Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases

Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence homology and may be evolutionarily linked to a common ancestor. To explore their individual specificity and similarity we performed two sets of experiments. We created a homology model of Cfr and explored the C2/C8 specificity using docking and binding energy calculations on the Cfr homology model and an X-ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore interchangeability between Cfr and RlmN we constructed various combinations of their genes. The function of the mixed genes was investigated by RNA primer extension analysis to reveal methylation at 23S rRNA position A2503 and by MIC analysis to reveal antibiotic resistance. The catalytic site is expected to be responsible for the C2/C8 specificity and most of the combinations involve interchanging segments at this site. Almost all replacements showed no function in the primer extension assay, apart from a few that had a weak effect. Thus Cfr and RlmN appear to be much less similar than expected from their sequence similarity and common target

Mechanisms of action of Cfr and RlmN.

<p>A simplified version of the mechanisms of action of Cfr and RlmN proposed by Grove <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145655#pone.0145655.ref001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145655#pone.0145655.ref002" target="_blank">2</a>] starting with the methylated Cys 338/355 (as used in our computational approach) that has been generated by attacking the activated methyl group of the first SAM. Reductive cleavage of a second SAM gives an 5’-deoxyadenosyl 5’ radical, as shown in the figure, that abstracts a hydrogen atom from the mCys338/355 group to yield a neutral, carbon-centered radical. The resulting methylene radical adds to C8/C2 of A2503 in 23S rRNA generating a protein-RNA crosslink that contains an unpaired electron (not shown). Loss of an electron and abstraction of the proton (shown in bold) from C8/C2 by a general base, results in the resolution of the covalent crosslink by disulfide bond formation that involves a second cysteine (Cys105/118).</p

The interchanged regions for the investigation of C2/C8 specificity.

<p>(A) Alignment of the amino acid sequences of Cfr (GI: 34328031 / NCBI: NP_899167.1) and RlmN (GI: 16130442 / NCBI: NP_417012.1). The seven coloured boxes depict the regions that encompass the active site of the enzymes. The dots above the sequence indicate the CX₃CX₂C motif. The black arrow shows the cysteine 338/355. The grey shading mark the 13 selectively conserved positions in Cfr- or RlmN-like proteins. (B) Representation of the X-ray crystal structure of RlmN (PDB file 3RFA) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145655#pone.0145655.ref026" target="_blank">26</a>] with the same region colouring as in the amino acid alignment and oriented similar to the Cfr model structure in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145655#pone.0145655.g002" target="_blank">Fig 2A</a>. Also, the SAM molecule and the [4Fe-4S] cluster are included as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145655#pone.0145655.g001" target="_blank">Fig 1A</a>. The three cysteines in the CX₃CX₂C as well as the Cys355 are shown in green sticks.</p

Summary of plasmid constructs and primer extension results from <i>E</i>. <i>coli</i> JW2501-1 harbouring the plasmids.

<p>Summary of plasmid constructs and primer extension results from <i>E</i>. <i>coli</i> JW2501-1 harbouring the plasmids.</p

Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides that target the <i>BRAF</i> V600E oncogene, with a library of rationally designed peptides employing CuAAC “click” chemistry. The peptide sequence has an influence on the specificity and affinity of target DNA/RNA binding. We also investigated the impact of locked nucleic acids (LNAs) on the latter. Lysine residues improve binding of POCs to target DNA and RNA, whereas the distance to lysine correlates exclusively with a decrease in binding of mismatched RNA targets. Glycine and tyrosine residues affect target binding as well. Importantly, the resistance of POCs to enzymatic degradation is dramatically improved by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media

Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr− strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m2A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules

Loss of bacterial cell pole stabilization in caulobacter crescentus sensitizes to outer membrane stress and peptidoglycan-directed antibiotics

Rod-shaped bacteria frequently localize proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the roles of polar factors in responses to extracellular stimuli have been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal resistance of cell envelope-directed antibiotics, including vancomycin which does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin sensitivity. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to the wild type under some, but not all, cell wall stress conditions that this strain was sensitized to, in particular cefixime and detergent exposure. Together, these results indicate that TipN contributes to cell envelope stress resistance in addition to its roles in intracellular development, and its loss influences signaling through the ChvIG two-component system which has been co-opted as a sensor of cell wall stress in CaulobacterIMPORTANCE Maintenance of an intact cell envelope is essential for free-living bacteria to protect themselves against their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and reform the cell envelope at the division plane as the cells divide. We have found that TipN, a factor required for correct division and cell pole development in Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilization of these weak points of the cell envelope as well as its other roles inside the cell