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
Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , ARN Ribosómico , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Subunidades Ribosómicas Grandes/metabolismo , Subunidades Ribosómicas Grandes/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/genética , Ribosomas/metabolismo , Ribosomas/genética , ARN Ribosómico/metabolismo , ARN Ribosómico/genética , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/químicaRESUMEN
The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Antibacterianos/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , ARN Ribosómico 23S/química , ARN Ribosómico 23S/metabolismo , Metiltransferasas/metabolismo , Metiltransferasas/química , Metiltransferasas/antagonistas & inhibidores , Metilación , Modelos Moleculares , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/genéticaRESUMEN
A slowly growing, non-chromogenic mycobacterial strain was isolated from sputum and bronchial lavage fluid samples of a patient presenting with productive cough, blood-tinged sputum, low-grade fever, and weakness. A positive acid-fast bacilli sputum smear result prompted the initiation of an anti-tuberculosis regimen. Multiplex real-time PCR showed a negative result for Mycobacterium tuberculosis complex and a positive result for nontuberculous mycobacteria. The DNA chip test confirmed this organism as a member of the genus Mycobacterium, but could not specify the species. Interestingly, the mycolic acid patterns obtained by HPLC nearly overlapped with those of M. simulans. The sequences of the Mycobacterium 16S rRNA gene and 16S-23S internal transcribed spacer region were unique and were found to have 100% similarity with those of M. riyadhense. After a review of the literature, we report this case as the first Korean case of M. riyadhense lung infection.
Asunto(s)
Adulto , Femenino , Humanos , Antituberculosos/farmacología , Cromatografía Líquida de Alta Presión , Enfermedades Pulmonares/microbiología , Pruebas de Sensibilidad Microbiana , Mycobacterium/clasificación , Infecciones por Mycobacterium/microbiología , Mycobacterium tuberculosis/genética , Ácidos Micólicos/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , ARN Ribosómico 16S/química , ARN Ribosómico 23S/química , República de Corea , Análisis de Secuencia de ADNRESUMEN
Using three reference strains of Brachyspira hyodysenteriae (B204, B234, B169), one B. pilosicoli (P43/6/78), one B. murdochii (56-150), one B. intermedia (PWS/A), one B. innocens (B256) and ten Korean isolates, PCR-RFLP analysis of DNA encoding 23S rRNA was performed to establish a rapid and accurate method for characterizing porcine intestinal spirochetes. Consequently, B. hyodysenteriae and B. pilosicoli revealed different restriction patterns; however, the other three species shared the same pattern. These findings are not consistent with a prior report. Differences in 23S rRNA gene sequences, between two B. murdochii strains, 56-150 and 155-20, were observed. These results indicate that 23S rRNA PCR-RFLP could be used as an identification method for pathogenic Brachyspira spp. (B. hyodysenteriae and B. pilosicoli) as well as an epidemiological tool for characterizing spirochetes isolated from swine.