title: Identifying active phage lysins through functional viral metagenomics.
abstract: 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.
authors.affiliation: Rockefeller University, Laboratory of Bacterial Pathogenesis and Immunology, 1230 York Ave., Box 172, New York, NY 10065, USA. jschmitz@rockefeller.edu
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Schmitz
authors.forename: Raymond
authors.initials: R
authors.lastname: Schuch
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Fischetti
mesh_terms: 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
chemical_list: D004279:DNA, Viral