Ribosomal resistance of an istamycin producer, Streptomycestenjimariensis, to aminoglycoside antibiotics

$\text{Streptomyces}\text{tenjimariensis}$ SS-939 was resistant to its own aminoglycoside antibiotics, istamycins, as well as kanamycin A, neamine, ribostamycin and butirosin A, but was susceptible to neomycin B, lividomycin A and streptomycin. This resistance to these antibiotics was found to be due to ribosomes of the strain.     

A genetic and biochemical study of streptomycin- and spectinomycin-resistance in Salmonella typhimurium

Summary In Salmonella typhimurium, streptomycin resistance can occur by mutation at the strA or the strB mutants have altered ribosomes which are refractory to the drug in cell-free amino acid incorporation systems, and in ³H-dihydrostreptomycin binding studies. StrB mutants, unlike the strA mutants, are resistant to several aminoglycoside antibiotics and resistance is not due to a mutational change in the cell's protein synthetic machinery. Spectinomycin resistant mutants of S. typhimurium also fall into two classes, only one of which is ribosomal in mechanism. The spcA and spcB loci are closely linked to strA, aroC, and argG on the S. typhimurium linkage map.     

Ribosomal subunits affected by antibiotic resistance mutations at seven chloroplast loci in Chlamydomonas reinhardtii

Summary Mutations at seven recombinationally distinct chloroplast loci confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii. Assays of polynucleotide-directed amino acid incorporation by ribosomes reconstituted from mutant and wild type subunits demonstrate that streptomycin, neamine/kanamycin and spectinomycin resistance mutations specifically affect the small ribosomal subunit, whereas mutations to erythromycin resistance affect the large subunit. Although in each case the subunit site of antibiotic resistance is the same as that observed in analogous mutations in Escherichia coli, the number of loci conferring resistance to a given antibiotic differs in the two organisms. We have previously shown that streptomycin resistance mutations in Chlamydomonas map at five discrete loci (one nuclear and four chloroplast), and that mutations to neamine/kanamycin and spectinomycin resistance appear to define a single chloroplast locus. Results presented here confirm our previous report that all chloroplast erythromycin resistance mutations isolated to date fall into two recombinationally distinct loci, and indicate that mutants at one of these loci may be further divided on the basis of their level of cross resistance to other macrolide antibiotics.     

Antibiotic sensitivity of the ribosomes of the ultra-thermophilic archaebacterium Pyrococcus woesei

The effect of several antibiotics on polypeptide synthesis and translational accuracy by ribosomes from the ultra-thermophilic archaebacterium Pyrococcus woesei was investigated. Pyrococcus was found to be unique among the sulfur-dependent extreme thermophiles in being sensitive to the miscoding-inducing action of aminoglycoside antibiotics (except streptomycin). On the whole, the antibiotic sensitivity patterns indicate phylogenetic closeness of Pyrococcus to the methanogenic-halophilic branch of the archaebacterial tree.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

On the interaction between heparin and some aminoglycoside antibiotics

An interaction between the aminoglycoside antibiotics and heparin wherein charge transfer complexes are formed has been investigated to determine the degree of inhibition of antibacterial function of the antibiotic in the complexed form.Minimum inhibitory concentration (MIC) values have been obtained for the action of the aminoclycoside antibiotics tobramycin, gentamicin, amikacin, kanamycin, and streptomycin, on a sensitive strain ofE. coli. Growth curves ofE. coli determined at concentrations of these antibiotics just below the MIC demonstrated significant lengthening of the lag phase relative to control growth curves generated in the absence of antibiotic. Heparin (1 U ml^–1 and 10 U ml^–1) had no effect on control growth curves; however, particularly at the higher concentration, it reduced the effect on the lag phase produced by the aminoglycoside antibiotics. Thus kanamycin, gentamicin, and tobramycin were most affected, while amikacin and streptomycin were least affected. The rank order of inhibition of antibiotic activity by interaction with heparin was in qualitative agreement with previously published figures for the degree of complexation between antibiotics and heparin.     

