An improved procedure for protoplast microelectrofusion and culture of Nicotiana tabacum intraspecific somatic hybrids: plant regeneration and initial proofs on organelle segregation

Somatic hybrids were produced between haploid Nicotiana tabacum L. cv. Petite Havana (wild type) and haploid streptomycin resistant (SR1) mutant by an improved version of microelectrofusion of preselected pairs of protoplasts and the culture of fusion products in a nurse culture. Resistance of diploid plants regenerated from 20 somatic hybrid clones was tested at low concentration of streptomycin in the light as well as at high concentrations of streptomycin in the dark. In two independent hybrid lines, plants resistant in the light but sensitive in the dark were found. The existence of this plant type indicates a segregation of chloroplasts and mitocondria in somatic hybrid clones. It is suggested that microelectrofusion of preselected pairs of protoplasts combined with a reliable nurse culture might be a good technique for controlled somatic hybridization, cell reconstitution and partial gene transfer to different plant species. It might also be used to follow and analyse organelle segregation in somatic hybrid clones. The possibility that mitochondria might be resistant to streptomycin in the SR1 mutant is also discussed.     

The biosynthesis of streptomycin. The origin of the C-formyl group of streptose

1. The biosynthesis of streptomycin in Streptomyces griseus has been studied by adding d-[3,4-(14)C(2)]glucose or d-[1,3-(14)C(2)]glucose to the growth medium and degrading the streptomycin produced. 2. The results suggest that the C-3' branch carbon atom of l-streptose arises from C-3 of d-glucose. 3. The mechanism of biosynthesis of streptose from glucose is discussed. It probably involves an intramolecular rearrangement of a 6-deoxy-4-oxyhexose derivative, and it is suggested that the nucleoside diphosphate sugar derivative hitherto recognized as an intermediate in the biosynthesis of l-rhamnose might participate in such a rearrangement.     

抗生素对BALB/C小鼠免疫机制的影响

本文给普通BALB/C小鼠口服不同剂量的链霉素、新霉素干扰动物肠道菌群平衡状态,初步研究了肠道菌群平衡紊乱对动物迟发型超敏反应、脾脏抗体形成细胞数、外周血白细胞化功能、腹腔巨噬细胞吞噬活性的影响,以初步探讨普通动物中正常菌群与免疫机制间的微生态关系。结果表明,口服两种抗生素破坏动物正常菌群平衡均可导致机体免疫机能水平降低;免疫机能降低程度与抗生素抗菌谱、药物剂量及用药时间有密切关系。提示,口服抗生素引起机体肠道正常菌群平衡紊乱是导致机体免疫机能下降的重要原因;正常菌群与机体保持稳定的平衡状态对机体免疫机能的正常发挥是必不可少的。     

Antibiotic sensitivity of plague microbe strains from foreign countries]

The method of serial dilutions on the Hottinger agar was applied to comparative assay of antibiotic sensitivity in 50 strains of the plague microbe isolated abroad and in 5 strains isolated in the plague focus in the Central Caucasus. The antibiotics used in the assay were the following: streptomycin, gentamicin, doxycycline, monomycin, kanamycin, tetracycline, erythromycin, ristomycin, lincomycin and polymyxin M. Irrespective of the origin, all the isolates were resistant to erythromycin, lincomycin and polymyxin M. The levels of the sensitivity to the other antibiotics were different. The data serve as a ground for the statement that there is no tendency to development of antibiotic resistance in the plague microbe in patients treated with high doses of the antibiotics and mainly streptomycin. Along with streptomycin, such antibiotics as gentamicin, tetracycline, doxycycline and kanamycin are useful in the therapy of plague and require further investigation.     

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.     

The mechanism of resistance to streptomycin inEscherichia coli. Functional analysis of the permeability barrier of cells harbouring the Rldrd-19Km plasmid

The mechanism of phenotypically altered SM resistance in mutants of Escherichia coli JC5455 (Rldrd-19Km-) lrs and JC5455 (pON5300) was compared with that of the standard strain JC5455 (Rldrd-19Km-). On analyzing the membrane polypeptides in polyacrylamide gel both mutants were found to possess a protein spectrum different from that of ths standard strain. Transport of D-xylose and L-arginine was the same in all strains, transport of L-proline was decreased in JC5455 (pON5300) which may indicate a mutational interference with energy metabolism. The basic uptake of dihydrostreptomycin was the same in all strains but there were differences after preincubation of cells with streptomycin or glucose. The increased resistance of JC5455 (Rldrd-19Km-) lrs may be due to observed quantitative differences in membrane polypeptides that might play a role in the binding and functional expression of aminoglycoside-3'adenylyl transferase which modifies streptomycin. The increased sensitivity toward streptomycin in JC5455 (pON5300) can be explained by a mutation due to N-methyl-N'-nitro-N-nitrosoguanidine in the host cell since this change of sensitivity to streptomycin could not be transferred by transformation into a nonmutagenized strain. The coincidence of inducibility of increased transport of streptomycin by this antibiotic and the altered frequency of reversion to high levels of streptomycin resistance in JC5455 (pON5300) and in the transformant JC5455 (pON5302) may indicate that the altered reversibility toward phenotypically high resistance to streptomycin is a property of pON5300 and is transferred by transformation.     

