Interaction of streptidin-dependent Actinomyces streptomycini (Streptomyces griseus) mutants No. 170 and 145 with mutants having blocks at various stages of streptomycin biosynthesis]

In complementation analysis of low active streptidine dependent strains of Act. streptomycini, 170 and 145 with mutants having different blocks in biosynthesis of streptomycin it was found that these strains were the donors of some thermostable substances and could reduce the biosynthesis of streptomycin in the mutants having impairements in biosynthesis of streptidine and streptobiosamine, as well as in a number of strains with unknown blocks. It is supposed that the substances produced by mutants 170 and 145 were intermediate products in streptomycin biosynthesis.     

Possible evolutionary relationships between streptomycin and bluensomycin biosynthetic pathways: detection of novel inositol kinase and O-carbamoyltransferase activities.

Bluensomycin (glebomycin) is an aminocyclitol antibiotic that differs structurally from dihydrostreptomycin in having bluensidine (1D-1-O-carbamoyl-3-guanidinodeoxy-scyllo-inositol) rather than streptidine (1,3-diguanidino-1,3-dideoxy-scyllo-inositol) as its aminocyclitol moiety. Extracts of the bluensomycin producer Streptomyces hygroscopicus form glebosus ATCC 14607 (S. glebosus) were found to have aminodeoxy-scyllo-inositol kinase activity but to lack 1D-1-guanidino-3-amino-1,3-dideoxy-scyllo-inositol kinase activity, showing for the first time that these two reactions in streptomycin producers must be catalyzed by different enzymes. S. glebosus extracts therefore possess the same five enzymes required for synthesis of guanidinodeoxy-scyllo-inositol from myo-inositol that are found in streptomycin producers but lack the next three of the four enzymes found in streptomycin producers that are required to synthesize the second guanidino group of streptidine-P. In place of a second guanidino group, S. glebosus extracts were found to catalyze a Mg2(+)-dependent carbamoylation of guanidinodeoxy-scyllo-inositol to form bluensidine, followed by a phosphorylation to form bluensidine-P. The novel carbamoyl-P:guanidinodeoxy-scyllo-inositol O-carbamoyltransferase and ATP:bluensidine phosphotransferase activities were not detected in streptomycin producers or in S. glebosus during its early rapid growth phase. Free bluensidine appears to be a normal intermediate in bluensomycin biosynthesis, in contrast to the case of streptomycin biosynthesis; in the latter, although exogenous streptidine can enter the pathway via streptidine-P, free streptidine is not an intermediate in the endogenous biosynthetic pathway. Comparison of the streptomycin and bluensomycin biosynthetic pathways provides a unique opportunity to evaluate those proposed mechanisms for the evolutionary acquisition of new biosynthetic capabilities that involve gene duplication and subsequent mutational changes in one member of the pair. In this model, there are at least five pairs of enzymes catalyzing analogous reactions that can be analyzed for homology at both the protein and DNA levels, including two putative pairs of inositol kinases detected in this study.     

Actinomyces streptomycini mutant blocked in streptidine biosynthesis]

Mutant 170 not capable of forming streptidine and streptomycin was obtained using chemical mutagenes. This mutant can produce streptomycin only with suplementation of exogenous streptidine. Experiment with labeled C14-streptidine showed its specific incorporation in streptidine moiety of streptomycin molecule.     

Vestibular histofluorescence could be due to accumulation of both the antibiotic and its derivative, streptidine, after acute streptomycin treatment in the guinea pig

Acute treatment with 300 mg/kg of pigmented guinea pigs with streptomycin sulfate induces an elevation of endogenous fluorescence in vestibular ampullary cristae. Fluorescence accumulates in all compartments of the epithelium, i.e., vestibular sensory and supporting cells and nerve fibers of the stroma and it was very intense 1 and 12 hours after its administration. Fluorescence decreased to control levels 24 hours following streptomycin injection. Fluorescence levels were very low either in untreated animals or in animals injected with saline physiological solution. To investigate whether this fluorescence was an intrinsic property of the antibiotic or whether it was due to a derivative of it, or both, an in vitro fluorescence spectrum was performed with 100 microM solutions of streptomycin or streptidine, or both, dissolved in various buffer solutions at 488 nm of excitation. A discrete level of fluorescence was observed in the spectrum regardless of media when separate solutions of both streptomycin or streptidine were studied. Fluorescence notably increased at 522-532 nm when the solutions contained both streptomycin and streptidine together. These results suggest that streptidine putatively derived from streptomycin may contribute to the observed fluorescence accumulation in vestibular preparations after acute treatment. Thus, these metabolic properties of the inner ear which transform streptomycin into streptidine, something never considered earlier, could be claimed as partially responsible for converting a therapeutic agent into a compound which could be as harmful as STP to the inner ear.     

