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The nucleotide sequence of the DNA fragment containing the streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.
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Three streptomycin-resistant mutants of group H streptococcus, strain Challis, were examined for ability to accumulate (14)C-streptomycin. Although the mutants exhibited different levels of transformation, only the streptomycin-sensitive parent Challis strain accumulated significant amounts of (14)C-streptomycin. It appears that impermeability to streptomycin does not necessarily result in reduction or loss of transformability. The amount of label accumulated by strain Challis was correlated with a loss of viability. In addition, accumulation of label was influenced by the concentration of (14)C-streptomycin, the time of exposure, and the type of medium employed.
The streptomycin sensitivity of ribosomes derived from a streptomycin-producing Streptomyces griseus was examined in a polyuridylic acid directed 14C-phenylalanine incorporating system. In order to get reproducible results it is essential to use cell-free extracts which do not inactivate streptomycin. This condition can be fulfilled by the combination of washed ribosomes of the streptomycin-producing strain and the 110 000 g supernatant of the streptomycin-nonproducing variant of S. griseus, because the streptomycin-phosphorylating activity can be washed out from ribosomes of younger streptomycin-producing cultures, and the streptomycin-nonproducing S. griseus does not have any streptomycin-inactivating capacity. In this amino acid polymerizing system the ribosomes of the streptomycin-producing strain were as sensitive to streptomycin as the ribosomes of the nonproducing variant or of Escherichia coli.
Several mutants of Escherichia coli affecting aerobic energy generation and energization of the bacterial membrane have been examined for their effect on streptomycin and gentamicin accumulation and susceptibility. A heme-deficient mutant (K207) and two mutants (CJ-8 [colicin K insensitive] and NR-70) associated with defective aerobic active transport were associated with decreased transport of streptomycin and gentamicin and increased resistance to those antibiotics. These mutants also exhibited increased resistance to several other aminoglycoside antibiotics, but not the aminocyclitol spectinomycin. The same observations were made with a ubiquinone-deficient mutant, but a strA derivative of this mutant was shown additionally to be saturable for streptomycin accumulation at a concentration four or more times lower than that required for saturation of the parent. A mutant uncoupled for adenosine 5'-triphosphate synthesis from electron transport and membrane Mg-adenosine 5'-triphosphatase deficient was hypersensitive to those aminoglycosides tested and spectinomycin, and showed enhanced transport of streptomycin and gentamicin. A variety of compounds structurally related to streptomycin were examined at high concentrations for inhibition of streptomycin uptake in a strA mutant of E. coli K-12 SA 1306, but no evidence for competition was detected, suggesting the absence of a common transport carrier. Four different divalent cations were shown to inhibit streptomycin and gentamicin accumulation in E. coli K-12 SA 1306. Divalent cations were shown to inhibit uptake of these two drugs in two bacterial species with distinct cell wall structures, Pseudomonas aeruginosa and Staphylococcus aureus, and to inhibit streptomycin uptake in spheroplasts of streptomycin-susceptible and -resistant E. coli. However, calcium had almost no inhibitory effect on streptomycin uptake by the ubiquinone-deficient mutant E. coli AN66. These and previous findings have been used to formulate a model for aminoglycoside entry into bacteria using a low-affinity membranous complex involved in membrane energization that includes respiratory quinones, which probably act to bind and transport aminoglycosides across the cell membrane. This phase of transport is associated with the lowest accumulation rate (termed energy-dependent phase I) that is rate limiting for susceptibility. It is further proposed that subsequent association of the membrane-bound aminoglycoside with higher-affinity binding sites on membrane-associated ribosomes carrying out a normal ribosomal cycle and protein synthesis results in a more rapid transport rate (termed energy-dependent phase II). The increased rate could result from a state of membrane energization analogous to that causing enhanced aminoglycoside transport rates seen in the uncoupled mutant, AN120. How this model explains the mechanism by which enzymatically modified aminoglycosides render cells resistant to unmodified aminoglycosides is also discussed.
