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ObjectivesTo determine the mechanism of induction of erm(47) and its atypical expression in the Gram-positive opportunistic pathogen Helcococcus kunzii, where it confers resistance to a subset of clinically important macrolide, lincosamide and streptogramin B (MLSB) antibiotics.MethodsThe resistant H. kunzii clinical isolate UCN99 was challenged with subinhibitory concentrations of a wide range of ribosome-targeting drugs. The methylation status of the H. kunzii ribosomal RNA at the MLSB binding site was then determined using an MS approach and was correlated with any increase in resistance to the drugs.ResultsThe H. kunzii erm(47) gene encodes a monomethyltransferase. Expression is induced by subinhibitory concentrations of the macrolide erythromycin, as is common for many erm genes, and surprisingly also by 16-membered macrolide, lincosamide, streptogramin, ketolide, chloramphenicol and linezolid antibiotics, all of which target the 50S ribosomal subunit. No induction was detected with spectinomycin, which targets the 30S subunit.ConclusionsThe structure of the erm(47) leader sequence functions as a hair trigger for the induction mechanism that expresses resistance. Consequently, translation of the erm(47) mRNA is tripped by MLSB compounds and also by drugs that target the 50S ribosomal subunit outside the MLSB site. Expression of erm(47) thus extends previous assumptions about how erm genes can be induced.

Place: Oxford Publisher: Oxford Univ Press WOS:000574288800042; The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m(5)U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m(5)U54 is subsequently thiolated to form m(5)s(2)U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m(1)s(4)Psi. The methyl and thiol groups in m(1)s(4)Psi and m(5)s(2)U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m(1)A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.

The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m5U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup, use the ancestral tetrahydrofolate-dependent TrmFO enzyme, to catalyze the formation of m5U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m5U1939 formation in 23S RNA. RlmFO has replaced the S-adenosyl-l-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m5U modifying enzymes that is highly dynamic and has undergone horizontal transfer. This emphasizes the necessity for combining bioinformatics predictions with empirical testing and structural information to get a reliable functional annotation of these enzymes.

Campylobacter jejuni is a bacterial pathogen that is generally acquired as a zoonotic infection from poultry and animals. Adhesion of C. jejuni to human colorectal epithelial cells is weakened after loss of its cj0588 gene. The Cj0588 protein belongs to the type I group of TlyA (TlyAI) enzymes, which 2′-O-methylate nucleotide C1920 in 23S rRNA. Slightly longer TlyAII versions of the methyltransferase are found in actinobacterial species including Mycobacterium tuberculosis, and methylate not only C1920 but also nucleotide C1409 in 16S rRNA. Loss of TlyA function attenuates virulence of both M. tuberculosis and C. jejuni. We show here that the traits impaired in C. jejuni null strains can be rescued by complementation not only with the original cj0588 (tlyAI) but also with a mycobacterial tlyAII gene. There are, however, significant differences in the recombinant phenotypes. While cj0588 restores motility, biofilm formation, adhesion to and invasion of human epithelial cells and stimulation of IL-8 production in a C. jejuni null strain, several of these properties are further enhanced by the mycobacterial tlyAII gene, in some cases to twice the original wild-type level. These findings strongly suggest that subtle changes in rRNA modification patterns can affect protein synthesis in a manner that has serious consequences for bacterial pathogenicity.

The chemically mutagenized Escherichia coli strain AS19 was isolated on the basis of its enhanced sensitivity to different antibiotics, in particular to actinomycin. The strain was later modified to study rRNA modifications that confer antibiotic resistance. Here, we present the genome sequence of the variant E. coli AS19-RrmA - .

Strains of the Pasteurellaceae bacteria Pasteurella multocida and Mannheimia haemolytica are major etiological agents of bovine respiratory disease (BRD). Treatment of BRD with antimicrobials is becoming more challenging due to the increasing occurrence of resistance in infecting strains. In Pasteurellaceae strains exhibiting resistance to multiple antimicrobials including aminoglycosides, beta-lactams, macrolides and sulfonamides, the resistance determinants are often chromosomally encoded within integrative and conjugative elements (ICEs). To gain a more comprehensive picture of ICE structures, we sequenced the genomes of six strains of P. multocida and four strains of M. haemolytica; all strains were independent isolates and eight of them were multiple-resistant. ICE sequences varied in size from 49 to 79 kb, and were comprised of an array of conserved genes within a core region and varieties of resistance genes within accessory regions. These latter regions mainly account for the variation in the overall ICE sizes. From the sequence data, we developed a multiplex PCR assay targeting four conserved core genes required for integration and maintenance of ICE structures. Application of this assay on 75 isolates of P. multocida and M. haemolytica reveals how the presence and structures of ICEs are related to their antibiotic resistance phenotypes. The assay is also applicable to other members of the Pasteurellaceae family including Histophilus somni and indicates how clustering and dissemination of the resistance genes came about.

Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2′-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campylobacter jejuni possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2′-O-methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both in vitro and also when expressed in Escherichia coli. Deletion of the cj0588 gene in C. jejuni or substitution with alanine of K80, D162, or K188 in the catalytic center of the enzyme cause complete loss of 2′-O-methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2′-O-methylation function of Cj0588/TlyA from any other putative roles that the protein might play. C. jejuni strains expressing catalytically inactive versions of Cj0588 have the same phenotype as cj0588-null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in C. jejuni is a consequence of the enzyme's ability to methylate its rRNA.

Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous rRNA methylase genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate, respectively, the N6 amino group in 23S rRNA nucleotide A2058. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high-level resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism.

In all free-living organisms a late-stage checkpoint in the biogenesis of the small ribosomal subunit involves rRNA modification by an RsmA/Dim1 methyltransferase. The hyperthermophilic archaeon Nanoarchaeum equitans, whose existence is confined to the surface of a second archaeon, Ignicoccus hospitalis, lacks an RsmA/Dim1 homolog. We demonstrate here that the I. hospitalis host possesses the homolog Igni_1059, which dimethylates the N(6)-positions of two invariant adenosines within helix 45 of 16S rRNA in a manner identical to other RsmA/Dim1 enzymes. However, Igni_1059 is not transferred from I. hospitalis to N. equitans across their fused cell membrane structures and the corresponding nucleotides in N. equitans 16S rRNA remain unmethylated. An alternative mechanism for ribosomal subunit maturation in N. equitans is suggested by sRNA interactions that span the redundant RsmA/Dim1 site to introduce 2'-O-ribose methylations within helices 44 and 45 of the rRNA.

Aminoglycoside antibiotics remain the drugs of choice for treatment of Pseudomonas aeruginosa infections, particularly for respiratory complications in cystic-fibrosis patients. Previous studies on...

Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida , that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC 50 ], 0.23 ± 0.01 μM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site.

The binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and C1920 in 23S rRNA. In Mycobacterium tuberculosis, the 2'-hydroxyls of both nucleotides are methylated by the enzyme TlyA. Loss of these methylations through inactivation of TlyA confers resistance to capreomycin and viomycin. We report here that TlyA orthologues occur in diverse bacteria and fall into two distinct groups. One group, now termed TlyA(I) , has shorter N- and C-termini and methylates only C1920; the second group (now TlyA(II) ) includes the mycobacterial enzyme, and these longer orthologues methylate at both C1409 and C1920. Ribosomal subunits are the preferred substrates for both groups of orthologues. Amino acid substitutions at the N-terminus of TlyA(II) reduce its ability to methylate these substrates. Growing pairs of recombinant TlyA(II) Escherichia coli strains in competition shows that even subtle changes in the level of rRNA methylation lead to significant differences in susceptibility to sub-inhibitory concentrations of capreomycin. The findings reveal that 2'-O-methyls at both C1409 and C1920 play a role in facilitating the inhibitory effects of capreomycin and viomycin on the bacterial ribosome.

The veterinary antibiotic tildipirosin (20,23-dipiperidinyl-mycaminosyl-tylonolide, Zuprevo) was developed recently to treat bovine and swine respiratory tract infections caused by bacterial pathogens such as Pasteurella multocida. Tildipirosin is a derivative of the naturally occurring compound tylosin. Here, we define drug-target interactions by combining chemical footprinting with structure modeling and show that tildipirosin, tylosin, and an earlier tylosin derivative, tilmicosin (20-dimethylpiperidinyl-mycaminosyl-tylonolide, Micotil), bind to the same macrolide site within the large subunit of P. multocida and Escherichia coli ribosomes. The drugs nevertheless differ in how they occupy this site. Interactions of the two piperidine components, which are unique to tildipirosin, distinguish this drug from tylosin and tilmicosin. The 23-piperidine of tildipirosin contacts ribosomal residues on the tunnel wall while its 20-piperidine is oriented into the tunnel lumen and is positioned to interfere with the growing nascent peptide.

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, ΔmU1915, 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.

