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1.  Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase 
The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the RlmN methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine in the enzyme and a transient cross-linking to the RNA, but they differ in which carbon atom in the adenine they methylate. Comparative sequence analysis identifies differentially conserved residues that indicate functional sequence divergence between the two classes of Cfr- and RlmN-like sequences. The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed.
doi:10.1128/AAC.00448-13
PMCID: PMC3719738  PMID: 23752511
2.  The Order Bacillales Hosts Functional Homologs of the Worrisome cfr Antibiotic Resistance Gene 
The cfr gene encodes the Cfr methyltransferase that methylates a single adenine in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to several classes of antibiotics that include drugs of clinical and veterinary importance. This paper describes a first step toward elucidating natural residences of the worrisome cfr gene and functionally similar genes. Three cfr-like genes from the order Bacillales were identified from BLAST searches and cloned into plasmids under the control of an inducible promoter. Expression of the genes was induced in Escherichia coli, and MICs for selected antibiotics indicate that the cfr-like genes confer resistance to PhLOPSa (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A) antibiotics in the same way as the cfr gene. In addition, modification at A2503 on 23S rRNA was confirmed by primer extension. Finally, expression of the Cfr-like proteins was verified by SDS gel electrophoresis of whole-cell extracts. The work shows that cfr-like genes exist in the environment and that Bacillales are natural residences of cfr-like genes.
doi:10.1128/AAC.00673-12
PMCID: PMC3393444  PMID: 22547628
3.  Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome 
Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms.
doi:10.1128/AAC.05702-11
PMCID: PMC3264260  PMID: 22143525
4.  Trends towards Lower Antimicrobial Susceptibility and Characterization of Acquired Resistance among Clinical Isolates of Brachyspira hyodysenteriae in Spain ▿ 
The antimicrobial susceptibility of clinical isolates of Brachyspira hyodysenteriae in Spain was monitored, and the underlying molecular mechanisms of resistance were investigated. MICs of tylosin, tiamulin, valnemulin, lincomycin, and tylvalosin were determined for 87 B. hyodysenteriae isolates recovered from 2008 to 2009 by broth dilution. Domain V of the 23S rRNA gene and the ribosomal protein L3 gene were sequenced in 20 isolates for which the tiamulin MIC was ≥4 μg/ml, presenting decreased susceptibility, and in 18 tiamulin-susceptible isolates (MIC ≤ 0.125 μg/ml), and all isolates were typed by multiple-locus variable-number tandem repeats analysis. A comparison with antimicrobial susceptibility data from 2000 to 2007 showed an increase in pleuromutilin resistance over time, doubling the number of isolates with decreased susceptibility to tiamulin. No alteration in susceptibility was detected for lincomycin, and the MIC of tylosin remained high (MIC50 > 128 μg/ml). The decreased susceptibility to tylosin and lincomycin can be explained by mutations at position A2058 of the 23S rRNA gene (Escherichia coli numbering). A2058T was the predominant mutation, but A2058G also was found together with a change of the neighboring base pair at positions 2057 to 2611. The role of additional point mutations in the vicinity of the peptidyl transferase center and mutations in the L3 at amino acids 148 and 149 and their possible involvement in antimicrobial susceptibility are considered. An association between G2032A and high levels of tiamulin and lincomycin MICs was found, suggesting an increasing importance of this mutation in antimicrobial resistance of clinical isolates of B. hyodysenteriae.
doi:10.1128/AAC.01749-10
PMCID: PMC3122432  PMID: 21555771
5.  Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides 
Artificial DNA, PNA & XNA  2012;3(1):14-21.
We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5′-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°Nm, Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.
doi:10.4161/adna.19272
PMCID: PMC3368812  PMID: 22679529
enzymatic synthesis; modified nucleotide; polymerase; triphosphate; α-L-LNA
6.  Mutations in 23S rRNA at the Peptidyl Transferase Center and Their Relationship to Linezolid Binding and Cross-Resistance▿  
Antimicrobial Agents and Chemotherapy  2010;54(11):4705-4713.
The oxazolidinone antibiotic linezolid targets the peptidyl transferase center (PTC) on the bacterial ribosome. Thirteen single and four double 23S rRNA mutations were introduced into a Mycobacterium smegmatis strain with a single rRNA operon. Converting bacterial base identity by single mutations at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid. The largest decrease in linezolid susceptibility for any of the introduced single mutations was observed with the G2576U mutation at a position that is 7.9 Å from linezolid. Smaller decreases were observed with the A2503G, U2504G, and G2505A mutations at nucleotides proximal to linezolid, showing that the degree of resistance conferred is not simply inversely proportional to the nucleotide-drug distance. The double mutations G2032A-C2499A, G2032A-U2504G, C2055A-U2504G, and C2055A-A2572U had remarkable synergistic effects on linezolid resistance relative to the effects of the corresponding single mutations. This study emphasizes that effects of rRNA mutations at the PTC are organism dependent. Moreover, the data show a nonpredictable cross-resistance pattern between linezolid, chloramphenicol, clindamycin, and valnemulin. The data underscore the significance of mutations at distal nucleotides, either alone or in combination with other mutated nucleotides, in contributing to linezolid resistance.
doi:10.1128/AAC.00644-10
PMCID: PMC2976117  PMID: 20696869
7.  Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer 
Nucleic Acids Research  2010;39(3):1155-1164.
