Indigenous and acquired modifications in the aminoglycoside binding sites of Pseudomonas aeruginosa rRNAs.
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.
Departamento de Sanidad Animal; Facultad de Veterinaria; Universidad Complutense de Madrid; Madrid, Spain; Centro de Vigilancia Sanitaria Veterinaria (VISAVET); Universidad Complutense de Madrid; Madrid, Spain.
Belen
B
Gutierrez
Stephen
S
Douthwaite
Bruno
B
Gonzalez-Zorn
D000617:Aminoglycosides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000617:Aminoglycosides; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D008780:Methyltransferases
RmtD; aminoglycosides; methyltransferases; pseudomonas; rRNA modifications