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.     

Isolation of mutants of Escherichia coli uncoupled in oxidative phosphorylation using hypersensitivity to streptomycin.

Mutants of Escherichia coli, harbouring the uncA401 or uncB402 alleles, were found to take up streptomycin more rapidly than the coupled parent strains. The increased rate of uptake results in greater sensitivity of the uncoupled strains, compared to the parent strains, to low concentrations of streptomycin. Studies with unc+ revertants showed that hypersensitivity to streptomycin is attributable to the mutation causing uncoupling. The uptake of streptomycin in an unc- strain is abolished by addition of the chemical uncoupler carbonylcyanide m-chlorophenylhydrazone. The phenotype of hypersensitivity to streptomycin can be used as a selection procedure for the isolation of uncoupled strains. In an experiment reported here, nine out of 12 strains isolated as being sensitive to streptomycin (at 2.5 micrograms/ml), were found to be unable to grow on succinate as a sole source of carbon. Five of the nine Suc- strains were found to be uncoupled in oxidative phosphorylation, and two of the five uncoupled strains lacked Mg2+-ATPase activity. The mutations causing uncoupling were cotransducible with the ilv genes.     

Spatial structure increases the benefits of antibiotic production in Streptomyces*

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.     

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.     

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.     

Mutation in Saccharomyces cerevisiae Conferring Streptomycin and Cold Sensitivity by Affecting Ribosome Formation and Function

A cold-sensitive, streptomycin-sensitive mutant of Saccharomyces cerevisiae accumulates a 28S ribonucleoprotein particle when grown at low temperature. This particle contains 17S ribosomal ribonculeic acid which is degraded when exposed to ribonuclease. The particle does not serve as a precursor to 60 and 40S ribosomal subunits nor is it turned over when growth is allowed to resume at the permissive temperature; rather it is only diluted by growth. That streptomycin sensitivity (allelic with cold sensitivity) is ribosomal is evidenced by the inhibition of protein synthesis in vitro by streptomycin and the binding of labeled streptomycin to the mutant but not the parental 40S ribosomal subunit.     

ANTIFUNGAL ACTION OF STREPTOMYCIN-COPPER CHELATE AGAINST PHYTOPHTHORA INFESTANS ON TOMATO (LYCOPERSICON ESCULENTUM)

The antifungal protection afforded by streptomycin-copper chelate in vivo in greenhouse tests against Phytophthora infestans on tomato was found to be of the order of six times greater than that of streptomycin sulphate. The copper chelate also showed much greater resistance to simulated rainfall. Similar tests showed that streptomycin-copper chelate (in terms of streptomycin content) was from 2 1/2 to 5 times more effective as an antifungal prophylactic spray than zinc ethylene-bis(dithiocarbamate) (zineb), and some fifty times more active than copper oxychloride formulated as a commercial 50% wettable powder. These three materials showed roughly similar resistance to weathering by 'rainfall' and it was assumed therefore that 250 i.u./ml. streptomycin as streptomycin-copper chelate might be expected to give the same degree of protection against Phytophthora infestans in the field as would the usual field rates of usage of zineb and copper oxychloride.     

