Alteration of 23 S ribosomal RNA and erythromycin-induced resistance to lincomycin and spiramycin in Staphylococcus aureus

Functionally active “hybrid” 50 S ribosomal subunits can be reconstituted using 23 S RNA from Staphylococcus aureus (strain 1206) and 5 S RNA, as well as 50 S ribosomal proteins from Bacillus stearothermophilus. Using this system, resistance of S. aureus 50 S subunits to lincomycin and spiramycin was analyzed. When 23 S RNA from either phenotypically resistant (“induced resistance”) S. aureuscells or derived genetically resistant (“constitutive resistance”) S. aureus cells, were used, the reconstituted 50 S subunits showed the resistant phenotype similar to that seen in native 50 S subunits obtained from resistant cells; only very weak inhibition by the antibiotics was observed in poly (U) - directed polyphenylalanine synthesis involving these 50 S subunits. In contrast, the 50 S particles reconstituted using 23 S RNA from uninduced (sensitive) S. aureus were subject to greater inhibition by the antibiotics in cell-free poly-peptide synthesis. It is concluded that modification of 23 S RNA, presumably the previously observed methylation to form dimethyladenine, is responsible for the resistance to the antibiotics in this strain of S. aureus.     

Deciphering the protein translation inhibition and coping mechanism of trichothecene toxin in resistant fungi

In modern times for combating the deleterious soil microbes for improved sustainable agricultural practices, there is a need to have a proper understanding of the plant-microbe interactions present in the rhizospheric microbiome of the plant roots. In the present study, the interactions of trichodermin with petidyltransferase centre of ribosomal complex was studied by molecular dynamics and in silico interaction methods to demonstrate its mechanism of action and to decipher the possible reason how it may inhibit protein synthesis at the ribosomal complex. Further we have illustrated how trichodermin resistance protein (60S ribosomal protein L3) helps to overcome the deleterious effects of trichothecene compounds like trichodermin. Normal mode analysis of trichodermin resistance protein and 25S rRNA that constitutes the petidyltransferase centre showed that the W-finger region of the protein moved towards 25S rRNA. Further analysis of molecular dynamics simulation time frames showed that several intermediate states of large motions of the protein molecules towards the 25S rRNA which finally blocks the binding pocket of the trichodermin. It indicated that this protein not only changes the local environment and conformation of the petidyltransferase centre but also restrain trichodermin from binding to the 25S rRNA at the petidyltransferase centre.     

Methylation of 23S ribosomal RNA due to carB, an antibiotic-resistance determinant from the carbomycin producer, Streptomyces thermotolerans.

A resistance gene, carB, originally isolated from the carbomycin-producing organism, Streptomyces thermotolerans, confers on Streptomyces lividans high-level resistance to the drug. However, ribosomes from S. lividans expressing carB show only moderate resistance to this macrolide in vitro, although they are highly resistant to the action of lincosamide antibiotics. The carB product monomethylates the amino group of the adenosine residue located at position 2058 in 23S ribosomal RNA. In contrast, ribosomes from S. lividans expressing ermE, in which 23S RNA is dimethylated at this same position, are much more highly resistant to macrolides and insensitive to lincosamides.     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.     

Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides

Methylation of either of two residues (G-1405 or A-1408) within bacterial 16 S ribosomal RNA results in high level resistance to specific combinations of aminoglycoside antibiotics. The product of a gene that originated in Micromonospora purpurea (an actinomycete that produces gentamicin) gives resistance to kanamycin plus gentamicin by converting residue G-1405 to 7-methylguanosine. Resistance to kanamycin plus apramycin results from conversion of residue A-1408 to 1-methyladenosine catalysed by the product of a gene from Streptomyces tenjimariensis.     

Effect of antibiotics on large ribosomal subunit assembly reveals possible function of 5 S rRNA.

Functional large ribosomal subunits of Thermus aquaticus can be reconstituted from ribosomal proteins and either natural or in vitro transcribed 23 S and 5 S rRNA. Omission of 5 S rRNA during subunit reconstitution results in dramatic decrease of the peptidyl transferase activity of the assembled subunits. However, the presence of some ribosome-targeted antibiotics of the macrolide, ketolide or streptogramin B groups during 50 S subunit reconstitution can partly restore the activity of ribosomal subunits assembled without 5 S rRNA. Among tested antibiotics, macrolide RU69874 was the most active: activity of the subunits assembled in the absence of 5 S rRNA was increased more than 30-fold if antibiotic was present during reconstitution procedure. Activity of the subunits assembled with 5 S rRNA was also slightly stimulated by RU69874, but to a much lesser extent, approximately 1.5-fold. Activity of the native T. aquaticus 50 S subunits incubated in the reconstitution conditions in the presence of RU69874 was, in contrast, slightly decreased. The presence of antibiotics was essential during the last incubation step of the in vitro assembly, indicating that drugs affect one of the last assembly steps. The 5 S rRNA was previously shown to form contacts with segments of domains II and V of 23 S rRNA. All the antibiotics which can functionally compensate for the lack of 5 S rRNA during subunit reconstitution interact simultaneously with the central loop in domain V (which is known to be a component of peptidyl transferase center) and a loop of the helix 35 in domain II of 23 S rRNA. It is proposed that simultaneous interaction of 5 S rRNA or of antibiotics with the two domains of 23 S rRNA is essential for the successful assembly of ribosomal peptidyl transferase center. Consequently, one of the functions of 5 S rRNA in the ribosome can be that of assisting the assembly of ribosomal peptidyl transferase by correctly positioning functionally important segments of domains II and V of 23 S rRNA.     

Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin

Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.     

Chloramphenicol-erythromycin resistance mutations in a 23S rRNA gene of Escherichia coli.

Two chloramphenicol resistance mutations were isolated in an Escherichia coli rRNA operon (rrnH) located on a multicopy plasmid. Both mutations also confer resistance to 14-atom lactone ring macrolide antibiotics, but they do not confer resistance to 16-atom lactone ring macrolide antibiotics or other inhibitors of the large ribosomal subunit. Classic genetic and recombinant DNA methods were used to map the two mutations to 154-base-pair regions of the 23S RNA genes. DNA sequencing of these regions revealed that chloramphenicol-erythromycin resistance results from a guanine-to-adenine transition at position 2057 of the 23S RNA genes of both independently isolated mutants. These mutations affect a region of 23S RNA strongly implicated in peptidyl transfer and known to interact with a variety of peptidyl transferase inhibitors.     

Erythromycin-inducible resistance in Staphylococcus aureus: survey of antibiotic classes involved

Certain erythromycin-resistant strains of Staphylococcus aureus remain sensitive to other macrolide antibiotics. If these strains are exposed to low levels of erythromycin, resistance to other antibiotics is induced. The antibiotics to which resistance is induced by erythromycin include: other macrolides as well as lincosaminide, streptogramin (group B) antibiotics but not chloramphenicol, amicetin, streptogramin (group A) antibiotics, tetracyclines, and aminoglycosides. Hence erythromycin induces resistance exclusively towards inhibitors of 50S ribosomal subunit function and, thus far, only with respect to three of six known classes of inhibitors which act on this subunit. In the four strains tested, erythromycin did not induce resistance to pactamycin or bottromycin, to fusidic acid (which inhibits a function involving both subunits), or to other antibiotics which do not inhibit ribosomal function. Thus, by inducing resistance erythromycin could antagonize the action of other antibiotics, and a consistent pattern of antagonism was observed to each antibiotic class in all of the strains in which this could be tested, as well as to other antibiotic members of the same chemical class in each bacterial strain.     

Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota

The widespread use of antibiotics for medical and veterinary purposes has led to an increase of microbial resistance. The antibiotic resistance of pathogenic bacteria has been studied extensively. However, antibiotics are not only selective for pathogens: they also affect all members of the gut microbiota. These microorganisms may constitute a reservoir of genes carrying resistance to specific antibiotics. This study was designed to characterize the gut microbiota with regard to the presence of genes encoding tetracycline resistance proteins (tet) in the gut of healthy exclusively breast-fed infants and their mothers. For this purpose we determined the prevalence of genes encoding ribosomal protection proteins (tet M, tet W, tet O, tet S, tet T and tet B) by PCR and characterized the gut microbiota by FISH in stools of infants and their mothers. The gene tet M was found in all the breast-fed infants and their mothers. tet O was found in all of the mothers' samples, whilst only 35% of the infants harboured this gene. tet W was less frequently found (85% of the mothers and 13% of the infants). None of the other genes analysed was found in any sample. Our results suggest that genes carrying antibiotic resistance are common in the environment, as even healthy breast-fed infants with no direct or indirect previous exposure to antibiotics harbour these genes.     

Mitochondrially encoded resistance to paromomycin in Saccharomyces cerevisiae: Reinvestigation of a controversy

