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 31 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 (31P) 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 31, 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 31, the 31P/LlaIR ؉ suicide cassette also inhibited four 31-derived recombinant phages at levels at least 10-fold greater than that of 31. The 31P/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.Many important industrial bioprocesses and food fermentations are susceptible to attack by bacteriophages. Control of phage problems in the commercial arena requires prevention of phage contamination, employment of strains that are resistant to phage infection, and minimization of opportunities for the appearance of new virulent phages. With the diversity of fermentation systems, it is not always possible to operate an aseptic fermentation or, if available, to maintain its integrity. Therefore, the longevity of any highly specialized strain in a commercial situation depends on the bacterium's relative phage resistance or sensitivity and how quickly defiant phages appear and proliferate against it. Modern biotechnology continues to provide increasing opportunities to create specialized strains with valuable processing or product characteristics. In dairy fermentations, which are plagued by phage attacks more often than any other bioprocessing industry, this problem has been traditionally approached with varying success by isolating phage-resistant mutants and incorporating the derivatives into the starter culture formulations. Over the last decade, a number of naturally occurring plasmid-encoded defenses in Lactococcus species have been discovered which prevent phage adsorption, prevent DNA injection, restrict unmodified phage DNA by resident restriction and modification (R/M) systems, or abort the phage infection following the early stages of phage development (21, 27). By using conjugation or transformation approaches, these phage...

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…It was proposed that the programmed cell death of infected or damaged clonal cells within the community is beneficial to the population as a whole (222). Artificially designed systems that trigger suicide by R-M components upon phage entry have been shown to function as a defense strategy against infection (223)(224)(225). However, the natural occurrence of such R-M systems has not been explored.…”

An Activator of Transcription Regulates Phage TP901-1 Late Gene Expression

“…In summary, our results suggest that within the P335 group of lactococcal phages at least two regulatory systems controlling transcription in the late stage of infection exist, the TP901-1 like (TP901-1, tuc2009, bIL285, bIL286, and bIL309) and the ⌽31 like (⌽31, r1t, and ⌽LC3) (31). A phage resistance mechanism based on the ⌽31 middle promoter and activator has already been constructed (12). The identification of a second regulatory system among the P335 phages may be exploited in newly designed phage resistance mechanisms effective against a larger group of the P335 phages.…”

Lactococcus lactis Lytic Bacteriophages of the P335 Group Are Inhibited by Overexpression of a Truncated CI Repressor

“…Sequencing over 14.3 kb of phage 31 has defined the following gene clusters: a locus involved in sensitivity to the phage resistance mechanism AbiA (10); a late promoter region and a transcriptional activator of the promoter (11,37,(44)(45)(46); the phage replication module (28,35); part of the lysis module (28); and a genetic switch region (28). The genetic switch region encodes two divergent promoters, P1 and P2, and homologues to cI and cro genes of temperate phages shown in Fig.…”