Pseudouridines in the stable RNAs of Bacteria are seldom subjected to further modification. There are 11 pseudouridine (Ψ) sites inEscherichia colirRNA, and further modification is found only at Ψ1915 in 23S rRNA, where the N-3 position of the base becomes methylated. Here, we report the identity of theE. colimethyltransferase that specifically catalyzes methyl group addition to form m3Ψ1915. Analyses ofE. colirRNAs using MALDI mass spectrometry showed that inactivation of theybeAgene leads to loss of methylation at nucleotide Ψ1915. Methylation is restored by complementing the knockout strain with a plasmid-encoded copy ofybeA. Homologs of theybeAgene, and thus presumably the ensuing methylation at nucleotide m3Ψ1915, are present in most bacterial lineages but are essentially absent in the Archaea and Eukaryota. Loss ofybeAfunction inE. colicauses a slight slowing of the growth rate. Phylogenetically,ybeAand its homologs are grouped with other putative S-adenosylmethionine-dependent, SPOUT methyltransferase genes in the Cluster of Orthologous Genes COG1576;ybeAis the first member to be functionally characterized. The YbeA methyltransferase is active as a homodimer and docks comfortably into the ribosomal A site without encroaching into the P site. YbeA makes extensive interface contacts with both the 30S and 50S subunits to align its active site cofactor adjacent to nucleotide Ψ1915. Methylation by YbeA (redesignated RlmH forrRNAlarge subunitmethyltransferaseH) possibly functions as a stamp of approval signifying that the 50S subunit has engaged in translational initiation.

A telithromycin-resistant clinical isolate of Streptococcus pneumoniae (strain P1501016) has been found to contain a version of erm (B) that is altered by a 136-bp deletion in the leader sequence. By allele replacement mutagenesis, a second strain of S. pneumoniae (PC13) with a wild-type erm (B) gene was transformed to the telithromycin-resistant phenotype by introduction of the mutant erm (B) gene. Whereas the wild-type PC13 strain showed slight telithromycin resistance only after induction by erythromycin (telithromycin MIC increased from 0.06 to 0.5 μg/ml), the transformed PC13 strain is constitutively resistant (MIC of 16 μg/ml). Expression of erm (B) was quantified by real-time reverse transcription-PCR in the presence of erythromycin or telithromycin; erm (B) expression was significantly higher in the transformed PC13 strain than the wild-type strain. Furthermore, the transformed strain had significantly higher levels of ribosomal methylation in the absence as well as in the presence of the antibiotics. Growth studies showed that the transformed PC13 strain had a shorter lag phase than the wild-type strain in the presence of erythromycin. Telithromycin resistance is conclusively shown to be conferred by the mutant erm (B) gene that is expressed at a constitutively higher level than the inducible wild-type gene. Elevated erm (B) expression results in a higher level of rRNA methylation that presumably hinders telithromycin binding to the ribosome.

The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli ). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10 −6 . Importantly, in the rsmG mutant background, high-level-streptomycin-resistant rpsL (encoding ribosomal protein S12) mutants emerged at a frequency 200 times greater than that seen for the wild-type strain. This elevated frequency in the emergence of high-level streptomycin resistance was facilitated by a mutation pattern in rpsL more varied than that obtained by selection of the wild-type strain.

Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment of multiple cellular processes. In contrast, it was recently reported that signaling induced by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce mitoribosomal hypermethylation and deafness. At variance with this model, we show here that 12S rRNA is near fully methylated in vivo in the mouse and thus cannot be further methylated to any significant extent. Furthermore, bacterial artificial chromosome transgenic mice overexpressing TFB1M have no increase of 12S rRNA methylation levels and hear normally. We thus conclude that therapies directed against mitoribosomal methylation are unlikely to be beneficial to patients with sensorineural hearing loss or other types of mitochondrial disease.

The cyclic peptide antibiotics capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to capreomycin and viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to capreomycin and viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of capreomycin and viomycin at the ribosome subunit interface.

Summary Macrolide and ketolide antibiotics inhibit protein synthesis on the bacterial ribosome. Resistance to these antibiotics is conferred by dimethylation at 23S rRNA nucleotide A2058 within the ribosomal binding site. This form of resistance is encoded by erm dimethyltransferase genes, and is found in many pathogenic bacteria. Clinical isolates of Streptococcus pneumoniae with constitutive erm(B) and Streptococcus pyogenes with constitutive erm(A) subtype (TR) are resistant to macrolides, but remain susceptible to ketolides such as telithromycin. Paradoxically, some strains of S. pyogenes that possess an identical erm(B) gene are clinically resistant to ketolides as well as macrolides. Here we explore the molecular basis for the differences in these streptococcal strains using mass spectrometry to determine the methylation status of their rRNAs. We find a correlation between the levels of A2058-dimethylation and ketolide resistance, and dimethylation is greatest in S. pyogenes strains expressing erm(B). In constitutive erm strains that are ketolide-sensitive, appreciable proportions of the rRNA remain monomethylated. Incubation of these strains with subinhibitory amounts of the macrolide erythromycin increases the proportion of dimethylated A2058 (in a manner comparable with inducible erm strains) and reduces ketolide susceptibility. The designation ‘constitutive’ should thus be applied with some reservation for most streptococcal erm strains. One strain worthy of the constitutive designation is S. pyogenes isolate KuoR21, which has lost part of the regulatory region upstream of erm(B). In S. pyogenes KuoR21, nucleotide A2058 is fully dimethylated under all growth conditions, and this strain displays the highest resistance to telithromycin (MIC > 64 µg ml−1).

Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.

Specific cleavage of RNA is catalysed by short oligodeoxynucleotides termed DNAzymes. DNAzymes consist of two binding arms that hybridize to a predetermined RNA sequence and a catalytic core that cleaves a phosphodiester bond held between the binding arms. DNAzymes are exemplified by the well-studied 10-23 DNAzyme, which compared with protein ribonucleases is highly specific, albeit slow. Here we report a significant improvement in cleavage kinetics, while maintaining specificity, by incorporation of LNA (locked nucleic acid) and α-l-LNA nucleotides into the binding arms of 10-23 DNAzyme. DNAzymes modified in this way (LNAzymes) enhance cleavage of a phosphodiester bond presented in a short RNA substrate as well as in longer and highly structured substrates, and efficient cleavage is maintained from single- to multiple-turnover conditions. Analysis of the cleavage reaction indicates that substrate hybridization is boosted by the presence of the locked residues within the LNAzymes, while no apparent change occurs in the catalytic strand-scission step.

There are three sites of m(5)U modification in Escherichia coli stable RNAs: one at the invariant tRNA position U54 and two in 23S rRNA at the phylogenetically conserved positions U747 and U1939. Each of these sites is modified by its own methyltransferase, and the tRNA methyltransferase, TrmA, is well-characterised. Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA, and here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry. A purified recombinant version of YgcA retains its activity and specificity, and methylates U1939 in an RNA transcript in vitro. We were unable to generate a recombinant version of YbjF that retained in vitro activity, so the function of this enzyme was defined in vivo by engineering a ybjF knockout strain. Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification. With this report, the functions of all the E.coli m(5)U RNA methyltransferases are identified, and a more appropriate designation for YbjF would be RumB (RNA uridine methyltransferases B), in line with the recent nomenclature change for YgcA (now RumA).

Avilamycin is an orthosomycin antibiotic that has shown considerable potential for clinical use, although it is presently used as a growth promoter in animal feed. Avilamycin inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit. The ribosomes of the producer strain, Streptomyces viridochromogenes Tu57, are protected from the drug by the action of three resistance factors located in the avilamycin biosynthetic gene cluster. Two of the resistance factors, aviRa and aviRb, encode rRNA methyltransferases that specifically target 23S rRNA. Recombinant AviRa and AviRb proteins retain their activity after purification, and both specifically methylate in vitro transcripts of 23S rRNA domain V. Reverse transcriptase primer extension indicated that AviRa is an N-methyltransferase that targets G2535 within helix 91 of the rRNA, whereas AviRb modified the 2'-O-ribose position of nucleotide U2479 within helix 89. MALDI mass spectrometry confirmed the exact positions of each of these modifications, and additionally established that a single methyl group is added at each nucleotide. Neither of these two nucleotides have previously been described as a target for enzymatic methylation. Molecular models of the 50S subunit crystal structure show that the N-1 of the G2535 base and the 2'-hydroxyl of U2479 are separated by approximately 10 A, a distance that can be spanned by avilamycin. In addition to defining new resistance mechanisms, these data refine our understanding of the probable ribosome contacts made by orthosomycins and of how these antibiotics inhibit protein synthesis.

The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA , tlrB , tlrC , and tlrD . The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5- and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit.

Summary Bacteria tune the function of their ribosomes by methylating specific rRNA nucleotides. Nucleotide G745 in Escherichia coli 23S rRNA is methylated by the methyltransferase enzyme RrmA, whereas in Streptomyces fradiae, the neighbouring nucleotide G748 is methylated by the enzyme TlrB. Both nucleotides line the peptide exit channel of the ribosome at the binding site of macrolide, lincosamide and streptogramin B antibiotics. Resistance to the macrolide tylosin, which is produced by S. fradiae, is conferred by methylation of G748. RrmA and TlrB are homologues (29% identical), and a database search against all presently available sequences revealed a further two dozen homologues from a wide variety of Bacteria. No homologues were found among the Archaea or Eukarya. The bacterial sequences adhere to the species phylogeny and segregate into two groups, in which the Gramnegative sequences align with RrmA and the Gram-positives with TlrB. Consistently, in more than 20 species tested, the distribution of methylation in the Gram-negative rRNAs (methylated at G745) and the Gram-positives (methylated at G748) perfectly matches the bacterial phylogeny. Cloning and ex-pression of representative methyltransferase genes showed that this specificity of methylation is determined solely by the methyltransferase enzyme and is independent of the origin of the rRNA substrate. This is the first case in which the position of an RNA methylation defines a sharp division between the Gram-negative and Gram-positive bacteria. Given the specificities and distribution of these methyltransferases, we propose a change in the nomenclature of RrmA to RlmAI (rRNA large subunit methyltransferase) and of TlrB to RlmAII.

