Mechanism of streptomycin resistance in Leptospira biflexa strain Urawa

The mechanism of streptomycin resistance of Leptospira biflexa was investigated. A streptomycin-resistance mutant of Leptospira showed cross-resistance to dihydrostreptomycin but not to other antibiotics. Enzymatic inactivation of the drug could not be demonstrated in this mutant. Protein synthesis on the ribosomes from the mutant was insensitive to streptomycin. These results suggest that ribosomal resistance is the reason for streptomycin resistance in Leptospira biflexa.     

Chloroplast and Cytoplasmic Ribosomes of Euglena: Selective Binding of Dihydrostreptomycin to Chloroplast Ribosomes

Dihydrostreptomycin binds preferentially to chloroplast ribosomes of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim. The K(diss) for the wild-type chloroplast ribosome-dihydrostreptomycin complex is 2 x 10(-7) M, a value comparable with that found for the Escherichia coli ribosome-dihydrostreptomycin complex. Chloroplast ribosomes isolated from the streptomycin-resistant mutant Sm(1) (r)BNgL and cytoplasmic ribosomes from wild-type have a much lower affinity for the antibiotic. The K(diss) for the chloroplast ribosome-dihydrostreptomycin complex of Sm(1) (r) is 387 x 10(-7) M, and the value for the cytoplasmic ribosome-dihydrostreptomycin complex of the wild type is 1,400 x 10(-7) M. Streptomycin competes with dihydrostreptomycin for the chloroplast ribosome binding site, and preincubation of streptomycin with hydroxylamine prevents the binding of streptomycin to the chloroplast ribosome. These results indicate that the inhibition of chloroplast development and replication in Euglena by streptomycin and dihydrostreptomycin is related to the specific inhibition of protein synthesis on the chloroplast ribosomes of Euglena.     

Autostimulation of dihydrostreptomycin uptake in Bacillus subtilis

In Bacillus subtilis it was shown that the membrane potential (delta psi) has to reach a threshold value of -180 to -190 mV for efficient uptake of dihydrostreptomycin to occur. The magnitude of delta psi is raised above this threshold, and dihydrostreptomycin uptake greatly enhanced, not only by dihydrostreptomycin itself (autostimulation) and by other miscoding aminoglycoside antibiotics, but also by puromycin, bacitracin and N,N'-dicyclohexylcarbodiimide. Stimulation of uptake by dihydrostreptomycin or puromycin was dependent on a specific interference with ongoing protein synthesis. Thus, chloramphenicol prevented the stimulating effect of puromycin by lowering the magnitude of delta psi. Although normally severely antagonizing streptomycin accumulation, K+, as well as spermidine and putrescine, which are known to stabilize ribosomes, consequently enhanced autostimulation of dihydrostreptomycin uptake in a K+-retention mutant with impaired protein synthesis. It is suggested that miscoding aminoglycosides and puromycin both enhance dihydrostreptomycin uptake by increasing delta psi due to ion fluxes, which are themselves caused by a dramatic stimulation of intracellular proteolysis of faulty proteins.     

Intracellular Distribution of 3H-Dihydrostreptomycin in a Streptomycin-dependent Strain of Bacillus megaterium

The cells of a streptomycin-dependent strain of Bacillus megaterium took up only 2 to 5% of the dihydrostreptomycin present in the medium when grown in the minimum concentration of streptomycin required for growth. During growth in the presence of (3)H-dihydrostreptomycin, radioactivity was accumulated intracellularly in three forms, namely, unbound, loosely bound to the ribosomes (removable by dialysis), and tightly bound to the ribosomes (retained after prolonged dialysis). More radioactivity for a given amount of ribonucleic acid was bound by ribosomes attached to the cell membrane than by supernatant ribosomes. Of the nondialyzable radioactivity associated with isolated ribonucleic acid, 40 to 60% was solubilized by treatment with ribonuclease or by dilute alkaline hydrolysis.     

Biosynthesis of streptomycin enzymic oxidation of dihydrostreptomycin (6-phosphate) to streptomycin (6-phosphate) with a particulate fraction of Streptomyces griseus

Resting cells and to a greater extent permeabilized cells of Streptomyces griseus can oxidize dihydrostreptomycin to streptomycin. The dihydrostreptomycin oxidoreductase activity was localized in the 100 000 × g particulate fraction. Sucrose density gradient centrifugation of the particulate suspension gave a band at a density of 1.09 which consisted mainly of membrane vesicles. This fraction had high dihydrostreptomycin oxidoreductase activity. S. griseus protoplasts also contain high oxidoreductase activity. These data are consistent with localization of the enzyme in the cell membrane. Dihydrostreptomycin and dihydrostreptomycin 6-phosphate can both serve as substrates for the oxidoreductase, but the phosphate was the better substrate in the cell free system. Addition of cofactors was not required for the bound dihydrostreptomycin oxidoreductase. The electron acceptor for the oxidation is unknown. Oxidation of dihydrostreptomycin 6-phosphate to streptomycin 6-phosphate very probably represents the penultimate step in the biosynthesis of streptomycin.     

