plasmids PSH2, RN1956 and pWA1 code for an aminoglycoside phosphotransferase; plasmid pWA1 also encodes an aminoglycoside-aminocyclitol adenylyltransferase. S. aureus plasmid pWA2 confers resistance to erythromycin and sulfonamide. Using plasmid ColE1-ApR (RSF2124) as a vehicle, we have transferred the genes determining aminoglycoside phosphotransferase and aminoglycoside-aminocyclitol adenylyltransferase activities from S. aureus to Escherichia coli. The new plasmids obtained confer aminoglycoside-aminocyclitol resistant phenotypes to E. coli, similar to, and by the same mechanisms as "naturally" occurring plasmids. By contrast, the results obtained after cloning of plasmid pWA2 indicate that certain S. aureus antibiotic resistance determinants (e.g. for erythromycin (Em) and sulfonamide (Su) cannot be phenotypically expressed in E. coli. The DNA of the constructed hybrid plasmids has been analysed by agarose gel electrophoresis following digestion with restriction endonucleases, by ultracentrifugation in cesium chloride, by hybridization, and by electron microscopy. Each hybrid is a cointegrate replicon, composed of an entire S. aureus plasmid covalently joined to ColE1-ApR.

Control by phospho-adenosinediphospho-ribose of NADP-dependent isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase in Streptomyces griseus.

The metabolic function of NAD(P)-glycohydrolase in the streptomycin-producing Streptomyces griseus was investigated. Phospho-adenosinediphospho-ribose, the product of NAD(P)-glycohydrolase reaction was shown to interfere as a competitive inhibitor not only with the glucose-6-phosphate dehydrogenase (VORONINA et al. 1978) but also with the NADP-dependent isocitrate and 6-phospho-gluconate dehydrogenases. Inhibition kinetics were studied with isocitrate dehydrogenase from pig heart and 6-phosphogluconate dehydrogenase from yeast as well as with mycelial extracts of a mutant of S. griseus lacking NAD(P)-glycohydrolase.

[Effect of Bacillus subtilis A-50 "streptomycin" mutations on the formation of molecular forms of subtilisin, an extracellular alkaline proteinase]. / Vliianie "streptomitsinovykh" mutatsii Bacillus subtilis A-50 na obrazovanie molekuliarnykh form subtilizina--vnekletochnoi shchelochnoi proteinazy.

The patterns of subtilisin molecular forms of streptomycin-resistant (Strr) and streptomycin-dependent (Strd) mutants of Bacillus subtilis A-50, as well as the revertants of Strd to streptomycin-independence (Str1) were studied. Strr mutants had different quantitative pattern of the same subtilisin molecular forms as compared with the initial strain A-50 (the forms with Rf 0.08, 0.16 and 0.3). In comparison with the initial strain A-50, Strd mutants and Str1 revertants revealed three additional forms of the active enzyme with Rf 0.02, 0.5 and 0.7 and the molecular weights less than 35,000, 28,000 and 20,000 respectively. It was suggested that the rate and character of the enzyme secretion of the degree of its post-translational modifications might result in the different pattern of subtilisin molecular forms produced by these streptomycin mutants.