Scintillation proximity assay for measurement of RNA methylation

Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA–protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay. We have adapted and optimized a scintillation proximity assay (SPA) to replace the more costly, wasteful and cumbersome filter binding assay and demonstrate its utility in studies of three distinct methyltransferases, RmtA, KsgA and ErmC’. In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC’ most efficiently methylates protein-depleted or protein-free 23S rRNA. This assay does not utilize engineered affinity tags that are often required in SPA, and is capable of detecting either radiolabeled RNA or RNA–protein complex. We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors. This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format

High prevalence of plasmid-mediated 16S rRNA methylase gene rmtB among Escherichia coli clinical isolates from a Chinese teaching hospital

<p>Abstract</p> <p>Background</p> <p>Recently, production of 16S rRNA methylases by Gram-negative bacilli has emerged as a novel mechanism for high-level resistance to aminoglycosides by these organisms in a variety of geographic locations. Therefore, the spread of high-level aminoglycoside resistance determinants has become a great concern.</p> <p>Methods</p> <p>Between January 2006 and July 2008, 680 distinct <it>Escherichia coli </it>clinical isolates were collected from a teaching hospital in Wenzhou, China. PCR and DNA sequencing were used to identify 16S rRNA methylase and extended-spectrum β-lactamase (ESBL) genes, including <it>armA </it>and <it>rmtB</it>, and in situ hybridization was performed to determine the location of 16S rRNA methylase genes. Conjugation experiments were subsequently performed to determine whether aminoglycoside resistance was transferable from the <it>E. coli </it>isolates via 16S rRNA methylase-bearing plasmids. Homology of the isolates harboring 16S rRNA methylase genes was determined using pulse-field gel electrophoresis (PFGE).</p> <p>Results</p> <p>Among the 680 <it>E. coli </it>isolates, 357 (52.5%), 346 (50.9%) and 44 (6.5%) isolates were resistant to gentamicin, tobramycin and amikacin, respectively. Thirty-seven of 44 amikacin-resistant isolates harbored 16S rRNA methylase genes, with 36 of 37 harboring the <it>rmtB </it>gene and only one harboring <it>armA</it>. The positive rates of 16S rRNA methylase genes among all isolates and amikacin-resistant isolates were 5.4% (37/680) and 84.1% (37/44), respectively. Thirty-one isolates harboring 16S rRNA methylase genes also produced ESBLs. In addition, high-level aminoglycoside resistance could be transferred by conjugation from four <it>rmtB</it>-positive donors. The plasmids of incompatibility groups IncF, IncK and IncN were detected in 34, 3 and 3 isolates, respectively. Upstream regions of the <it>armA </it>gene contained <it>IS</it>CR1 and <it>tnpU</it>, the latter a putative transposase gene,. Another putative transposase gene, <it>tnpD</it>, was located within a region downstream of <it>armA</it>. Moreover, a transposon, Tn<it>3</it>, was located upstream of the <it>rmtB</it>. Nineteen clonal patterns were obtained by PFGE, with type H representing the prevailing pattern.</p> <p>Conclusion</p> <p>A high prevalence of plasmid-mediated <it>rmtB </it>gene was found among clinical <it>E. coli </it>isolates from a Chinese teaching hospital. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the <it>rmtB </it>gene.</p

blaKPC and rmtB on a single plasmid in Enterobacter amnigenus and Klebsiella pneumoniae isolates from the same patient

Enterobacter amnigenus (EA76) and Klebsiella pneumoniae (KP76) isolates with multidrug-resistant (MDR) patterns were identified from the same patient in the neurosurgery department of our hospital. An outbreak of MDR K. pneumoniae had also occurred in this department. To characterize the resistance mechanism and molecular epidemiology of these isolates, sequential experiments including antimicrobial susceptibility testing, polymerase chain reaction (PCR), plasmid analysis, pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. EA76 and KP76 were resistant to all of the antibiotics tested, except colistin and tigecycline. blaKPC-2, blaTEM-1, blaSHV-12, blaCTX-M-3, blaCTX-M-14, and rmtB genes were identified in both isolates, with blaKPC-2, blaTEM-1, blaCTX-M-14, and rmtB being co-carried on one plasmid in each isolate. Further analysis showed different restriction patterns between the two KPC-carrying plasmids. Of the 11 carbapenem-resistant isolates found in the outbreak, all were resistant to all of the β-lactams tested, with 63.64% (7/11) also exhibiting resistance to aminoglycosides and 72.73% (8/11) exhibiting resistance to quinolones. PCR analysis and molecular typing of the 11 K. pneumoniae strains revealed that the seven aminoglycoside-resistant isolates shared the same antibiotic-resistant gene pattern and identical or one-band-difference PFGE profiles relative to KP76. In addition, all of the eight aminoglycoside-resistant isolates, including KP76, belonged to the national epidemic clone ST11. The overall results indicate the emergence of E. amnigenus and outbreak of ST11 K. pneumoniae, with both co-harboring blaKPC and rmtB genes on a single plasmid in our neurosurgery wards

Kinetic characterization of GES-22 beta-lactamase harboring the M169L clinical mutation

The class A p-lactamase GES-22 has been identified in Acinetobacter baumannii isolates in Turkey, and subsequently shown to differ from GES-11 by a single substitution (M169L). Because M169 is part of the omega loop, a structure that is known to have major effects on substrate selectivity in class A beta-lactamases, we expressed, purified and kinetically characterized this novel variant. Our results show that compared with GES-11(6xHis), GES-22(6xHis) displays more efficient hydrolysis of penicillins, and aztreonam, but a loss of efficiency against ceftazidime. In addition, the M169L substitution confers on GES-22 more efficient hydrolysis of the mechanistic inhibitors clavulanic acid and sulbactam. These effects are highly similar to other mutations at the homologous position in other class A beta-lactamases, suggesting that this methionine has a key structural role in aligning active site residues and in substrate selectivity across the class.Recep Tayyip Erdogan University:BAP-2013.102.03.12 Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK): TUBITAK-113Z054 United States Department of Health & Human Services National Institutes of Health (NIH) - USA 1R15AI082416 Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) 2214-

Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics

New Disturbing Trend in Antimicrobial Resistance of Gram-Negative Pathogens

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Genome evolution and plasticity of <em>Serratia marcescens</em>:an important multidrug resistant nosocomial pathogen

Serratia marcescens is an important nosocomial pathogen that can cause an array of infections, most notably of the urinary tract and bloodstream. Naturally, it is found in many environmental niches, and is capable of infecting plants and animals. The emergence and spread of multidrug-resistant strains producing extended-spectrum or metallo beta-lactamases now pose a threat to public health worldwide. Here we report the complete genome sequences of two carefully selected S. marcescens strains, a multidrug-resistant clinical isolate (strain SM39) and an insect isolate (strain Db11). Our comparative analyses reveal the core genome of S. marcescens and define the potential metabolic capacity, virulence, and multidrug resistance of this species. We show a remarkable intraspecies genetic diversity, both at the sequence level and with regards genome flexibility, which may reflect the diversity of niches inhabited by members of this species. A broader analysis with other Serratia species identifies a set of approximately 3,000 genes that characterize the genus. Within this apparent genetic diversity, we identified many genes implicated in the high virulence potential and antibiotic resistance of SM39, including the metallo beta-lactamase and multiple other drug resistance determinants carried on plasmid pSMC1. We further show that pSMC1 is most closely related to plasmids circulating in Pseudomonas species. Our data will provide a valuable basis for future studies on S. marcescens and new insights into the genetic mechanisms that underlie the emergence of pathogens highly resistant to multiple antimicrobial agents