Studies on the Biosynthesis of Streptomycin

Myo-inositol, especially in combination with arginine, enhances streptomycin production. Compounds which show structural relationship with myo-inositol are ineffective.

Myo-inositol decreases the incorporation of C¹⁴-glucose into streptomycin, particularly into streptidine. This effect suggests that myo-inositol is a precursor of the streptidine ring.

Methionine stimulates antibiotic production in a synthetic medium but proves to be unfavorable in a complex medium.

The γ- and δ-isomers of hexachlorocyclohexane inhibit streptomycin formation.

The formation of streptomycin by washed mycelium was studied. Essentially the same results were here obtained as with growing cultures.

     

Coordination compounds derived from the interaction of streptomycin and cobalt, nickel, copper, and calcium salts characterized by 13C NMR and spectroscopic studies. Structure and bonding properties of the streptidine fraction

The coordination compounds of streptomycin (St), Co2(St)Cl4.13H2O (2), Co2(St)(NO3)4.7H2O (3), Ni2(St)Cl4.14H2O (4), Ni2(St)(NO3)4.14H2O (5), Cu2(St)Cl4.6H2O (6), and Ca(St)Cl2.8H2O (7) have been synthesized by the reaction of streptomycin sulfate (1) with three equivalents of the corresponding inorganic salt. The compounds (2)-(7) were characterized by electronic spectroscopy (in the solid state and in solution) by conductivity measurements and by 13C NMR in solution. The reaction of streptomycin with CuCl2 in water hydrolyzed the molecule giving the copper complex of the streptidine fraction (Std), Cu(Std)Cl.H2O (8). This compound was characterized by the same techniques. Detailed x-ray diffraction and 13C NMR studies of streptidine sulfate (9) were carried out.     

Streptidine, a metabolic derivative produced after administration of streptomycin in vivo, is vestibulotoxic in rats

Streptomycin is the treatment of choice in developing countries for patients suffering from tuberculosis or other infectious diseases. However, it produces incapacitating vestibular symptoms whose onset is delayed and gradual. This observation led to the notion that a streptomycin metabolic derivative and not the antibiotic itself is the damaging agent for the inner ear. To study further the existence of this ototoxic metabolite, chronic treatment with streptomycin or its putative derivative streptidine was carried out in young male Long Evans rats. The presence of streptomycin or streptidine in the blood of animals of either experimental group was assessed by high performance liquid chromatography and analysis of swimming behavior was used to evaluate vestibular damage. Features of the sensory epithelium and quantification of hair cells were attained in sections of the utricular organ of all groups by light microscopy. After 25, 35 and 45 days of treatment with streptomycin, a metabolite with the same chromatographic properties as the streptidine standard run in parallel was identified in the blood of rats. Concentrations of the metabolite were 2.26 microg/ml on the 25th day and around 8.0 microg/ml in both the 35th and the 45th day of treatment, while streptomycin was below its detection level at either period. In streptidine-treated rats, the concentration of this compound was 1.0, 1.84 and 4.94 microg/ml on the 25th, 35th and 45th treatment days, respectively. Treatment with either streptomycin or streptidine resulted in similar abnormal swimming patterns and histological alterations of the utricular epithelium. Loss of hair cells was roughly equivalent even though streptidine was administered in a dose 90% lower than streptomycin. The gradual appearance of streptidine as a metabolic derivative of the antibiotic in the blood of rats or the administration of this compound alone, causing similar functional and structural vestibular deterioration seen in streptomycin-treated animals, supports the notion that streptidine is a potential contributor to ototoxicity after prolonged antibiotic administration.     

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.     

Accumulation of streptomycin-phosphate in cultures of streptomycin producers grown on a high-phosphate medium

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.     

Phosphorylation of Streptomycin at C6-OH of Streptidine Moiety by an Intracellular Enzyme of Streptomyces griseus

In streptomycin-producing Streptomyces griseus HUT 6037, an enzyme which phosphorylated streptomycin appeared in old mycelium at stationary to autolyzing stage. This enzyme phosphorylated streptomycin with equimolar ATP at C₆-OH in the streptidine moiety. This phosphomonoester of streptomycin was identified with the phosphorylated streptomycin (referred to as L compound) which was previously reported to accumulate in the culture broth when the pH was controlled below neutral.     