OBJECTIVES: To determine how resistance to macrolides is conferred in field isolates of Pasteurella multocida and Mannheimia haemolytica that lack previously identified resistance determinants for rRNA methylation, efflux and macrolide-modifying enzymes.METHODS: Isolates of P. multocida and M. haemolytica identified as being highly resistant (MICs >64 mg/L) to the macrolides erythromycin, gamithromycin, tilmicosin, tildipirosin and tulathromycin were screened by multiplex PCR for the previously identified resistance genes erm(42), msr(E) and mph(E). Strains lacking these determinants were analysed by genome sequencing and primer extension on the rRNAs.RESULTS: Macrolide resistance in one M. haemolytica isolate was conferred by the 23S rRNA mutation A2058G; resistance in three P. multocida isolates were caused by mutations at the neighbouring nucleotide A2059G. In each strain, all six copies of the rrn operons encoded the respective mutations. There were no mutations in the ribosomal protein genes rplD or rplV, and no other macrolide resistance mechanism was evident.CONCLUSIONS: High-level macrolide resistance can arise from 23S rRNA mutations in P. multocida and M. haemolytica despite their multiple copies of rrn. Selective pressures from exposure to different macrolide or lincosamide drugs presumably resulted in consolidation of either the A2058G or the A2059G mutation.

This chapter concentrates on resistance to a subset of antibiotics, the 14-membered ring macrolides, and considers their fate as effective antibacterial agents now that resistance is widespread among bacterial pathogens. The ribosome target site for macrolides lies within the 23S rRNA at the peptidyltransferase center of the 50S subunit. Shortly after the introduction of erythromycin in therapy in the 1950s, resistance to the drug became widespread in numerous pathogens. The degree of resistance that is conferred seems to depend on the proportion of ribosomes that contain a modification (or mutation) at position 2058. Recent technical advances (not the least being reverse transcription-PCR methods) have made it possible to identify rRNA mutations that arise in pathogens during macrolide therapy. Pathogens that attain macrolide resistance by spontaneous rRNA mutations generally contain only one or two rrn operons. The occurrence of MLS resistance mutations is not limited to erythromycin derivatives nor to human pathogens but can occur in any species with a low rrn copy number that is exposed to macrolides. The details of the molecular contacts made between the Erm methyltransferases and the conserved rRNA motif await resolution by X-ray diffraction or NMR techniques. It can be hoped that determination of the three-dimensional structure of this motif will facilitate the design of compounds that will act as specific and effective competitive inhibitors of Erm methyltransferases.

International audience; Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m(5)U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N(5),N(10)-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses two homologs of trmFO, but surprisingly lacks the m(5)U54 tRNA modification. We created single and dual deletions of the trmFO homologs using a novel synthetic biology approach. Subsequent analysis of the M. capricolum RNAs by mass spectrometry shows that the TrmFO homolog encoded by Mcap0476 specifically modifies m(5)U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.

Ketolides are the most recent generation of antimicrobials derived from the 14-membered ring macrolide, erythromycin A. The main structural feature that differentiates ketolides from erythromycin is the keto group, which replaces the l-cladinose moiety at position 3 of the macrolactone ring. The keto group bestows greater acid stability on the drugs, and enables them to bind to their ribosomal target without causing expression of MLSB resistance in inducible strains. Several ketolides are described here, including ABT 773 and telithromycin (HMR 3647), both of which possess a carbamate at C11/C12 of the macrolactone ring. In telithromycin, which is the first ketolide to be approved for clinical use, the carbamate is linked to an alkyl-aryl extension, which is responsible for the increased potency of this compound relative to macrolides. This review examines how the structural differences between macrolides and the new ketolides are related to their antimicrobial activities in inhibiting protein synthesis and blocking the assembly of new ribosomal subunits.

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.

