title: A triggered-suicide system designed as a defense against bacteriophages.
abstract: A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.
authors.affiliation: Department of Microbiology, North Carolina State University, Raleigh 27695-7624, USA.
authors.forename: G M
authors.initials: GM
authors.lastname: Djordjevic
authors.forename: D J
authors.initials: DJ
authors.lastname: O'Sullivan
authors.forename: S A
authors.initials: SA
authors.lastname: Walker
authors.forename: M A
authors.initials: MA
authors.lastname: Conkling
authors.forename: T R
authors.initials: TR
authors.lastname: Klaenhammer
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics / Q000254:growth & development*; D015280:DNA Restriction-Modification Enzymes / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D007218:Industrial Microbiology / Q000379:methods; D013294:Lactococcus lactis / Q000821:virology*; D008969:Molecular Sequence Data; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*
chemical_list: D015280:DNA Restriction-Modification Enzymes; C069779:DNA modification methylase LlaI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)