Streptomycin action to the mammalian inner ear vestibular organs: comparison between pigmented guinea pigs and rats

Streptomycin is the antibiotic of choice to treat tuberculosis and other infectious diseases but it causes vestibular malfunction and hipoacusia. Rodents are usually employed as models of drug action to the inner ear and results are extrapolated to what happens in humans. In rats, streptomycin destroys macular sensory cells and does not affect cochlear ones, whereas in guinea pigs the contrary is true. Action on the vestibular cristae cells involved in vestibulo-ocular reflex integrity is less clear. Thus, we compared this response in both pigmented guinea pigs (Cavia cobaya) and rats (Rattus norvegicus) after parallel streptomycin chronic treatment. In guinea pigs, the reflex was obliterated along treatment time; in rats this behavior was not observed, suggesting that the end organ target was diverse. In recent studies, streptidine, a streptomycin derivative found in the blood of humans and rats treated with streptomycin, was the actual ototoxic agent. The putative streptomycin vestibular organ target observed in humans corresponds with the guinea pig observations. Results observed in rats are controversial: streptidine did not cause any damage either to vestibular cristae nor auditory cells. We hypothesize differential drug metabolism and distribution and conclude that results in laboratory animals may not always be applicable in the human situation.     

Enzymatic Phosphorylation of Streptomycin by Extracts of Streptomycin-producing Strains of Streptomyces

Extracts of post-exponential phase mycelia of Streptomyces bikiniensis ATCC 11062, and other streptomycin-producers, catalyze phosphorylation of streptomycin and dihydrostreptomycin with adenosine-5'-triphosphate. The phosphate is esterified with an -OH group of the streptidine moiety. It is suggested that O-phosphoryl-streptomycin might serve as an intracellular precursor of extracellular streptomycin or as a detoxification product of streptomycin or that it might serve an unknown physiological function in the producing organism.     

Slightly active Actinomyces streptomycini (Streptomyces griseus) mutants having a protein in the biosynthesis of the streptidine portion of the streptomycin molecule]

Twelve mutants having a block in biosynthesis of the streptidine part of the streptomyciu molecule were selected under the effect of nitrozomethylbiuret. These 12 strains responded by an increase in the level of the antibiotic production to the addition of streptidine to the cultivation medium. The complementation analysis showed that every streptidine-dependent mutant interacted at least with 2 other mutants. On the basis of the data obtained it is possible to conclude that all 12 mutants had blocks in streptidine biosynthesis but at different stages of this complicated process.     

Preliminary studies on streptomutin A

Summary Supplementation of the culture medium with 2-deoxystreptidine allowsStreptomyces griseus MIT-5, an idiotrophic mutant, to produce an antibiotic which we have called streptomutin A. Degradation studies on the antibiotic showed that the aminocyclitol moiety is different from streptidine. Streptomutin A also is distinct from streptomycin in having a much different ratio of biological to chemical activity.This paper is Contribution No. 2746 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U.S.A.     

Mistranslation and genetic variability: the effect of streptomycin

Streptomycin is an aminoglycoside antibiotic that acts at the level of protein synthesis. Exposure to sublethal concentrations of this antibiotic increased significantly the number of Arg+ mutants derived from an Escherichia coli argE3 (ochre) rpsL31 (streptomycin-resistant) strain. The vast majority of these mutants appeared on selective minimal medium plates with streptomycin (200 micro g/ml) during stationary phase, after 6-10 days incubation at 37 degrees C. Derivative mutD5 or mutL or mutS mutants, carrying a faulty varepsilon subunit of DNA polymerase or a defective mismatch DNA-repair protein, respectively, also showed higher numbers of Arg+ mutants on selective medium with streptomycin than on medium without streptomycin. Interestingly, with these DNA-repair mutants about 50% of the Arg+ mutants generated in the presence of streptomycin appeared during the first 5 days of incubation. These observations suggest that the activities of these fidelity-repair proteins prevent in the parental strain the early appearance of the supernumerary Arg+ mutants on the selective medium with streptomycin. The appearance of Arg+ mutants on the plates with streptomycin was not significantly altered by recA, rpoS or dps mutations. A high percentage of the Arg+ mutants arising in the presence of streptomycin were streptomycin-dependent for growth without arginine (Arg+ St-D). These types of mutants displayed a Ram (for ribosomal ambiguity) phenotype, manifested by increased misreading, assayed by in vitro and in vivo experiments and by leakiness on several selective minimal media. Genetic data indicated that these mutants carry a mutation located at about 74 min of the E.coli map that relieves the high translational fidelity conferred by the rpsL mutation. These studies suggest that the growth-limiting conditions of the assay system used, as well as the presence of streptomycin, which causes an increased production of altered proteins, favours the appearance and growth of compensatory Arg+ mutants.     