Udgivelsesdato: 2000-Apr The macrolide antibiotic erythromycin and its 6-O-methyl derivative (clarithromycin) bind to bacterial ribosomes primarily through interactions with nucleotides in domains II and V of 23S rRNA. The domain II interaction occurs between nucleotide A752 and the macrolide 3-cladinose moiety. Removal of the cladinose, and substitution of a 3-keto group (forming the ketolide RU 56006), results in loss of the A752 interaction and an approximately 100-fold drop in drug binding affinity. Within domain V, the key determinant of drug binding is nucleotide A2058 and substitution of G at this position is the major cause of drug resistance in some clinical pathogens. The 2058G mutation disrupts the drug-domain V contact and leads to a further > 25 000-fold decrease in the binding of RU 56006. Drug binding to resistant ribosomes can be improved over 3000-fold by forming an alternative and more effective contact to A752 via alkyl-aryl groups linked to a carbamate at the drug 11/12 position (in the ketolide antibiotics HMR 3647 and HMR 3004). The data indicate that simultaneous drug interactions with domains II and V strengthen binding and that the domain II contact is of particular importance to achieve binding to the ribosomes of resistant pathogens in which the domain V interaction is perturbed.

Udgivelsesdato: 1999-Aug Erm methyltransferases modify bacterial 23S ribosomal RNA at adenosine 2058 (A2058, Escherichia coli numbering) conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics. The motif that is recognized by Erm methyltransferases is contained within helix 73 of 23S rRNA and the adjacent single-stranded region around A2058. An RNA transcript of 72 nt that displays this motif functions as an efficient substrate for the ErmE methyltransferase. Pools of degenerate RNAs were formed by doping 34-nt positions that extend over and beyond the putative Erm recognition motif within the 72-mer RNA. The RNAs were passed through a series of rounds of methylation with ErmE. After each round, RNAs were selected that had partially or completely lost their ability to be methylated. After several rounds of methylation/selection, 187 subclones were analyzed. Forty-three of the subclones contained substitutions at single sites, and these are confined to 12 nucleotide positions. These nucleotides, corresponding to A2051-A2060, C2611, and A2614 in 23S rRNA, presumably comprise the RNA recognition motif for ErmE methyltransferase. The structure formed by these nucleotides is highly conserved throughout bacterial rRNAs, and is proposed to constitute the motif that is recognized by all the Erm methyltransferases.

The Erm methyltransferases confer resistance to macrolide, lincosamide and streptogramin B (MLS) antibiotics by methylation of a single adenosine base within bacterial 23 S ribosomal RNA. The ErmE methyltransferase, from the macrolide-producing bacterium Saccharopolyspora erythraea, recognizes a motif within domain V of the rRNA that specifically targets adenosine 2058 (A2058) for methylation. Here, we define the structure of the RNA motif by a combination of molecular genetics and biochemical probing. The core of the motif has the primary sequence 2056-GG A HA-2060, where H is any nucleotide except guanosine, and ErmE methylates at the adenosine in bold. For efficient recognition by ErmE, this sequence must be displayed within a particular secondary structure. An irregular stem (helix 73) is required immediately 5′ to A2058, with an unpaired nucleotide, preferably a cytidine residue, at position 2055. Nucleotides 2611 to 2616 are collectively required to form part of the 3′-side of helix 73, but there is little or no restriction on the identities of individual nucleotides here. There are minor preferences in the identities of nucleotides 2051 to 2055 that are adjacent to the motif core, although their main role is in maintaining the irregular secondary structure. The essential elements of the ErmE motif are conserved in bacterial 23 S rRNAs, and thus presumably also form the recognition motif for other Erm methyltransferases.

Under physiological conditions, the ErmE methyltransferase specifically modifies a single adenosine within ribosomal RNA (rRNA), and thereby confers resistance to multiple antibiotics. The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated efficiently, and with equally high fidelity, in rRNAs from phylogenetically diverse bacteria. However, the fidelity of ErmE is reduced when magnesium is removed, and over twenty new sites of ErmE methylation appear in E. coli 16S and 23S rRNAs. These sites show widely different degrees of reactivity to ErmE. The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA. This suggests that methylation at the new sites results from changes in the RNA substrate rather than the methyltransferase. Chemical probing confirms that the rRNA structure opens upon magnesium depletion, exposing potential new interaction sites to the enzyme. The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclusively at adenosines (underlined); these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are slight preferences for the nucleotides shown in lower case). This consensus is believed to represent the core of the motif that Erm methyltransferases recognize at their canonical A2058 site. The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recognition.

