Image_1_Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.TIF

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 μM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5′ UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.</p

Table_1_Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.DOC

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 μM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5′ UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.</p

Table_3_Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.DOC

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 μM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5′ UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.</p

Image_3_Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.TIF

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 μM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5′ UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.</p

Substrate Recognition and Modification by the Nosiheptide Resistance Methyltransferase

<div><p>Background</p><p>The proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR) confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2ʹO-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR) in complex with the cofactor S-Adenosyl-L-methionine (SAM) are available, the principles behind NHR substrate recognition and catalysis remain unclear.</p><p>Methodology/Principal Findings</p><p>We have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA) and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066) and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5ʹ) to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation.</p><p>Conclusion/Significance</p><p>Taken together these data suggest that local base-base interactions play an important role in aligning the substrate 2’ hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an increasingly important role in fundamental biological processes and we anticipate that the approach outlined in this manuscript may be useful for investigating other classes of nucleic acid methyltransferases.</p></div

Table_4_Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.XLS

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 μM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5′ UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.</p

Table_5_Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.XLS

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 μM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5′ UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.</p

Image_2_Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.TIF

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 μM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5′ UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.</p

Table_7_Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli.XLS

Aminoglycosides are not only antibiotics but also have wider and diverse non-antibiotic cellular functions. To elucidate the understanding of non-antibiotic cellular functions, here we report transcriptome-profiling analysis of Escherichia coli in the absence or presence of 0.5 and 1 μM of Kanamycin B, concentrations that are neither lethal nor inhibit growth, and identified the differentially expressed genes (DEGs) at two given concentrations of Kanamycin B. Functional classification of the DEGs revealed that they were mainly related to microbial metabolism including two-component systems, biofilm formation, oxidative phosphorylation and nitrogen metabolism in diverse environments. We further showed that Kanamycin B and other aminoglycosides can induce reporter gene expression through the 5′ UTR of napF gene or narK gene (both identified as DEG) and Kanamycin B can directly bind to the RNA. The results provide new insights into a better understanding of the wider aminoglycosides cellular function in E. coli rather than its known antibiotics function.</p

Investigation of neighbouring group influence on methylation of 29 nt substrate RNA.

<p><b>A.</b> The 29 nt hairpin substrate, with A1067 and positions in the loop marked. <b>B.</b> The relative specificity for methylation of mutated nucleotides positioned 3′ to A1067. <b>C.</b> The relative specificity for methylation of mutated nucleotides located 5′ to A1067. <b>D.</b> Structure of the abasic deoxy (ds-linker) linker, that was used to replace U1066 (1066ds) and G1068 (1068ds). <b>E.</b> The relative specificity for methylation of linker modified 1066ds and 1068ds RNA.</p