We used two strains of streptomycin-susceptible enterococci (MIC, 64 and 128 micrograms of streptomycin per ml, respectively) isolated from patients with infective endocarditis. When combined with penicillin, 20 micrograms of streptomycin per ml killed both strains synergistically in vitro whereas combinations of 5 and 10 micrograms of streptomycin per ml did not act synergistically against either strain. By using the rabbit model of enterococcal experimental endocarditis, animals were treated for 3 days with procaine penicillin (1.2 X 10(6) U intramuscularly three times daily) together with low-dose streptomycin (3.5 mg/kg) or high-dose streptomycin (10 mg/kg) intramuscularly three times daily. The peak concentrations of streptomycin in serum at 0.5 h were 9.2 and 26.8 micrograms/ml in the low- or high-dose group, respectively. When combined with procaine penicillin, both dosages of streptomycin were more effective (P less than 0.01) than procaine penicillin alone for the treatment of enterococcal experimental endocarditis. There was no significant difference in the efficacy of procaine penicillin plus low-dose streptomycin versus procaine penicillin plus high-dose streptomycin therapy of enterococcal experimental endocarditis.
An efficient in vitro amino acid-incorporating system from Mycobacterium tuberculosis H37Rv was standardized. Ribonucleic acid (RNA) isolated from phage-infected M. smegmatis cells served as natural messenger RNA and directed the incorporation of (14)C-amino acids into protein. The effects of various antitubercular drugs and "known inhibitors" of protein synthesis on amino acid incorporation were studied. Antibiotics like chloramphenicol and tetracycline inhibited mycobacterial protein synthesis, though they failed to prevent the growth of the organism. This failure was shown to be due to the impermeability of mycobacteria to these drugs by use of "membrane-active" agents along with the antibiotics in growth inhibition studies. Several independent streptomycin-resistant mutants of M. tuberculosis H37Rv were isolated. Streptomycin inhibited the incorporation of (14)C-amino acids into proteins by whole cells of a streptomycin-susceptible strain by more than 90%, whereas very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains. In vitro, streptomycin was an effective inhibitor of susceptible strains, whereas in streptomycin-resistant strains the concentration of streptomycin at which half-maximal inhibition was produced varied according to the resistance of whole cells, and there was a correlation between the two. In one low-level streptomycin-resistant mutant, the in vitro amino acid-incorporating system was as sensitive to various concentrations of streptomycin as the parental type, and a possible involvement of a membrane site in the development of low-level resistance was indicated. Streptomycin susceptibility and high-level resistance were shown to be ribosomal in nature.
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.
The gene encoding streptomycin 6-kinase involved in the self-resistance of the streptomycin-producing Streptomyces griseus HUT 6037 was cloned in the plasmid vector pIJ703. The resulting plasmid, pSP6, contained 2.5 kb inserts of S. griseus DNA. When streptomycin-susceptible S. lividans 1326 was retransformed with pSP6, all transformants produced streptomycin 6-kinase. Addition of streptomycin to the culture medium of S. lividans carrying pSP6 plasmid brought about a remarkable increase in streptomycin 6-kinase activity in the cell extracts. It is suggested from the results that the production of streptomycin 6-kinase in streptomycin producer was induced by streptomycin accumulated during cultivation.
A phosphorylated derivative of streptomycin accumulated in cultures of Streptomyces griseus ATCC 12475 and SC2376 grown on complex media containing an excess of inorganic phosphate (0.01 m). This compound did not accumulate significantly in the absence of added inorganic phosphate. The phosphorylated derivative did not inhibit growth of Bacillus subtilis or support growth of a streptomycin-dependent strain of Escherichia coli; however, incubation of the derivative with alkaline phosphatase gave a compound which was active with both systems. In the phosphorylated derivative, phosphate is esterified with an -OH group of the streptidine moiety of streptomycin. It is suggested that the phosphoryl group is introduced during biosynthesis of the streptidine moiety of streptomycin or by the action of streptomycin kinase on preformed streptomycin (or both), and subsequent dephosphorylation by streptomycin-phosphate phosphatase is inhibited by high concentrations of inorganic phosphate. A column chromatographic procedure for separation of streptidine-phosphate, streptomycin-phosphate, and streptomycin is described.
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