Paromomycin and dihydrostreptomycin binding to Escherichia coli ribosomes.

Paromomycin binds specifically to a single type of binding site on the 70-S streptomycin-sensitive Escherichia coli ribosome. This site is different from that of dihydrostreptomycin since paromomycin binds to streptomycin-resistant ribosomes and sine dihydrostreptomycin does not compete for paromomycin binding. Paromomycin binding, unlike dihydrostreptomycin binding, is independent of changes in ribosome concentration but influenced by magnesium ion concentration. Moreover, paromomycin does not bind to the 30-S subunit of the streptomycin-sensitive ribosome, except in the presence of dihydrostreptomycin, which probably induces the conformational changes necessary for a paromomycin binding site. This induction does not occur with streptomycin-resistant ribosomes. Neither antibiotic binds to the 50-S subunit. In general, binding of the one antibiotic increases the number of sites available for binding of the other. Both antibiotics exhibit marked non-specific binding at high antibiotic/ribosome ratios. Competition studies have enabled the classification of other aminoglycosides according to their ability to compete for the paromomycin and dihydrostreptomycin binding sites. Derivatives structurally related to paromomycin compete for its binding, the degree of competition being related to antibacterial activity, but do not compete for dihydrostreptomycin binding; they, on the contrary, increase the number of dihydrostreptomycin binding sites. Neither gentamicin nor kanamycin derivatives, which induce a high level of misreading, nor kasugamycin and spectinomycin, which do not induce misreading, compete for paromomycin or dihydrostreptomycin binding sites. Other sites may be involved in the binding of these aminoglycosides and in inducing misreading.     

Autoradiographic Evidence for the Impermeability of Mouse Peritoneal Macrophages to Tritiated Streptomycin

Cultured mouse peritoneal macrophages were found to be relatively impermeable to streptomycin. Based on radioactivity measurements and radioautographic evidence, macrophages were impermeable to tritiated dihydrostreptomycin for periods up to 20 hr of incubation. Little or no intracellular streptomycin could be detected even when incubation was carried out in the presence of therapeutic blood levels of carrier dihydrostreptomycin. When the cultured mouse macrophages were allowed to phagocytize staphylococci, yeast cells, or polystyrene latex particles in the presence of tritiated streptomycin, the impermeability of the cells to the antibiotic was not affected. These observations suggested that the process of phagocytosis does not facilitate the intracellular accumulation of streptomycin, as seems to be the case for the fixed phagocytic cells of the liver.     

Relation of aerobiosis and ionic strength to the uptake of dihydrostreptomycin in Escherichia coli

Aminoglycoside antibiotics exhibit a markedly reduced antibacterial activity under anaerobic conditions. Anaerobiosis or inhibitors of electron transport produced an extensive decrease in the uptake of dihydrostreptomycin in Escherichia coli K-12. Uptake of proline or putrescine were only slightly impaired under anaerobic conditions in the presence of glucose. Both the susceptibility to and the uptake of dihydrostreptomycin under anaerobic conditions were partially restored by addition of the alternative electron acceptor, nitrate. This stimulation required functional nitrate reductase activity. Abolition of uptake by 2,4-dinitrophenol under both aerobic and anaerobic conditions indicates that streptomycin uptake requires electron transport as well as a sufficient membrane potential. In addition, the initial rate of dihydrostreptomycin uptake was competitively and reversibly inhibited by added salts. The inhibition was relatively nonspecific with respect to the identity of salt added, being approximately dependent on the ionic strength. Although dihydrostreptomycin and polyamines mutually inhibited each other's uptake, several conditions (polyamine limitation, streptomycin uptake-deficient mutants) were found in which uptake of these two substrates was oppositely affected. Aminoglycosides thus do not appear to enter on one of the usual cellular transport systems, but perhaps utilize a component of the electron transport system.     

