Studies on a 50S Ribosomal Precursor Particle as a Substrate for <em>erm </em>E Methyltransferase Enzyme in <em>Staphylococcus aureus </em>.

<p>Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by <em>erm</em> methyl transferase enzymes. We are interested in investigating the true substrate for methylation because it is known from our work and the work of others that fully assembled 50S subunits are not substrates for methylation. We have published a model for 50S ribosomal subunit formation where, the precursor particle that accumulates in erythromycin treated cells is a target for methyl transferase activity. Current studies are aimed at investigating the role of the precursor particle as substrate for <em>erm</em>E methyltransferase activity and the competition between this enzyme and erythromycin for the 50S precursor particle.</p><p>Slot-blot hybridization experiments have identified the presence of 23S rRNA in the 50S precursor region. Quantitation of the 23S rRNA in these blots also revealed that the percentage of the precursor increased as the concentration of erythromycin was increased in the growth media. Ribosomal proteins of <em>S. aureus</em> were studied by two-dimensional gel electrophoresis. Protein content of the 50S precursor particle was analyzed by MALDI-TOF. These studies have identified 16 50S ribosomal proteins in the precursor region.</p><p>Methyltransferase assays showed that 50S precursor particle was a substrate for <em>erm</em>E methyltransferase. Importantly, RNA that is already assembled into 50S subunits was not a substrate for the enzyme. Inhibition curves showed that macrolide, lincosamide, and streptogramin B (MLS_B) drugs bound to the precursor particle with similar affinity and inhibited the <em>erm</em>E methyltransferase activity. Competition experiments suggested that the enzyme can displace erythromycin from the 50S precursor particle and that <em>erm</em> methyltransferase has a lower association constant for the precursor particle compared to that of the erythromycin. This suggests that higher concentrations of erythromycin are needed to combat <em>erm</em> induced resistance.</p><p>These studies shed light on the interaction of <em>erm</em>E methyltransferase and erythromycin in the clinically important pathogen <em>S. aureus</em>.</p>

Name thesis:degree_name

PhD (Doctor of Philosophy)

Level thesis:degree_level

Dissertation - unrestricted

Discipline thesis:degree_discipline

Biomedical Sciences

Year dc:date.issued

2007

Author dc:creator

• Pokkunuri, Indira

• 50S precursor particle
• ermE methyltransferase
• MLSB antibiotics
• Chemical and Pharmacologic Phenomena
• Medical Sciences
• Medicine and Health Sciences

Statement dc:rights

• Copyright by the authors.

Repository record dc:identifier

https://dc.etsu.edu/etd/2092

OAI identifier oai:identifier

oai:dc.etsu.edu:etd-3453