We have reinvestigated the nature of mitochondrially inherited resistance to paromomycin in Saccharomyces cerevisiae. Resistance to this antibiotic can arise by a nucleotide alteration in the gene coding for 15 S ribosomal RNA at a recognition site for the restriction endonuclease ThaI (CGCG), as has been observed by Li (M. Li, K. Lyon, N. Martin and A. Tzagoloff (1981). “Abstracts, Cold Spring Harbor Meeting on Mitochondrial Genes,” p. 56. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.). We have confirmed this finding and document here also a new type of paromomycin resistance that is unrelated to this ThaI restriction site. Certain petite mutants derived from different locations of the mtDNA of S. cerevisiae KL14-4A can elicit resistance to paromomycin when crossed with a wild-type sensitive strain. These petite mutants lack detectable sequence homology with the 15 S ribosomal RNA gene and they have no extensive sequence homology with each other. We have constructed paromomycin-resistant diploids by crossing such KL14-4A petite mutants with a sensitive wild-type strain. The diploids that receive the paromomycin-resistant allele from a petite mutant retaining the 15 S ribosomal RNA gene no longer contained the ThaI site. However, diploids that become resistant after a cross with petite mutants retaining fragments from other mtDNA regions than the 15 S ribosomal RNA, still contain the ThaI site. This shows that paromomycin resistance can occur in the presence of the ThaI site. After sporulation, suitable paromomycin-resistant haploids were crossed with each other and sensitive recombinant diploids were found, indicating the existence of more than one form of paromomycin resistance. Possible explanations for this novel type of paromomycin resistance and the unorthodox way in which it arises, are presented.     

A family of r-determinants in Streptomyces spp. that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics.

Inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics in Streptomyces spp. comprises a family of diverse phenotypes in which characteristic subsets of the macrolide-lincosamide-streptogramin antibiotics induce resistance mediated by mono- or dimethylation of adenine, or both, in 23S ribosomal ribonucleic acid. In these studies, diverse patterns of induction specificity in Streptomyces and associated ribosomal ribonucleic acid changes are described. In Streptomyces fradiae NRRL 2702 erythromycin induced resistance to vernamycin B, whereas in Streptomyces hygroscopicus IFO 12995, the reverse was found: vernamycin B induced resistance to erythromycin. In a Streptomyces viridochromogenes (NRRL 2860) model system studied in detail, tylosin induced resistance to erythromycin associated with N6-monomethylation of 23S ribosomal ribonucleic acid, whereas in Staphylococcus aureus, erythromycin induced resistance to tylosin mediated by N6-dimethylation of adenine. Inducible macrolide-lincosamide-streptogramin resistance was found in S. fradiae NRRL 2702 and S. hygroscopicus IFO 12995, which synthesize the macrolides tylosin and maridomycin, respectively, as well as in the lincosamide producer Streptomyces lincolnensis NRRL 2936 and the streptogramin type B producer Streptomyces diastaticus NRRL 2560. A wide range of different macrolides including chalcomycin, tylosin, and cirramycin induced resistance when tested in an appropriate system. Lincomycin was active as inducer in S. lincolnensis, the organism by which it is produced, and streptogramin type B antibiotics induced resistance in S. fradiae, S. hygroscopicus, and the streptogramin type B producer S. diastaticus. Patterns of adenine methylation found included (i) lincomycin-induced monomethylation in S. lincolnensis (and constitutive monomethylation in a mutant selected with maridomycin), (ii) concurrent equimolar levels of adenine mono- plus dimethylation in S. hygroscopicus, (iii) monomethylation in S. fradiae (and dimethylation in a mutant selected with erythromycin), and (iv) adenine dimethylation in S. diastaticus induced by ostreogrycin B.     

Mutations in conserved helix 69 of 23S rRNA of Thermus thermophilus that affect capreomycin resistance but not posttranscriptional modifications

Translocation during the elongation phase of protein synthesis involves the relative movement of the 30S and 50S ribosomal subunits. This movement is the target of tuberactinomycin antibiotics. Here, we describe the isolation and characterization of mutants of Thermus thermophilus selected for resistance to the tuberactinomycin antibiotic capreomycin. Two base substitutions, A1913U and mU1915G, and a single base deletion, DeltamU1915, were identified in helix 69 of 23S rRNA, a structural element that forms part of an interribosomal subunit bridge with the decoding center of 16S rRNA, the site of previously reported capreomycin resistance base substitutions. Capreomycin resistance in other bacteria has been shown to result from inactivation of the TlyA methyltransferase which 2'-O methylates C1920 of 23S rRNA. Inactivation of the tlyA gene in T. thermophilus does not affect its sensitivity to capreomycin. Finally, none of the mutations in helix 69 interferes with methylation at C1920 or with pseudouridylation at positions 1911 and 1917. We conclude that the resistance phenotype is a consequence of structural changes introduced by the mutations.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Antibiotic Resistance Mutations in the Chloroplast 16s and 23s Rrna Genes of Chlamydomonas Reinhardtii: Correlation of Genetic and Physical Maps of the Chloroplast Genome

