Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium |
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| Institution: | 1. Department of Human Nutrition, Faculty of Contemporary Life Science, Chugokugakuen University, Okayama, Japan;2. Department of Microbiology, Kawasaki Medical School, Kurashiki, Japan;3. Laboratory of Gene Regulation Study, Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan;1. Department of Biological Sciences, University of Ulsan, Ulsan, 680-749, South Korea;3. Meta-Inflammation Research Center, University of Ulsan, Ulsan, 680-749, South Korea;2. Department of Radiology, Kangbuk Samsung Hospital, Seoul, South Korea;4. Department of Surgery, Ulsan University Hospital, University of Ulsan, Ulsan, South Korea;1. School of Biological Sciences, Department of Molecular & Biomedical Science, Research Centre for Infectious Diseases, University of Adelaide, Adelaide, 5005, Australia;2. Institute for Glycomics, Griffith University Gold Coast Campus, Queensland, 4222, Australia;1. University of Chinese Academy of Sciences, Beijing, 100049, China;2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China;3. Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan;4. State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, China;1. Department of Life Sciences and Biotechnology, Ferrara, Italy;2. Department of Pathology and Diagnostics, University Hospital of Verona, Italy;3. Department of Cellular and Molecular Medicine & Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Denmark |
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| Abstract: | We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G16, -G18, -G20, -G22, and -G24). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G20 clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs. |
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| Keywords: | Antimicrobial agent Random sequence Bacterial growth inhibition Guanine-rich RNA Poly-G sRNA sRNA"} {"#name":"keyword" "$":{"id":"kwrd0040"} "$$":[{"#name":"text" "_":"small RNA |
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