Dimethylation by Erm methyltransferases at the N-6 position of adenine 2058 (A2058, Escherichia coli numbering) in domain V of bacterial 23 S rRNA confers resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics. The ErmE methyltransferase from Saccharopolyspora erythraea methylates a 625 nucleotide transcript of domain V as efficiently as it methylates intact 23 S rRNA. By progressively truncating domain V, the motif required for specific recognition by the enzyme has been localized to a helix and single-stranded region adjacent to A2058. The smallest RNA transcript that shows methyl-accepting activity is a 27-nucleotide stem-loop, corresponding to the 23 S rRNA sequences 2048 to 2063 and 2610 to 2620 (helix 73), with A2058 situated within the hairpin loop. Methylation of A2058 in the truncated RNAs is optimal in the absence of magnesium, and the efficiency of methylation is halved by the presence of 2 to 3 mM magnesium. Magnesium serves to stabilize a conformation in the truncated RNA that prevents efficient methylation. This contrasts to the intact domain V RNA, where 2 mM magnesium ions support a conformation at A2058 that is most readily recognized by ErmE. Methylation of domain V RNA is generally far less susceptible to ionic conditions than the truncated RNAs. The effects of monovalent cations on the methylation of truncated transcripts suggest that RNA structures outside helix 73 support the ErmE interaction. However, interaction with these structures is not essential for specific ErmE recognition of A2058.

The mechanisms by which rRNA mutations confer clindamycin resistance were examined in Mycobacterium smegmatis strains containing homogeneous populations of ribosomes with base substitutions at nucleotides A2058 and A2059. Computer graphic predictions based on structural studies correlate with the resistance phenotypes for six of seven strains with unique rRNA mutations.

Objectives: 16S rRNA methyltransferases are an emerging mechanism conferring high-level resistance to clinically relevant aminoglycosides and have been associated with important mechanisms such as NDM-1. We sought genes encoding these enzymes in isolates highly resistant (MIC > 200 mg/L) to gentamicin and amikacin from an Indian hospital and we additionally screened for the novel RmtF enzyme in 132 UK isolates containing NDM. Methods: All highly aminoglycoside-resistant isolates were screened for armA and rmtA-E by PCR, with cloning experiments performed for isolates negative for these genes. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to determine the methylation target of the novel RmtF methyltransferase. RmtF-bearing strains were characterized further, including susceptibility testing, PFGE, electroporation, PCR-based replicon typing and multilocus sequence typing of rmtF-bearing plasmids. Results: High-level aminoglycoside resistance was detected in 140/1000 (14%) consecutive isolates of Enterobacteriaceae from India. ArmA, RmtB and RmtC were identified among 46%, 20% and 27% of these isolates, respectively. The novel rmtF gene was detected in 34 aminoglycoside-resistant isolates (overall prevalence 3.4%), most (59%) of which also possessed a bla NDM gene; rmtF was detected in 6 NDM producers from the UK. It was found on different plasmid backbones. Four and two isolates showed resistance to tigecycline and colistin, respectively. Conclusions: RmtF was often found in association with NDM in members of the Enterobacteriaceae and on diverse plasmids. It is of clinical concern that the RmtF- and NDM-positive strains identified here show additional resistance to tigecycline and colistin, current drugs of last resort for the treatment of serious bacterial infections.

Udgivelsesdato: 1995-May-26 The structure of domain II in all 23 S (and 23 S-like) rRNAs is constrained by a pseudoknot formed between nucleotides 1005 and 1138, and between 1006 and 1137 (Escherichia coli numbering). These nucleotides are exclusively conserved as 1005C.1138G and 1006C.1137G pairs in all Bacteria, Archaea and chloroplasts, whereas 1005G.1138C and 1006U.1137A pairs occur in Eukarya. We have mutagenized nucleotides 1005C-->G, 1006C-->U, 1137G-->A and 1138G-->C, both individually and in combinations, in a 23 S rRNA gene from the bacterium E. coli. The ability of 23 S rRNA to support cell growth is reduced when either of these base-pairs is disrupted, and it is completely abolished upon disruption of both base-pairs. Each mutant 23 S rRNA is assembled into 50 S subunits, but the mutant subunits do not stably interact with 30 S to engage in protein synthesis. Enzymatic and chemical probing of ribosomal particles reveals increased accessibility in the rRNA structure close to the sites of the mutations. The degree to which the mutations increase rRNA accessibility correlates with the severity of their phenotypic effects. Nucleotide 1131G is extremely reactive to dimethyl sulphate modification in wild-type subunits and ribosomes, but is rendered unreactive when either the pseudoknot is broken or when the r-proteins are removed. The structure of the pseudoknot region is possibly influenced by interaction of an r-protein at or close to the pseudoknot. Re-establishing the pseudoknot Watson-Crick interactions with one "eukaryal" (1005G.1138C or 1006U.1137A) pair and one "bacterial" C.G pair largely restores the structure and function of the rRNA. Bacterial ribosomes containing both these eukaryal pairs also participate in protein synthesis, although at much reduced efficiency, and the structure of their pseudoknot region is partially open and accessible.