On the mechanism of translocation of dihydrostreptomycin across the bacterial cytoplasmic membrane

This review examines two mechanisms, the channel and the uniport, proposed to explain the rapid, energy-dependent (EDP-II) phase of transport of dihydrostreptomycin (and streptomycin) across the bacterial cytoplasmic membrane. Bioenergetic and kinetic predictions are made from these two mechanisms and compared with available experimental data. Both the above mechanisms would be expected to lead to reversible transport kinetics, and to observable uptake of dihydrostreptomycin by respiring cytoplasmic membrane vesicles. However, transport is kinetically irreversible and is not observed in membrane vesicles (although the membrane vesicle findings need further confirmation), so the author rejects the proposed channel and uniport mechanisms. A possible mechanism of dihydrostreptomycin transport that would be consistent with the above experimental data, would be one in which a chemical reaction occurred as an obligatory part of the translocation cycle. Such a mechanism could be classified as primary translocation. The author emphasizes that this hypothesis is put forward to stimulate further experimental testing; it is not proposed to be a definitive explanation of the mechanism of energy-dependent dihydrostreptomycin transport.     

Streptomycin sensitivity of ribosomes isolated from a streptomycin-producing Streptomyces griseus.

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.     

Streptomycin-induced formation of 70-S monosomes and oligomers in Chlamydomonas reinhardi

Under ionic conditions, where the 70 S ribosomes but not the 80 S ribosomes partly dissociate into the subunits, in three mutants of Chlamydomonas reinhardi streptomycin causes in vivo at first an increase, later a decrease of the 70 S ribosome fraction. This behaviour can be explained, if streptomycin acts on the ribosome cycle of the organelle ribosomes of eukaryotes in the same way as on the ribosome cycle of E. coli.Streptomycin also induces the formation of dimers and oligomers from 80 S cytoplasmic ribosomes. The kinetics of this formation is similar to that of the 70 S ribosomes. However, this effect of streptomycin does not seem to influence the functional capacity of the 80 S ribosomes.     

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.     

Fluorescence studies on a streptomycin-induced conformational change in ribosomes which correlates with misreading

The fluorescent reagent N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) was employed to detect and study the previously reported conformational change in the Escherichia coli ribosome induced by streptomycin. Labeling of ribosomes with this probe, which results in the derivatization of proteins S18 and L31', described earlier, inhibits neither their ribosomal protein synthesizing nor misreading ability. To calculate the amount of streptomycin bound to the ribosome, we determined the K'D for streptomycin, which is 0.24 micron, indicating that under our conditions, bound streptomycin/ribosome molar ratios are low, not in excess of 1. Under these conditions, streptomycin addition induces fluorescence quenching by 15% but does not affect streptomycin-resistant ribosomes. Maximal misreading occurs at these same ratios. Removal of AEDANS-L31' from the ribosomes drastically reduces streptomycin-induced quenching indicating the involvement of the environment of this protein in streptomycin action. The finding that streptomycin decreases AEDANS-L31' affinity for the ribosome supports this view. Streptomycin has been shown to bind to the 30 S subunit protein S4 while the 50 S protein L31' has been shown to be localized at the subunit interface. Thus, the observation that streptomycin influences this 50 S subunit protein L31', combined with the tight correlation between the effects of streptomycin on quenching and on misreading, strongly suggests that this antibiotic induces a conformational change at the subunit interface of the ribosome, and that this results in misreading. Polyuridylic acid also induces a conformational change in the ribosome but the polynucleotide and streptomycin seem to act independently. Streptomycin-resistant ribosomes, which undergo neither streptomycin-induced fluorescence nor streptomycin-induced misreading, are resistant to misreading induced by high Mg2+ as well.     

Interaction of aminoglycosides and the other antibiotic on selected bacterial strains

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.     

Mechanism of Resistance to Antibiotic Synergism in Enterococci

Enterococci exhibit two types of resistance to streptomycin. Moderately high-level resistance is observed in most naturally occurring strains and can be overcome by simultaneous exposure to penicillin. In addition, very high-level resistance is found in those strains against which penicillin plus streptomycin fail to produce synergism in vitro. To study the mechanism of streptomycin resistance in enterococci, ribosomes from a wild-type strain and from a highly streptomycin-resistant mutant were isolated, characterized, and studied in an in vitro amino acid incorporation system. The ribosomes from the organism with moderately high-level streptomycin resistance were sensitive to streptomycin in vitro, suggesting that this type of resistance is caused by failure of streptomycin to reach the ribosomes. Very high-level resistance (and lack of penicillin-streptomycin synergism), on the other hand, appears to be due to ribosomally mediated streptomycin resistance.     

