RESUMEN
Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.
Asunto(s)
Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/química , Uridina/análogos & derivados , Uridina/metabolismo , Uridina/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/químicaRESUMEN
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 m5U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m5U54 is subsequently thiolated to form m5s2U54. 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 m1s4Ψ. The methyl and thiol groups in m1s4Ψ and m5s2U 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 m1A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.
Asunto(s)
Desulfurococcaceae/genética , Nanoarchaeota/genética , Conformación de Ácido Nucleico , ARN de Transferencia/ultraestructura , Archaea/genética , Archaea/ultraestructura , Escherichia coli/genética , Metilación , Filogenia , ARN de Transferencia/genética , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/ultraestructuraRESUMEN
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 (tlyA I ) but also with a mycobacterial tlyA II 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 tlyA II 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Campylobacter jejuni/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Biopelículas , Células CACO-2 , Campylobacter jejuni/genética , Capreomicina , Células Epiteliales , Regulación Bacteriana de la Expresión Génica , Genes de ARNr/genética , Humanos , Macrófagos , Metilación , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Células RAW 264.7 , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Virulencia , Factores de Virulencia/genéticaRESUMEN
OBJECTIVES: To 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The 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.
Asunto(s)
Firmicutes , Lincosamidas , Macrólidos , Metiltransferasas , Estreptogramina B , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Firmicutes/efectos de los fármacos , Firmicutes/enzimología , Lincosamidas/farmacología , Macrólidos/farmacología , Metiltransferasas/genética , Ribosomas , Estreptogramina B/farmacologíaRESUMEN
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 N6-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 2Î-O-ribose methylations within helices 44 and 45 of the rRNA.
Asunto(s)
Adenosina/metabolismo , Metiltransferasas/metabolismo , Nanoarchaeota/genética , ARN Ribosómico 16S/metabolismo , Desulfurococcaceae/enzimología , Desulfurococcaceae/genética , Escherichia coli/genética , Metilación , Metiltransferasas/genética , Nanoarchaeota/enzimología , ARN Ribosómico 16S/química , Subunidades Ribosómicas Pequeñas de Archaea/metabolismoRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Cetólidos/farmacología , Metiltransferasas/genética , ARN Ribosómico 23S/genéticaRESUMEN
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.
Asunto(s)
ADN Mitocondrial/genética , Audición/genética , Ribosomas Mitocondriales/metabolismo , Factores de Transcripción/genética , Animales , Sordera/genética , Femenino , Pérdida Auditiva Sensorineural/inducido químicamente , Pérdida Auditiva Sensorineural/genética , Masculino , Metilación , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Mutación Puntual/genética , ARN Ribosómico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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 m5U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N5,N10-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses two homologs of trmFO, but surprisingly lacks the m5U54 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 m5U1939 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flavoproteínas/metabolismo , Metiltransferasas/metabolismo , Mycoplasma capricolum/enzimología , ARN Ribosómico 23S/metabolismo , Proteínas Bacterianas/genética , Biocatálisis , Flavoproteínas/genética , Metilación , Metiltransferasas/genética , Mycoplasma capricolum/genética , ARN Ribosómico 23S/química , ARN de Transferencia/metabolismo , Uridina/metabolismoRESUMEN
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.
Asunto(s)
Farmacorresistencia Bacteriana , Genes de ARNr , Macrólidos/farmacología , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/genética , Mutación , Pasteurella multocida/efectos de los fármacos , Pasteurella multocida/genética , Antibacterianos/farmacología , Genoma Bacteriano , Humanos , Mannheimia haemolytica/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Operón , Pasteurella multocida/aislamiento & purificación , ARN Ribosómico 23S/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli/química , Metiltransferasas/química , ARN Ribosómico 23S/metabolismo , Dominio Catalítico , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Metiltransferasas/metabolismo , Modelos Moleculares , ARN Ribosómico 23S/química , Streptococcus mutans/enzimologíaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Capreomicina/farmacología , ARN Ribosómico/metabolismo , ARNt Metiltransferasas/metabolismo , Proteínas Bacterianas/genética , Metilación , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación de Ácido Nucleico , Subunidades Ribosómicas/metabolismo , Viomicina/farmacología , ARNt Metiltransferasas/genéticaRESUMEN
Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m5C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m5C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m5C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/química , Enterococcus faecium/enzimología , Metiltransferasas/química , ARN Ribosómico 16S/química , Secuencia de Aminoácidos , Anticodón/química , Proteínas Bacterianas/metabolismo , Codón/química , Farmacorresistencia Bacteriana , Enterococcus faecium/efectos de los fármacos , Metiltransferasas/genética , Datos de Secuencia Molecular , ARN Ribosómico 16S/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m(5)U methyltransferases appear to have arisen in Bacteria and were then dispersed by horizontal transfer of an rlmD-type gene to the Archaea and Eukaryota. The bacterium Escherichia coli has three gene paralogs and these encode the methyltransferases TrmA that targets m(5)U54 in tRNAs, RlmC (formerly RumB) that modifies m(5)U747 in 23S rRNA, and RlmD (formerly RumA) the archetypical enzyme that is specific for m(5)U1939 in 23S rRNA. The thermococcale archaeon Pyrococcus abyssi possesses two m(5)U methyltransferase paralogs, PAB0719 and PAB0760, with sequences most closely related to the bacterial RlmD. Surprisingly, however, neither of the two P. abyssi enzymes displays RlmD-like activity in vitro. PAB0719 acts in a TrmA-like manner to catalyze m(5)U54 methylation in P. abyssi tRNAs, and here we show that PAB0760 possesses RlmC-like activity and specifically methylates the nucleotide equivalent to U747 in P. abyssi 23S rRNA. The findings indicate that PAB0719 and PAB0760 originated as RlmD-type m(5)U methyltransferases and underwent changes in target specificity after their acquisition by a Thermococcales ancestor from a bacterial source.