Mutants resistant to streptomycin, spectinomycin, neamine/kanamycin and erythromycin define eight genetic loci in a linear linkage group corresponding to about 21 kb of the circular chloroplast genome of Chlamydomonas reinhardtii. With one exception, all of these mutants represent single base-pair changes in conserved regions of the genes encoding the 16S and 23S chloroplast ribosomal RNAs. Streptomycin resistance can result from changes at the bases equivalent to Escherichia coli 13, 523, and 912-915 in the 16S gene, or from mutations in the rps12 gene encoding chloroplast ribosomal protein S12. In the 912-915 region of the 16S gene, three mutations were identified that resulted in different levels of streptomycin resistance in vitro. Although the three regions of the 16S rRNA mutable to streptomycin resistance are widely separated in the primary sequence, studies by other laboratories of RNA secondary structure and protein cross-linking suggest that all three regions are involved in a common ribosomal neighborhood that interacts with ribosomal proteins S4, S5 and S12. Three different changes within a conserved region of the 16S gene, equivalent to E. coli bases 1191-1193, confer varying levels of spectinomycin resistance, while resistance to neamine and kanamycin results from mutations in the 16S gene at bases equivalent to E. coli 1408 and 1409. Five mutations in two genetically distinct erythromycin resistance loci map in the 23S rDNA of C. reinhardtii, at positions equivalent to E. coli 2057-2058 and 2611, corresponding to the rib3 and rib2 loci of yeast mitochondria respectively. Although all five mutants are highly resistant to erythromycin, they differ in levels of cross-resistance to lincomycin and clindamycin. The order and spacing of all these mutations in the physical map are entirely consistent with our genetic map of the same loci and thereby validate the zygote clone method of analysis used to generate this map. These results are discussed in comparison with other published maps of chloroplast genes based on analysis by different methods using many of the same mutants.     

The gene for the S7 ribosomal protein of Chlamydia trachomatis: characterization within the chlamydial sfr operon

The prokaryotic ribosomal operon, str, contains open reading frames for the two elongation factors, elongation factor G (EF-G) and elongation factor Tu (EF-Tu), and ribosomal proteins S7 and S12. The DNA sequence and predicted amino acid sequence for S7 from Chlamydia trachomatis are presented and compared with homologues from other prokaryotes. Also, the relationship of the S7 gene to the open reading frames for ribosomal protein S12 and EF-G is described. Significant amino acid homology is also noted when the amino-terminal sequence of chlamydial EF-G is compared with the cytoplasmic tetracycline resistance factors, tetM and tetO, from streptococci and Campylobacter jejuni. Related findings and possible resistance mechanisms for the newly recognized tetracycline-resistant clinical isolates of C. trachomatis are discussed.     

Toxin-dependent utilization of engineered ribosomal protein L3 limits trichothecene resistance in transgenic plants

The contamination of agricultural products with Fusarium mycotoxins is a problem of world-wide importance. Fusarium graminearum and related species, which are important pathogens of small grain cereals and maize, produce an economically important and structurally diverse class of toxins designated trichothecenes. Trichothecenes inhibit eukaryotic protein synthesis. Therefore, a proposed role for these fungal toxins in plant disease development is to block or delay the expression of defence-related proteins induced by the plant. Using yeast as a model system, we have identified several mutations in the gene encoding ribosomal protein L3 (Rpl3), which confer semi-dominant resistance to trichothecenes. Expression of an engineered tomato RPL3 (LeRPL3) cDNA, into which one of the amino acid changes identified in yeast was introduced, improved the ability of transgenic tobacco plants to adapt to the trichothecene deoxynivalenol (DON), but did not result in constitutive resistance. We show here that, in the presence of wild-type Rpl3 protein, the engineered Rpl3 protein is not utilized, unless yeast transformants or the transgenic plants are challenged with sublethal amounts of toxin. Our data from yeast two-hybrid experiments suggest that affinity for the ribosome assembly factor Rrb1p could be altered by the toxin resistance-conferring mutation. This toxin-dependent utilization of the resistance-conferring Rpl3 protein could seriously limit efforts to utilize the identified target alterations in transgenic crops to increase trichothecene tolerance and Fusarium resistance.     

Genetic and biochemical manipulations of the small ribosomal subunit from Thermus thermophilus HB8

Crystals of the small ribosomal subunit from Thermus thermophilus diffract to 3A and exhibit reasonable isomorphism and moderate resistance to irradiation. A 5A MIR map of this particle shows a similar shape to the part assigned to this particle within the cryo-EM reconstructions of the whole ribosome and contains regions interpretable either as RNA chains or as protein motifs. To assist phasing at higher resolution we introduced recombinant methods aimed at extensive selenation for MAD phasing. We are focusing on several ribosomal proteins that can be quantitatively detached by chemical means. These proteins can be modified and subsequently reconstituted into depleted ribosomal cores. They also can be used for binding heavy atoms, by incorporating chemically reactive binding sites, such as -SH groups, into them. In parallel we are co-crystallizing the ribosomal particles with tailor made ligands, such as antibiotics or cDNA to which heavy-atoms have been attached or diffuse the latter compounds into already formed crystals.