Antibiotic stress-induced modulation of the endoribonucleolytic activity of RNase III and RNase G confers resistance to aminoglycoside antibiotics in Escherichia coli

Here, we report a resistance mechanism that is induced through the modulation of 16S ribosomal RNA (rRNA) processing on the exposure of Escherichia coli cells to aminoglycoside antibiotics. We observed decreased expression levels of RNase G associated with increased RNase III activity on rng mRNA in a subgroup of E. coli isolates that transiently acquired resistance to low levels of kanamycin or streptomycin. Analyses of 16S rRNA from the aminoglycoside-resistant E. coli cells, in addition to mutagenesis studies, demonstrated that the accumulation of 16S rRNA precursors containing 3–8 extra nucleotides at the 5’ terminus, which results from incomplete processing by RNase G, is responsible for the observed aminoglycoside resistance. Chemical protection, mass spectrometry analysis and cell-free translation assays revealed that the ribosomes from rng-deleted E. coli have decreased binding capacity for, and diminished sensitivity to, streptomycin and neomycin, compared with wild-type cells. It was observed that the deletion of rng had similar effects in Salmonella enterica serovar Typhimurium strain SL1344. Our findings suggest that modulation of the endoribonucleolytic activity of RNase III and RNase G constitutes a previously uncharacterized regulatory pathway for adaptive resistance in E. coli and related gram-negative bacteria to aminoglycoside antibiotics.     

Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples

Antibiotic resistance in 40 Staphylococcus aureus clinical isolates from 110 diabetic patients (36%) was evaluated. Of these, 32 (80%) of the isolates showed multidrug-resistance to more than eight antibiotics and 35% isolates were found to be methicillin resistant S. aureus (MRSA). All 40 S. aureus strains (100%) screened from diabetic clinical specimens were resistant to penicillin, 63% to ampicillin, 55% to streptomycin, 50% to tetracycline and 50% to gentamicin. Where as low resistance rate was observed to ciprofloxacin (20%) and rifampicin (8%). In contrast, all (100%) S. aureus strains recorded susceptibility to teicoplanin, which was followed by vancomycin (95%). Genotypical examination revealed that 80% of the aminoglycoside resistant S. aureus (ARSA) have aminoglycoside modifying enzyme (AME) coding genes; however, 20% of ARSA which showed non-AME mediated (adaptive) aminoglycoside resistance lacked these genes in their genome. In contrast all MRSA isolates possessed mecA, femA genetic determinants in their genome.

     

Effect of aminoglycoside antibiotics on the autolytic enzyme of Streptomyces griseus

The isolated cell wall of Streptomyces griseus 52–1 strain labelled with fluorescein isothiocyanate (FITC) and containing wall-bound autolytic enzyme was lysed as a function of different cations. The autolysis was accelerated by aminoglycoside antibiotics (streptomycin and the structurally closely related neomycin) which have a polycationic character. Since this strain is a streptomycin producer it is suggested that streptomycin may have a regulatory function on autolysis.     

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.

     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase

Summary The wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.Abbreviations bp base pairs - EDTA ethylenediaminetetraacetic acid - kb kilobases' - TES n-tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid     

Aminoglycoside sensitivity of ribosomes from the archaebacterium Methanococcus vannielii: Structure-activity relationship

Abstract The effect of about 20 aminoglycoside antibiotics comprising compounds with specific 70S or with 70S plus 80S activity on polypeptide synthesis and translational misreading by ribosomes from the archaebacterium Methanococcus vannielii was investigated. A clear structure-activity relationship was found: sensitivity was observed only to the class of 4,5-disubstituted deoxystreptamine compounds, with neomycin and paromomycin as the most active ones. The streptomycin class aminoglycosides were completely inactive whereas the gentamicin group compounds solely affected misreading and only at high concentrations. Viomycin, a specific inhibitor of the translocation reaction at the eubacterial ribosome which competes with binding of 2-deoxystreptamine aminoglycosides was inactive as well.     

