Identifying active phage lysins through functional viral metagenomics.
Recent metagenomic sequencing studies of uncultured viral populations have provided novel insights into the ecology of environmental bacteriophage. At the same time, viral metagenomes could also represent a potential source of recombinant proteins with biotechnological value. In order to identify such proteins, a novel two-step screening technique was devised for cloning phage lytic enzymes from uncultured viral DNA. This plasmid-based approach first involves a primary screen in which transformed Escherichia coli clones that demonstrate colony lysis following exposure to inducing agent are identified. This effect, which can be due to the expression of membrane-permeabilizing phage holins, is discerned by the development a hemolytic effect in surrounding blood agar. In a secondary step, the clones identified in the primary screen are overlaid with autoclaved Gram-negative bacteria (specifically Pseudomonas aeruginosa) to assay directly for recombinant expression of lytic enzymes, which are often encoded proximally to holins in phage genomes. As proof-of-principle, the method was applied to a viral metagenomic library constructed from mixed animal feces, and 26 actively expressed lytic enzymes were cloned. These proteins include both Gram-positive-like and Gram-negative-like enzymes, as well as several atypical lysins whose predicted structures are less common among known phage. Overall, this study represents one of the first functional screens of a viral metagenomic population, and it provides a general approach for characterizing lysins from uncultured phage.
Schmitz
Jonathan E
JE
Rockefeller University, Laboratory of Bacterial Pathogenesis and Immunology, 1230 York Ave., Box 172, New York, NY 10065, USA. jschmitz@rockefeller.edu
Schuch
Raymond
R
Fischetti
Vincent A
VA
D001408:Bacillus anthracis / Q000235:genetics / Q000821:virology; D001433:Bacteriolysis / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D015723:Gene Library; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000821:virology; D012689:Sequence Homology, Nucleic Acid
D004279:DNA, Viral