A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15–0.50 kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by Tm versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation.
doi:10.1093/nar/gkq823
PMCID: PMC3035450  PMID: 20870750
8.  Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria 
Nucleic Acids Research  2009;38(5):1652-1663.
The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity.
doi:10.1093/nar/gkp1142
PMCID: PMC2836569  PMID: 20007606
9.  The Cfr rRNA Methyltransferase Confers Resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A Antibiotics 
A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones.
doi:10.1128/AAC.00131-06
PMCID: PMC1489768  PMID: 16801432
10.  Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets 
Background
DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes.
Results
We investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1).
Conclusion
The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.
doi:10.1186/1471-2199-7-19
PMCID: PMC1501032  PMID: 16753066
11.  Interaction of Pleuromutilin Derivatives with the Ribosomal Peptidyl Transferase Center 
Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity.
doi:10.1128/AAC.50.4.1458-1462.2006
PMCID: PMC1426994  PMID: 16569865
12.  Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start 
Nucleic Acids Research  2005;33(22):7129-7137.
The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0°C) than the wild-type double-stranded DNA (dsDNA, 26.0°C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.5 and 39.0°C). Zipping of pyrene moieties in an easily denaturing duplex gave formation of a strong excimer band at 480 nm upon excitation at 343 nm in the steady-state fluorescence spectra. The excimer band disappeared upon addition of a similar short dsDNA, but remained when adding a 128mer dsDNA containing the same sequence. When P was inserted into 2′-OMe-RNA strands, the duplex with zipping P was found to be more stable (42.0°C) than duplexes with the complementary ssDNAs (31.5 and 19.5°C). The excimer band observed in the ds2′-OMe-RNA with zipping P had marginal changes upon addition of both 8 and 128mer dsDNA. Synthesized oligonucleotides were tested in a transcriptional inhibition assay for targeting of the open complex formed by Escherichia coli RNA polymerase with the lac UV-5 promoter using the above mentioned 128mer dsDNA. Inhibition of transcription was observed for 8mer DNAs possessing pyrene intercalators and designed to target both template and non-template DNA strands within the open complex. The observed inhibition was partly a result of unspecific binding of the modified DNAs to the RNA polymerase. Furthermore, the addition of 8mer DNA with three bulged insertions of P designed to be complementary to the template strand at the +36 to +43 position downstream of the transcription start resulted in a specific halt of transcription producing a truncated RNA transcript. This is to our knowledge the first report of an RNA elongation stop mediated by a small DNA sequence possessing intercalators. The insertions of P opposite to each other in ds2′-OMe-RNA showed inhibition efficiency of 96% compared with 25% for unmodified ds2′-OMe-RNA.
doi:10.1093/nar/gki1019
PMCID: PMC1322271  PMID: 16377781
13.  Resistance to the Peptidyl Transferase Inhibitor Tiamulin Caused by Mutation of Ribosomal Protein L3 
The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.
doi:10.1128/AAC.47.9.2892-2896.2003
PMCID: PMC182624  PMID: 12936991
14.  Interaction of Avilamycin with Ribosomes and Resistance Caused by Mutations in 23S rRNA 
Antimicrobial Agents and Chemotherapy  2002;46(11):3339-3342.
The antibiotic growth promoter avilamycin inhibits protein synthesis by binding to bacterial ribosomes. Here the binding site is further characterized on Escherichia coli ribosomes. The drug interacts with domain V of 23S rRNA, giving a chemical footprint at nucleotides A2482 and A2534. Selection of avilamycin-resistant Halobacterium halobium cells revealed mutations in helix 89 of 23S rRNA. Furthermore, mutations in helices 89 and 91, which have previously been shown to confer resistance to evernimicin, give cross-resistance to avilamycin. These data place the binding site of avilamycin on 23S rRNA close to the elbow of A-site tRNA. It is inferred that avilamycin interacts with the ribosomes at the ribosomal A-site interfering with initiation factor IF2 and tRNA binding in a manner similar to evernimicin.
doi:10.1128/AAC.46.11.3339-3342.2002
PMCID: PMC128742  PMID: 12384333
15.  Macrolide Resistance Conferred by Base Substitutions in 23S rRNA 
doi:10.1128/AAC.45.1.1-12.2001
PMCID: PMC90232  PMID: 11120937

Results 1-15 (15)