Kinetic mechanism of streptomycin adenylyltransferase from a recombinant<Emphasis Type="BoldItalic">Escherichia coli</Emphasis>

Bacterial resistance to the aminoglycoside antibiotics is manifested primarily by enzymic modification of these drugs. One important mechanism of streptomycin modification is through ATP-dependent O-adenylation, catalyzed by streptomycin adenylyltransferase. Initial velocity patterns deduced from steady state kinetics indicate a sequential mechanism. Dead-end inhibition by tobramycin and neomycin is non-competitive versus streptomycin and uncompetitive versus ATP, indicative of ordered substrate binding where ATP binds first and then streptomycin. These results surmise that streptomycin adenylyltransferase follows an ordered, sequential kinetic mechanism in which one substrate (ATP) binds prior to the antibiotic and pyrophosphate is released prior to formation of AMP-streptomycin.     

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin.Biochemical analysis shows that the mutants each have two modified 30S ribosomal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).     

Unique antibiotic sensitivity of an in vitro polypeptide synthesis system from the archaebacterium Thermoplasma acidophilum. Phylogenetic implications

Summary The susceptibility of Thermoplasma acidophilum (an extremely acidophilic, moderately thermophilic, wall-less sulphur-oxidizing archaebacterium) to 50 ribosome-specific inhibitors of polypeptide elongation was surveyed using efficient poly(U)-and poly(UG)-directed cell-free systems and comparable reference systems derived from eubacterial (Bacillus stearothermophilus, Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) species. Under optimum temperature (58° C) and ionic conditions for polypeptide synthesis Thermoplasma ribosomes are only sensitive to the 70 S/80 S ribosome-directed aminoglycoside neomycin, and to five 80 S ribosome-directed inhibitors all of which (-sarcin, mitogillin, restrictocin, dianthin and gelonin) impair the functioning of the large (60 S) ribosomal subunit. Sensitivity of the three structurally related compounds -sarcin, mitogillin and restrictocin and susceptibility to neomycin place Thermoplasma ribosomes between those of Sulfolobus solfataricus (only sensitive to -sarcin) and Methanococcus vannielli (sensitive to -sarcin, mitogillin, restrictocin and neomycin but also affected by a variety of 70 S ribosome-directed drugs). The phylogenetic significance of the greatly diversified antibiotic sensitivity spectra displayed by archaebacteria in general, as opposed to the uniform ones exhibited by eubacteria and eukaryotes, is discussed.     

Non-target host risk assessment of the idiobiont parasitoid Bracon celer (Hymenoptera: Braconidae) for biological control of olive fruit fly in California

Non-target risk posed by an African parasitoid, Bracon celer Szépligeti (Hymenoptera: Braconidae), was assessed for a classical biological control program against olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae: Dacinae), in California, USA. Behavioral and reproductive responses to non-target tephritid species were tested with beneficial (Chaetorellia succinea [Costa] and Parafreutreta regalis Munro) (Tephritidae: Tephritinae) and native (Rhagoletis fausta [Osten Sacken]) (Tephritidae: Trypetinae) fruit fly species in successive no-choice and choice experiments under close confinement in quarantine. Non-target host-plant substrates exposed to B. celer were yellow-starthistle flower heads containing C. succinea, Cape ivy stem galls containing P. regalis, and bitter-cherry fruit containing R. fausta. The parasitoid probed all three infested non-target plant substrates, but significantly less than olives infested with B. oleae. It produced offspring from P. regalis in Cape ivy stem galls, but appeared unable to penetrate yellow-starthistle flower heads with its ovipositor. Bracon celer killed some B. oleae and R. fausta larvae without parasitism. Reproduction on P. regalis indicates that B. celer has a broad physiological host range, which, combined with the parasitoid's acceptance of all three host-plant substrates, indicates a strong potential to negatively impact non-target species. Although physical and temporal barriers to host attack may reduce risk to most non-target tephritids by B. celer in California, the parasitoid should not be released due to its risk of harming the beneficial P. regalis. Release of P. regalis is still under consideration, however, and final risk assessment should depend on whether the fly proves useful for weed control.     

