Bacteriophage-triggered defense systems: Phage adaptation and design improvements

Abstract

A novel bacteriophage defense system, based on an inducible suicide gene, was challenged with a lactococcal bacteriophage to investigate the potential for phage adaptation. The defense system was encoded by pTRK414H, a high-copy-number replicon encoding a tightly regulated φ31p trigger promoter fused to the lethal L/aIR+ restriction endonuclease cassette. Repeated transfers of Lactococcus lactis NCK690(pTRK414H) in the presence of φ31 selected for phage φ31 derivatives which were markedly less sensitive to φ31p-LlaIR+-encoded restriction than the parental phage, φ31. The efficiency of plaquing (EOP) on L. lactis NCK690(pTRK414H) was 10-4 for φ31 versus 0.4 for the derived phages. The mutant phages remained fully sensitive to L/aIR+ restriction, suggesting an alteration in the recognition or firing of the φ31p promoter. Sequencing over the promoter region in four mutant phages revealed the identical C-to-A transversion, generating a Phe- to-Leu substitution, in a transcriptional activator of the φ31p promoter, designated ORF2. The mutant phages were analyzed for their ability to induce the native φ31p promoter element fused to a lacZ.st reporter gene. Compared to the parental phage, φ31, lower levels of β-galactosidase activity were induced throughout the lytic cycle, indicating that the strength at which the mutant phages activated the φ31p promoter was altered. Based on these observations, improvements were made in promoter strength and restriction activity in an attempt to elevate the effectiveness of the phage-triggered suicide system. When the φ31p-LlaIR+ cassette was paired with other abortive defense systems, Per31 and AbiA, the EOP of φ31 was reduced to <10-10 and the level of phage in the culture was lowered below the detection limits of the assay.