Conformational changes induced in Escherichia coli ribosomes by streptomycin. A spin label study

A spin-labeling study, with a nitroxide analog of N-ethylmaleimide, was carried out to investigate the effect of streptomycin on the conformation of ribosomes from E. coli. Spin-labeling of 70S ribosomes and 30S subunits was performed before and after the addition of streptomycin. Streptomycin has no effect if added after labeling, which confirms that the blocking of sulfhydryl groups of ribosomal proteins interferes with the binding of the antibiotic. However, when the antibiotic is added before labeling, there is a decrease in the rotational correlation time of the labels. This result indicates that the binding of streptomycin to ribosomes loosens the ribosomal structure.     

Reduction of Aliphatic α-Hydroxycarbonyl Compounds

The reduction of streptomycin with aluminum amalgam to dihydrodesoxystreptomycin (α-hydroxyaldehyde to monohydric alcohol, in its streptose moiety) is reproduced at dropping mercury electrode and at controlled-potential mercury pool electrode. The accompanying formation of dihydrostreptomycin, of which α-hydroxyl group is intact, depends upon the cathode potential and pH. Electrolysis at a potential corresponding to the relatively negative part of polarographic reduction wave and at relatively higher pH favors the formation of dihydro derivative and vice versa. This explains, from polarographic standpoint, the characteristic mode of reduction with aluminum amalgam. Electroreduction of streptomycin is applicable for the preparation of dihydrodesoxyderivative but not so for that of dihydro derivative.     

Suboptimal growth with hyper-accurate ribosomes

Mutant bacteria with hyperaccurate ribosomes support their excessive accuracy of translation in vitro by dissipating 1.5 to 2.5 cognate ternary complexes per peptide bond formed. This is to be compared with a dissipation rate close to 1.1 for wild-type ribosomes. Here, we have tested the hypothesis that a corresponding loss of translational efficiency in vivo would lower the growth rate of the mutants. Such a growth inhibitory effect would explain why the lower accuracy of wild-type ribosomes is more fit. Our data show that as expected the of the hyperaccurate mutants is smaller than that of wild-type bacteria. In contrast, during glucose-limited growth in chemostats there is not the same simple correlation between growth yield and ribosomal efficiency for the hyperaccurate mutants.Abbreviations SmR streptomycin resistant - SmP streptomycin pseudodependent - SmD streptomycin dependent - EF-Tu elongation factor Tu - EF-Ts elongation factor Ts     

[3H] dihydrostreptomycin accumulation and binding to ribosomes in Rhizobium mutants with different levels of streptomycin resistance.

Rhizobium trifolii B1, a symbiotic nitrogen fixer, is sensitive to streptomycin (10 microgram/ml) and spontaneously produces spheroplast-like forms during cultivation. Streptomycin-resistant mutants selected with high doses of antibiotic (1,000 microgram/ml) showed pleiotropic changes, including loss of spheroplast formation and infectivity to plants, whereas mutants selected with low doses of streptomycin (10 to 100 microgram/ml) retained properties of parent strain B1 (I. Zelazna-Kowalska, Acta Microbiol. Pol., in press). The present studies revealed that strain B1 and its mutant with a high level of streptomycin resistance, B1 strH, accumulated the antibiotic at similar rates. Mutant B1 strL, with a low level of streptomycin resistance (up to 100 microgram/ml), accumulated the antibiotic at a lower rate. Ribosomes isolated from strains B1 and B2 strL bound [3H]dihydrostreptomycin, whereas those from strain B1 strH did not. These observations indicate that, in R. trifolii B1, mutation to a high level of streptomycin resistance affects ribosomal structure, whereas low-level resistance involves a change in membrane permeability.     

Resistance and Sensitivity of Chloroplast Ribosomes to Streptomycin in Mutants of Chlamydomonas reinhardi

• 1 In a mendelian (sr₃) and an uniparental (sr₃₅) streptomycin resistant mutant of Chlamydomonas reinhardi the influence of streptomycin on protein synthesis on the chloroplast and cytoplasmic ribosomes was investigated in vitro. Hetero-, mixo- and phototrophic agar cultures and heterotrophic liquid cultures were used.
• 2 Protein synthesis on the cytoplasmic ribosomes, measured by the activity of glyceraldehyde-3-phosphate: NADP dehydrogenase (EC 1.2.1.9), was not inhibited, but rather stimulated by streptomycin.
• 3 Protein synthesis on the chloroplast ribosomes of sr₃, measured by the activity of ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39), was greatly inhibited by streptomycin, especially in hetero- and mixotrophic cultures. In sr₃₅ the chloroplast ribosomes were resistant to streptomycin.
• 4 Heterotrophically grown cultures of sr₃ and of a streptomycin-sensitive strain are yellow in the presence of streptomycin and form no or only reduced thylakoids on solid media. But 70-S organelle-ribosomes are present in a normal amount.
• 5 The relationship between chloroplast protein synthesis and thylakoid formation is discussed.