Phenotypic suppression by streptomycin of amber mutants in the ribonucleic Acid bacteriophage coat protein cistron

After mutagenesis with nitrosoguanidine or ultraviolet light, 298 streptomycin high-resistant and 98 streptomycin high-dependent mutants were isolated from HfrC Su⁻. They were tested for their ability to phenotypically suppress five different amber ribonucleic acid (RNA) bacteriophage mutants in the presence of streptomycin. The phage mutants are all in the coat protein, which is 129 amino acids long; the uracil-adenine-guanine codons were at the following positions: sus3 and amB2, 6; amB11, 50; amB21, 54; sus11, 70. Only sus3 and amB2 could be phenotypically suppressed by streptomycin; this was clearly demonstrated in nine mutant strains, seven str-HR and two str-HD. The suppression was always dependent upon added streptomycin and was dose-dependent in all cases. None of the mutants showed measurable suppression in absence of the drug. Among revertants to streptomycin independence from streptomycin-dependent strains that could show phenotypic suppression, most of those that were still resistant to streptomycin (10 μg or more) retained the capacity to show phenotypic suppression; whereas among those revertants sensitive to 10 μg of streptomycin or less, none retained the capacity. Eight different amber polar mutants (strong and weak) in gene 34 of phage T4 were also tested for pleiotypic suppression by streptomycin in all the streptomycin-resistant and -dependent strains isolated. No suppression was found in any of the 396 strains tested.     

Properties of streptomycin dependent non-nodulating mutants of cowpea rhizobia and Bradyrhizobium japonicum

We have isolated and characterized mutants from cowpea rhizobia strains JRW3 and IRC256 and Bradyrhizobium japonicum USDA110, which show dependence on streptomycin (Sm) for growth. In the presence of Sm, the majority of the Sm^D (streptomycin dependent) mutants showed cross-resistance to other aminoglycoside antibiotics and some showed no growth at 37°C and 40°C. When nodulation abilities of Sm^D mutants (derived from all three strains) were examined, most of them (> 91%) showed non-nodulating phenotypes to their respective hosts. Preliminary biochemical and genetic characterization indicated that drug-uptake function was altered in Sm^D mutant, and the wild type strain JRW3 could be transformed to streptomycin dependent by Sm^D DNA.     

Variations in Strains of Streptomyces griseus Isolated from a Degenerating Streptomycin-Producing Culture

Streptomycin-resistant strains were isolated from a degenerated streptomycin-producing culture of Streptomyces griseus. From 250 resistant strains, 3 low, 2 intermediate, and 2 high potency strains were selected; these were compared in their morphological, cultural, physiological, and streptomycin-producing properties. Though no definite correlation between streptomycin production and the other properties could be obtained, the following correlations were considered as distinct differences among the low, intermediate, and high potency strains. (i) When streptomycin-producing ability degenerates, more submerged spore formation or fragmentation of mycelium into shorter filaments appears to occur. (ii) On agar medium, low and intermediate potency strains often show finely wrinkled growth; high potency strains do not show such characteristics. (iii) High potency strains excrete a distinct yellow soluble pigment on synthetic agar medium and on glucose-yeast extract agar, but low and intermediate potency strains show little or no ability to form this soluble pigment. (iv) In low and intermediate potency strains, inositol and arginine did not stimulate streptomycin production as they did in high potency strains. Streptamine showed some stimulating effect in the high potency strains and, in contrast, a depressive effect in intermediate potency strains, though streptidine showed a distinctly stimulating effect in all groups of strains employed.     

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.     

Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin

Orias, E. (University of California, Santa Barbara), and T. K. Gartner. Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin. J. Bacteriol. 91:2210-2215. 1966.-Streptomycin-induced suppression of amber and ochre rII mutants of phage T4 was studied in a streptomycin-sensitive strain of Escherichia coli and four nearly isogenic streptomycin-resistant derivatives of this strain, in the presence and in the absence of an ochre suppressor. Most of the 12 rII mutants tested were suppressed by streptomycin in the streptomycin-sensitive su(-) strain. This streptomycin-induced suppression in the su(-) strain was eliminated by the independent action of at least two of the four nonidentical mutations to streptomycin resistance. In two of the su(+)str-r strains, streptomycin markedly augmented the suppression caused by the ochre suppressor. In those su(-)str-r hosts in which significant streptomycin-induced suppression could be measured, the amber mutants were more suppressible than the ochre mutants.