A novel genomic approach identifies bacterial DNA-dependent RNA polymerase as the target of an antibacterial oligodeoxynucleotide, RBL1.
Rapid emergence of antibiotic-resistant bacterial pathogens limits the applicability of existing drugs, which has created an urgent need for novel antibiotics preferably with entirely new mechanisms of action. Oligodeoxynucleotides (ODNs) and their modified forms have been shown to inhibit bacterial gene expression, representing a potential for developing highly specific and efficacious antibacterial agents. In this study, a tetracycline (Tet)-inducible, randomized single-stranded DNA (ssDNA) expression library was constructed and screened for conditional growth-defective or lethal phenotypes in an Escherichia coli system. From approximately 5000 transformants screened, 12 bacterial colonies were identified with either growth-defective or lethal phenotypes. One clone, CY01, with a lethal phenotype was selected and sequenced, and the ODN sequence that it generates was designated as RBL-1. Because RBL-1 shows no significant homologies to any bacterial gene sequence, a potential RBL-1 targeting protein was isolated by affinity purification. Using mass spectrometry analysis, this protein was identified as bacterial DNA-dependent RNA polymerase (RNAP). RBL-1 was further shown to effectively inhibit RNA polymerase activity in vitro. The usage of this randomized ssDNA expression library screening technology to selectively modulate production and/or function of proteins may provide a powerful strategy in both identifying novel gene targets for antibiotic discovery and developing novel antibacterial agents.
Tan
Xin-Xing
XX
Cytogenix, Inc., 3100 Wilcrest Drive, Suite 140, Houston, Texas 77042, USA.
Chen
Yin
Y
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013752:Tetracycline / Q000737:chemistry
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012321:DNA-Directed RNA Polymerases; D013752:Tetracycline