Bleaching euglena gracilis with antihistamines and streptomycin-type antibiotics

Summary Kanamycin, paromomycin, and neomycin, like streptomycin, permanently bleach Euglena gracilis. This effect, along with general toxicity, is opposed by Mg, histidine or a combination of pantothenate, nicotinic acid, and threonine. Such opposition is thought to be peripheral effects centered on uptake and transport.Certain antihistamines, notably tripelennamine, methapyrilene, and pyrilamine induce permanent bleaching. Diphenylhydramine and phenindamine induced temporary bleaching. Doxylamine, antazoline, pyrathiazine, pheniramine, prophenpyridamine, and promethazine did not bleach when tested up to inhibitory concentrations.Bleaching by streptomycin+heat was additive, not synergistic.The evidence at hand for the mode of action of ultraviolet irradiation, streptomycin antibiotics, and erythromycin suggests, as a working hypothesis, that the common factor may be interference with nucleic acid metabolism; the common factor in bleaching by antibiotics may be simultaneous provision of a molecular grouping favoring uptake and transport of the active moiety, which in turn may be rare sugars interfering with ribose and desoxyribose in the photosynthetic apparatus.New antibiotics of the streptomycin family might well be screened for bleaching activity as a possible index of damage to the 8th cranial nerve, for so far the correlation is excellent for this class of antibiotic.This paper is dedicated to Professor Dr. E. G. Pringsheim on the occasion of his 80th birthday.     

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.     

Streptomycin resistance in chloramphenicol-producing strains of Streptomyces species 3022a.

Resistance to low (5 mug/ml) concentrations of streptomycin in agar media was not inherited by all of the surviving population. Outgrowth of cultures in liquid media supplemented with the antibiotic depended upon inoculum size. Antibiotic titers in the supplemented cultures decreased during incubation, and an inactive radioactive product was detected when [14C] streptomycin was used. This low-level resistance is, therefore, attributed to enzymic inactivation of the antibiotic. Growth 10 mug/ml or higher concentrations of streptomycin on agar media was due to selection of resistant variants present in the parent strain. A range of such variants existed, decreasing in frequency as their degree of resistance increased. Examination of one that was resistant to moderate concentrations of streptomycin, (25 mug/ml) and a second that was resistant to high (100 mug/ml) concentrations of streptomycin suggested that both possed ribosomes which had lower affinity for the antibiotic than those of the parent strain, and that tolerance to high levels of streptomycin was due to a resistant ribosomal system for protein biosynthesis.     

Transfer of class 1 integron-mediated antibiotic resistance genes from shiga toxin-producing Escherichia coli to a susceptible E. coli K-12 strain in storm water and bovine feces

Transfer of class 1 integron-mediated antibiotic resistance genes has been demonstrated under laboratory conditions. However, there is no information concerning the transfer of these genes in an agricultural environment. The present study sought to determine if integron-mediated streptomycin and sulfisoxazole resistance genes could be transferred from Shiga toxin-producing Escherichia coli (STEC) strains 6-20 (O157:H7) and 7-63 (O111:H8) to the susceptible strain E. coli K-12 MG1655 in bovine feces (pH 5.5, 6.0, or 6.5) and storm water (pH 5, 6, 7, or 8) at 4, 15, and 28 degrees C, which are average seasonal temperatures for winter, spring-fall, and summer, respectively, in the Griffin, GA, area. The results indicated that at 28 degrees C, the integron-mediated antibiotic resistance genes were transferred from both of the STEC donors in bovine feces. Higher conjugation efficiencies were, however, observed in the conjugation experiments involving STEC strain 6-20. In storm water, the resistance genes were transferred only from STEC strain 6-20. Greater numbers of transconjugants were recovered in the conjugation experiments performed with pH 6.5 bovine feces and with pH 7 storm water. Antibiotic susceptibility tests confirmed the transfer of integron-mediated streptomycin resistance and sulfisoxazole resistance, as well as the transfer of non-integron-mediated oxytetracycline resistance and tetracycline resistance in the transconjugant cells. These results suggest that the antibiotic resistance genes in STEC could serve as a source of antibiotic resistance genes disseminated via conjugation to susceptible cells of other E. coli strains in an agricultural environment.     

Modulation of RNA function by aminoglycoside antibiotics

One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.     

A-factor and streptomycin biosynthesis inStreptomyces griseus

Accumulating data have shown that the metabolites with a -butyrolactone ring functions as an autoregulatory factor or a microbial hormone for the expression of various phenotypes not only in a variety ofStreptomyces spp. but also in the distantly related bacteria. A-factor, as a representative of this type of autoregulators, triggers streptomycin biosynthesis and cellular differentiation inStreptomyces griseus. A model for the A-factor regulatory cascade on the basis of recent work is as follows. At an early step in the A-factor regulatory relay, the positive A-factor signal is first received by an A-factor receptor protein that is comparable in every aspect to eukaryotic hormone receptors, and then, via one or more regulatory steps, transmitted to an A-factor-responsive protein that binds to the upstream activation sequence of thestrR gene, a regulatory gene in the streptomycin biosynthetic gene cluster. The StrR protein thus induced appears to activate the other streptomycin biosynthetic genes. This review summarizes the characteristics of A-factor as a microbial hormone and the A-factor regulatory relay leading to streptomycin production.