Asunto(s)
Archaea/enzimología , Archaea/genética , Metiltransferasas/metabolismo , Pyrococcus abyssi/enzimología , Pyrococcus abyssi/genética , ARN Ribosómico/genética , ARN de Transferencia/genética , Archaea/metabolismo , Metilación , Pyrococcus abyssi/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Uridina/metabolismoRESUMEN
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.
Asunto(s)
Aminoglicósidos/farmacología , Proteínas Bacterianas/genética , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana Múltiple , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Metiltransferasas/genética , beta-Lactamasas/genética , Amicacina/farmacología , ADN Bacteriano/química , ADN Bacteriano/genética , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Gentamicinas/farmacología , Humanos , India , Datos de Secuencia Molecular , Plásmidos , Análisis de Secuencia de ADN , Reino UnidoRESUMEN
Aminoglycoside antibiotics remain the drugs of choice for treatment of Pseudomonas aeruginosa infections, particularly for respiratory complications in cystic-fibrosis patients. Previous studies on other bacteria have shown that aminoglycosides have their primary target within the decoding region of 16S rRNA helix 44 with a secondary target in 23S rRNA helix 69. Here, we have mapped P. aeruginosa rRNAs using MALDI mass spectrometry and reverse transcriptase primer extension to identify nucleotide modifications that could influence aminoglycoside interactions. Helices 44 and 45 contain indigenous (housekeeping) modifications at m (4)Cm1402, m (3)U1498, m (2)G1516, m (6) 2A1518, and m (6) 2A1519; helix 69 is modified at m (3)Ψ1915, with m (5)U1939 and m (5)C1962 modification in adjacent sequences. All modifications were close to stoichiometric, with the exception of m (3)Ψ1915, where about 80% of rRNA molecules were methylated. The modification status of a virulent clinical strain expressing the acquired methyltransferase RmtD was altered in two important respects: RmtD stoichiometrically modified m (7)G1405 conferring high resistance to the aminoglycoside tobramycin and, in doing so, impeded one of the methylation reactions at C1402. Mapping the nucleotide methylations in P. aeruginosa rRNAs is an essential step toward understanding the architecture of the aminoglycoside binding sites and the rational design of improved drugs against this bacterial pathogen.
Asunto(s)
Aminoglicósidos/química , Pseudomonas aeruginosa/genética , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , ARN Ribosómico 23S/química , ARN Ribosómico 23S/metabolismo , Aminoglicósidos/genética , Aminoglicósidos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dicroismo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Metiltransferasas/metabolismo , ARN Ribosómico 23S/metabolismo , Uridina/análogos & derivados , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Biocatálisis , Metiltransferasas/química , Datos de Secuencia Molecular , ARN Ribosómico 23S/química , Alineación de Secuencia , Uridina/metabolismoRESUMEN
Mannheimia haemolytica and Pasteurella multocida are aetiological agents commonly associated with respiratory tract infections in cattle. Recent isolates of these pathogens have been shown to be resistant to macrolides and other ribosome-targeting antibiotics. Direct analysis of the 23S rRNAs by mass spectrometry revealed that nucleotide A2058 is monomethylated, consistent with a Type I erm phenotype conferring macrolide-lincosamide resistance. The erm resistance determinant was identified by full genome sequencing of isolates. The sequence of this resistance determinant, now termed erm(42), has diverged greatly from all previously characterized erm genes, explaining why it has remained undetected in PCR screening surveys. The sequence of erm(42) is, however, completely conserved in six independent M. haemolytica and P. multocida isolates, suggesting relatively recent gene transfer between these species. Furthermore, the composition of neighbouring chromosomal sequences indicates that erm(42) was acquired from other members of the Pasteurellaceae. Expression of recombinant erm(42) in Escherichia coli demonstrated that the enzyme retains its properties as a monomethyltransferase without any dimethyltransferase activity. Erm(42) is a novel addition to the Erm family: it is phylogenetically distant from the other Erm family members and it is unique in being a bona fide monomethyltransferase that is disseminated between bacterial pathogens.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/enzimología , Metiltransferasas/metabolismo , Pasteurella multocida/efectos de los fármacos , Pasteurella multocida/enzimología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Bacteriana/fisiología , Mannheimia haemolytica/genética , Metiltransferasas/genética , Pruebas de Sensibilidad Microbiana , Pasteurella multocida/química , Pasteurella multocida/genética , Reacción en Cadena de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
Asunto(s)
Antibacterianos/química , Mannheimia haemolytica/efectos de los fármacos , Pasteurella multocida/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Tilosina/análogos & derivados , Drogas Veterinarias/química , Antibacterianos/farmacología , Sitios de Unión , Disacáridos/química , Disacáridos/farmacología , Escherichia coli/química , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Mannheimia haemolytica/química , Mannheimia haemolytica/genética , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mutación , Nucleótidos/genética , Pasteurella multocida/química , Pasteurella multocida/genética , ARN Ribosómico/química , ARN Ribosómico/genética , Ribosomas/química , Fracciones Subcelulares/química , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Transcripción Genética/efectos de los fármacos , Tilosina/química , Tilosina/farmacología , Drogas Veterinarias/farmacologíaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Macrólidos/farmacología , Mannheimia haemolytica/efectos de los fármacos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Pasteurella multocida/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Lincosamidas/farmacología , Mannheimia haemolytica/genética , Pasteurella multocida/genética , Estreptogramina B/farmacologíaRESUMEN
Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2'-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2'-O-methylation at position 34 in both tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). Loss of YibK methylation reduces the efficiency of codon-wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/ß knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2'-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N6-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.