The DNA sequence that encodes 23S rRNA domain V of Bacilus subtilis, nucleotides 2036 to 2672 (C. J. Green, G. C. Stewart, M. A. Hollis, B. S. Vold, and K. F. Bott, Gene 37:261-266, 1985), was cloned and used as a template from which to transcribe defined domain V RNA in vitro. The RNA transcripts served as a substrate in vitro for specific methylation of B. subtilis adenine 2085 (adenine 2058 in Escherichia coli 23S rRNA) by the ErmSF methyltransferase, an enzyme that confers resistance to the macrolide-lincosamidestreptogramin B group of antibiotics on Streptomycesfiradiae NRRL 2702, the host from which it was cloned.Thus, neither RNA sequences belonging to domains other than V nor the association of 23S rRNA with ribosomal proteins is needed for the specific methylation of adenine that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics.The ern N-methyltransferases (methylases) constitute a group of resistance factor enzymes that confer resistance to the macrolide, lincosamide, and streptogramin B antibiotics by methylating a specific adenine residue in 23S rRNA within the sequence GAAAG (11)(12)(13)(14). The precise location of the methylated adenine residue in Bacillus stearothermophilus 23S rRNA corresponds to that of adenine 2058 (A2058) in Escherichia coli 23S rRNA, as shown by Skinner et al. (23), and this location, in turn, corresponds to that of A2085 of Bacillus subtilis 23S rRNA, whose sequence was determined by Green et al. (6). Pulse-chase experiments with labeled adenosine (11) reveal that the methylase utilizes nascent 23S rRNA rather than mature 50S subunits as the substrate. Pulse-chase experiments, however, cannot rule out the possibility that the nascent 23S rRNA is utilized by the methylase in vivo in the form of partially assembled subparticles of the 50S subunit instead. Phenol-extracted 23S rRNA from ribosomes sensitive to macrolide, lincosamide, and streptogramin B antibiotics can serve as a substrate for a partially purified methylase preparation (22); however, the RNA thus prepared could contain an additional active structural component, present in the ribosome from which the rRNA substrate was extracted.The simplest model to describe the requirements for methylation of 23S rRNA postulates that rRNA alone can function as a substrate. If this is so, which sequence characteristics of 23S rRNA confer the specificity that enables its recognition by erm methylases? To evaluate the possibilities would require that a truncated rRNA be used as a substrate for in vitro methylation by a purified enn methylase preparation. MATERUILS AND METHODSStrains, plasmids, and primers. Bacterial strains and plasmids that were used in this study are listed in Table 1.Oligonucleotide primers that were used are listed in Table 2. ermSF cloning and expression in E. coli. PCR was performed (15), with modifications for the use of Streptomyces fradiae NRRL 2702 chromosomal DNA as the template. The two oligonucleotides 8803, a 45-mer, and 8800, a 24-mer, were used as upstream (sen...
ermSF (synonym tlrA) from Streptomyces fradiae NRRL 2702 confers resistance to the macrolide-lincosamide- streptogramin type B (MLS) superfamily of antibiotics. ErmSF specifically methylates Bacillus subtilis 23S rRNA in vitro at A2085 (B. subtilis coordinate, which is equivalent to the Escherichia coli coordinate A2058). In the present studies, partial B. subtilis 23S rRNA sequences containing portions of the peptidyltransferase circle which include A2085 were constructed in order to identify structural requirements needed for RNA to function as substrate of ErmSF. A model methylase substrate based on the 41-nucleotide construct DK111, ggCCUAUCCGUCGCGGGUUCGCCCGCGACAGGACGGA*AAGA, had methyl-acceptor activity. This sequence contains 23S rRNA stem 73 [Stade, K., et al. (1994) Nucleic Acids Res. 22, 1394-1399] underlined, flanking a tetraloop-like (UUCG), and the impaired sequence AAAGA, at the 3' end containing A2085 (A*). A set of systematic alterations introduced into the sequence suggested that the four unpaired nucleotides in stem 73 are necessary for methyl-acceptor activity, whereas inversion of 11 out 13 paired bases in stem 73 conferred no significant reduction in methyl-acceptor activity.
Combinatorial peptide display on phage M13 protein pIII was used to discover peptide sequences that selectively bind to ErmC methyltransferase from Bacillus subtilis. One peptide, Ac-LSGVIAT-NH 2 , inhibited methylation in vitro with a 50% inhibitory concentration of 20 M. Interestingly, the set of six peptides which inhibited ErmC stimulated ErmSF, a homologous methyltransferase from Streptomyces fradiae. Thus, Ac-LSGVIAT-NH 2 may not act directly at the catalytic center of ErmC, but may modulate its activity by binding at a structurally unrelated, but functionally linked, site.The ErmC N-methyltransferase of Staphylococcus aureus and its close relative ErmCЈ from Bacillus subtilis confer resistance to erythromycin and related macrolide antibiotics. These enzymes act by specifically methylating a single adenine residue in the peptidyl transferase center of bacterial 23S rRNA (for reviews, see references 7 and 8). The same methylation confers coresistance to structurally unrelated lincosamide and streptogramin type B antibiotics. The three groups are collectively known as the macrolide-lincosamide-streptogramin B (MLS) antibiotics, and the form of resistance based on the Erm group of methyltransferases is found in a wide range of pathogens. The three-dimensional structures of the ErmAM and ErmCЈ methyltransferases have recently been solved by nuclear magnetic resonance (3, 9) and X-ray crystallography (1, 5), respectively. These developments bring us closer to rationally devising ligands that will selectively bind to Erm enzymes and possibly reduce the efficiency with which they confer MLS resistance.Macrolide antibiotics have served as a mainstay of antimicrobial therapy for approximately the last half century, especially in instances where the recipient of the antibiotic was allergic to beta-lactam antibiotics. Attempts have been made to discover Erm methyltransferase inhibitors that maintain the effectiveness of macrolide antibiotics in the face of the increasing frequency of resistant isolates (2, 3). The latter work (3) was part of a series of major structural studies of Erm methyltransferases (1, 5, 9).Nonpeptide ligands which displace an inhibitory peptide from its binding site on the enzyme might, themselves, have inhibitory activity. The use of inhibitory peptides might thus serve as a platform for the discovery of nonpeptide inhibitors of Erm enzymes. A proposed way to achieve this would be based on the discovery of test ligands to displace an inhibitory peptide from its association with its cognate Erm target. The displacement of an inhibitory ligand would also be easier to measure than methyltransferase activity.Large-scale screening of inhibitory ligands by a direct assay of methyltransferase catalytic activity is cumbersome since it requires the separation of product (methyl-labeled 23S rRNA) from substrate (unreacted S-adenosyl-L-methionine). We report the use of combinatorial phage display to discover peptides that inhibit ErmCЈ methylase activity and which might serve as displaceable ligands in ...
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