Screening and cloning of antimicrobial DNA sequences using a vital staining method.
A novel method for cloning of genes coding for cytotoxic molecules based on a cell viability assay is described. The working hypothesis is that expression of DNA sequences coding for cytotoxic molecules in bacterial cells will lead to cell death or impairment, and the isolation of the impaired or dead cells could lead to identification of DNA sequences responsible for debilitating the host cells. We verified this concept by isolating the well known antimicrobial Puroindoline b gene in Escherichia coli cells. We further demonstrated the feasibility to use this approach for isolating DNA encoding for antimicrobials from cDNA expression libraries. Sequence analysis and bioassay indicated that the isolated clones encoded previously characterized antimicrobial proteins (AMPs), proteins not previously characterized as AMPs, as well as novel antimicrobial peptides. In addition, clones harboring ribosomal protein encoding cDNA were also identified. Therefore, this method could also be used to identify host genes important in maintaining bacterial cell viability.
Cheng
Xiongying
X
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. xiongying.cheng@vitegen.com
Liu
Guozhen
G
Ye
Gongyin
G
Wang
Haijiao
H
Shen
Xiaojing
X
Wu
Kechun
K
Xie
Jiahua
J
Altosaar
Illimar
I
D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000494:pharmacology; D001483:Base Sequence; D002999:Clone Cells; D003001:Cloning, Molecular; D004058:Diffusion / Q000187:drug effects; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000254:growth & development; D015723:Gene Library; D005820:Genetic Testing; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry; D012689:Sequence Homology, Nucleic Acid; D013194:Staining and Labeling / Q000379:methods*; D013312:Stress, Physiological / Q000187:drug effects
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D010455:Peptides; D010940:Plant Proteins; D012269:Ribosomal Proteins; C082942:puroindoline protein, Triticum aestivum