Udgivelsesdato: 1995-Jul-11 The ribosomal protein L11 binds to the region of 23S rRNA associated with the GTPase-dependent steps of protein synthesis. Nucleotides 1054-1107 within this region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite. Twenty point mutations (G-->A and C-->T transitions) and numerous multiple mutations were generated. Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability. Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions. These effects are relieved by point mutations at other sites, indicating functional interconnections within the higher order structure of this 23S rRNA region. Several mutations prevent direct binding of r-protein L11 to 23S rRNA in vitro. These mutations are mainly in a short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where the protein probably makes nucleotide contacts. Some of these mutations also interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site on the rRNA. When added together to rRNA, proteins L10.(L12)4 and L11 bind cooperatively to overcome the effects of mutations at 1091 and 1099. The proteins also stimulate each others binding to rRNA mutated at 1087 or 1092, although in these cases binding remains clearly substoichiometric. Surprisingly, none of the mutations prevents incorporation of L11 into ribosomes in vivo, indicating that other, as yet unidentified, factors are involved in the cooperative assembly process.

The bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the family Pasteurellaceae is conferred by combinations of at least three genes: erm (42), which encodes a monomethyltransferase and confers a type I MLS B (macrolide, lincosamide, and streptogramin B) phenotype; msr (E), which encodes a macrolide efflux pump; and mph (E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence of erm (42), msr (E), and mph (E) and differentiates between these genes. In addition, the assay distinguishes P. multocida from M. haemolytica by amplifying distinctive fragments of the 23S rRNA ( rrl ) genes. One rrl fragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative for erm (42), msr (E), and mph (E). The multiplex system has been tested on more than 40 selected isolates of P. multocida and M. haemolytica and correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing.

The 23S rRNA nucleotide m(2)G2445 is highly conserved in bacteria, and in Escherichia coli this modification is added by the enzyme YcbY. With lengths of around 700 amino acids, YcbY orthologs are the largest rRNA methyltransferases identified in Gram-negative bacteria, and they appear to be fusions from two separate proteins found in Gram-positives. The crystal structures described here show that both the N- and C-terminal halves of E. coli YcbY have a methyltransferase active site and their folding patterns respectively resemble the Streptococcus mutans proteins Smu472 and Smu776. Mass spectrometric analyses of 23S rRNAs showed that the N-terminal region of YcbY and Smu472 are functionally equivalent and add the m(2)G2445 modification, while the C-terminal region of YcbY is responsible for the m(7)G2069 methylation on the opposite side of the same helix (H74). Smu776 does not target G2069, and this nucleotide remains unmodified in Gram-positive rRNAs. The E.coli YcbY enzyme is the first example of a methyltransferase catalyzing two mechanistically different types of RNA modification, and has been renamed as the Ribosomal large subunit methyltransferase, RlmKL. Our structural and functional data provide insights into how this bifunctional enzyme evolved.

Udgivelsesdato: 1994-Feb-11 The antibiotics thiostrepton and micrococcin bind to the GTPase region in domain II of 23S rRNA, and inhibit ribosomal A-site associated reactions. When bound to the ribosome, these antibiotics alter the accessibility of nucleotides 1067A and 1095A towards chemical reagents. Plasmid-coded Escherichia coli 23S rRNAs with single mutations at positions 1067 or 1095 were expressed in vivo. Mutant ribosomes are functional in protein synthesis, although those with transversion mutations function less effectively. Antibiotics were bound under conditions where wild-type and mutant ribosomes compete in the same reaction for drug molecules; binding was analysed by allele-specific footprinting. Transversion mutations at 1067 reduce thiostrepton binding more than 1000-fold. The 1067G substitution gives a more modest decrease in thiostrepton binding. The changes at 1095 slightly, but significantly, lower the affinity of ribosomes for thiostrepton, again with the G mutation having the smallest effect. Micrococcin binding to ribosomes is reduced to a far greater extent than thiostrepton by all the 1067 and 1095 mutations. Extrapolating these results to growing cells, mutation of nucleotide 1067A confers resistance towards micrococcin and thiostrepton, while substitutions at 1095A confer micrococcin resistance, and increase tolerance towards thiostrepton. These data support an rRNA tertiary structure model in which 1067A and 1095A lie in close proximity, and are key components in the drug binding site. None of the mutations alters either the higher order rRNA structure or the binding of r-proteins. We therefore conclude that thiostrepton and micrococcin interact directly with 1067A and 1095A.

Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m(5)U modifications. However, as previously shown, the m(5)U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m(5)U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m(5)U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.