Intralineage diversity of archaebacterial ribosomes: A dichotomy of ribosome features separates sulfur-dependent archaebacteria and methanococcaceae from other archaebacterial taxa

Summary The aggregate masses and relative protein contents of eubacterial and archaebacterial ribosomes have been estimated from the buoyant densities of the ribosomal subunits and the anhydrous weights of the rRNA species. In contrast to the situation in eubacteria, archaebacterial ribosomes fall into two size classes that differ only in the relative abundances of their protein moieties. One class comprises eubacterial-sized particles (2.3-megadalton (Mdal) monomer; 1.5-Mdal and 0.8-Mdal large and small subunits, respectively) having a eubacterial composition of roughly one-third protein and two-thirds RNA. The other class comprises ribosomes heavier than those of eubacteria (3-Mdal monomer; 1.8-Mdal and 1.2-Mdal subunits) and having the same protein/RNA ratio as eukaryotic ribosomes (55% and 50% protein for the small and large subunits, respectively). Eubacterialsized ribosomes are harbored by extreme halophiles and all methanogens but the Methanococcaceae. Ribosomes heavier than those of eubacteria are found in the Methanococcaceae and all sulfur-dependent thermophiles. The data indicate that a change in ribosome structure occurred within the methanogen branch; therefore, although ribosome composition is distributed in archaebacteria, its distribution does not break them into two separate kingdoms: The Methanococcaceae and Methanobacteriaceae are related to each other far more closely than either is to the sulfur-dependent thermophiles, and the root of the archaebacterial tree definitely does not lie betweenMethanobacterium andMethanococcus. We surmise that ribosomes larger than those of eubacteria represent a more rudimentary organelle structure that became fixed owing to nonparallel evolution of the translational machinery in archaebacteria.     

Ribosomes of the extremely thermophilic eubacterium Thermotoga maritima are uniquely insensitive to the miscoding-inducing action of aminoglycoside antibiotics.

Poly(U)- and poly(UG)-programmed cell-free systems were developed from the extreme thermophilic, anaerobic eubacterium Thermotoga maritima, and their susceptibility to aminoglycoside and other antibiotics was assayed at a temperature (75 degrees C) close to the physiological optimum (80 degrees C) for cell growth and in vitro polypeptide synthesis, using a Bacillus stearothermophilus system as the reference. The synthetic capacity of the Thermotoga assay mixture was abolished by the eubacterium-targeted drugs chloramphenicol, thiostrepton, and kirromycin. However, streptomycin, the disubstituted 2-deoxystreptamines (kanamycin, gentamicin, neomycin, and paromomycin), and the monosubstituted 2-deoxystreptamine (hygromycin) all failed to promote translational misreading of poly(U) on Thermotoga ribosomes; they also failed to block polyphenylalanine synthesis at a low (less than 10(-4) M) concentration and did not inhibit Thermotoga cell growth at a high (10 micrograms/ml) concentration even though Thermotoga ribosomes possess the 16S rRNA sequences required for aminoglycoside action. In contrast to the other eubacteria, Thermotoga elongation factor G was also refractory to the steroid inhibitor of peptidyl-tRNA translocation fusidic acid.     

tmRNA of Streptomyces collinus and Streptomyces griseus during the growth and in the presence of antibiotics

Streptomycetes are soil microorganisms with the potential to produce a broad spectrum of secondary metabolities. The production of antibiotics is accompanied by a decrease in protein synthesis, which raises the question of how these bacteria survived the transition from the primary to the secondary metabolism. Translating ribosomes incapable to properly elongate or terminate polypeptide chain activate bacterial trans‐translation system. Abundance and stability of the tmRNA during growth of Streptomyces collinus and Streptomyces griseus producing kirromycin and streptomycin, respectively, was analysed. The level of tmRNA is mostly proportional to the activity of the translational system. We demonstrate that the addition of sub‐inhibitory concentrations of produced antibiotics to the cultures from the beginning of the exponential phase of growth leads to an increase in tmRNA levels and to an incorporation of amino acids into the tag‐peptides at trans‐translation of stalled ribosomes. These findings suggest that produced antibiotics induce tmRNA that facilitate reactivation of stalled complex of ribosomes and maintain viability. The effect of antibiotics that inhibit the cell‐wall turnover, DNA, RNA or protein synthesis on the level of tmRNA was examined. Antibiotics interfering with ribosomal target sites are more effective at stimulation of the tmRNA level in streptomycetes examined than those affecting the synthesis of DNA, RNA or the cell wall.