Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics

ABSTRACT Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites. </jats:p

A <i>cfr</i>-Like Gene from <i>Clostridium difficile</i> Confers Multiple Antibiotic Resistance by the Same Mechanism as the <i>cfr</i> Gene

The Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Many cfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from a Peptoclostridium difficile (formerly Clostridium difficile) strain does function as a Cfr protein. The enzyme is expressed in Escherichia coli and shows elevated MICs for five classes of antibiotics. A primer extension stop indicates a modification at A2503 in 23S rRNA

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

Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance

ABSTRACT Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli , although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin. </jats:p

Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start

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

The Maltase Involved in Starch Metabolism in Barley Endosperm Is Encoded by a Single Gene

During germination and early seedling growth of barley (Hordeum vulgare), maltase is responsible for the conversion of maltose produced by starch degradation in the endosperm to glucose for seedling growth. Despite the potential relevance of this enzyme for malting and the production of alcoholic beverages, neither the nature nor the role of maltase is fully understood. Although only one gene encoding maltase has been identified with certainty, there is evidence for the existence of other genes and for multiple forms of the enzyme. It has been proposed that maltase may be involved directly in starch granule degradation as well as in maltose hydrolysis. The aim of our work was to discover the nature of maltase in barley endosperm. We used ion exchange chromatography to fractionate maltase activity from endosperm of young seedlings, and we partially purified activity for protein identification. We compared maltase activity in wild-type barley and transgenic lines with reduced expression of the previously-characterised maltase gene Agl97, and we used genomic and transcriptomic information to search for further maltase genes. We show that all of the maltase activity in the barley endosperm can be accounted for by a single gene, Agl97. Multiple forms of the enzyme most likely arise from proteolysis and other post-translational modifications

Investigating the Antiproliferative Activity of High Affinity DNA Aptamer on Cancer Cells

10.1371/journal.pone.0050964PLoS ONE81

Mapping of ribosomal 23S ribosomal RNA modifications in Clostridium sporogenes

All organisms contain RNA modifications in their ribosomal RNA (rRNA), but the importance, positions and exact function of these are still not fully elucidated. Various functions such as stabilizing structures, controlling ribosome assembly and facilitating interactions have been suggested and in some cases substantiated. Bacterial rRNA contains much fewer modifications than eukaryotic rRNA. The rRNA modification patterns in bacteria differ from each other, but too few organisms have been mapped to draw general conclusions. This study maps 23S ribosomal RNA modifications in Clostridium sporogenes that can be characterized as a non-toxin producing Clostridium botulinum. Clostridia are able to sporulate and thereby survive harsh conditions, and are in general considered to be resilient to antibiotics. Selected regions of the 23S rRNA were investigated by mass spectrometry and by primer extension analysis to pinpoint modified sites and the nature of the modifications. Apparently, C. sporogenes 23S rRNA contains few modifications compared to other investigated bacteria. No modifications were identified in domain II and III of 23S rRNA. Three modifications were identified in domain IV, all of which have also been found in other organisms. Two unusual modifications were identified in domain V, methylated dihydrouridine at position U2449 and dihydrouridine at position U2500 (Escherichia coli numbering), in addition to four previously known modified positions. The enzymes responsible for the modifications were searched for in the C. sporogenes genome using BLAST with characterized enzymes as query. The search identified genes potentially coding for RNA modifying enzymes responsible for most of the found modifications.</p

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 (k(obs )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