Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis

Abstract

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.

FIG. 1

Southern blot analysis showing hybridization of labeled Tuc2009 DNA probes to DNA fragments of bacteriophages Q30, Q33, and u136. (a) Ethidium bromide-stained agarose gel with EcoRI-restricted phage DNA. (b through g) Southern blot analysis. The Tuc2009 DNA probe used is indicated above each panel. Lane M contained DNA molecular weight marker X (Boehringer). Lanes A, Tuc2009; lanes B, Q30; lanes C, Q33; lanes D, ul36.

FIG. 2

Lysis-in-broth experiment, demonstrating the increased levels of resistance conferred by increasing the numbers of copies of ori₂₀₀₉ cloned. Phage was added at zero time at a multiplicity of infection of approximately 1, and the OD₆₀₀ was measured over time. Symbols: ⧫, UC509.9(pNZ8048); ■, UC509.9(pNZRep-Cii); ▴, UC509.9(pSMG-1); ×, UC509.9(pSMG-2). The data are averages based on three independent experiments.

FIG. 3

Northern blot analysis showing antisense rep₂₀₀₉ mRNA transcribed from pNZ44-rep₂₀₀₉rev. Lane 1, total RNA isolated from SMQ86 harboring pNZRep-3; lane 2, total RNA isolated from SMQ86 harboring pNZ44-rep₂₀₀₉rev. RNA samples were prepared and blotted as described in Materials and Methods. An 800-bp PCR DNA fragment representing rep₂₀₀₉ was used as a hybridization probe. Transcript size was estimated by using the 0.24- to 9.5-kb RNA ladder from Gibco BRL (Life Technologies Ltd., Paisley, United Kingdom). Sizes (in kilobases) are indicated on the left. The position of the main transcript representing antisense rep₂₀₀₉ mRNA is indicated on the right.

FIG. 4

Southern blot analysis showing the effects of antisense constructs on intracellular Tuc2009 DNA replication. Total cellular DNA was isolated at 20-min intervals from Tuc2009-infected UC509.9 harboring antisense constructs. Lanes A, zero time; lanes B, 20 min; lanes C, 40 min; lanes D, 60 min. The gene targeted by each antisense construct is indicated.

FIG. 5

Restriction profiles of phage genomes comparing Per2009-sensitive and Per2009^r phages. (A) Lanes 1 and 3, Per2009-sensitive phage Q30; lanes 2 and 4, Per2009-insensitive phage Q30^r. (B) Lanes 1 and 3, Per2009-sensitive phage Q33; lanes 2 and 4, Per2009-insensitive phage Q33^r. (C) Lanes 1 and 3, Per2009-sensitive phage ul36; lanes 2 and 4, Per2009-insensitive phage ul36^r. In all panels lanes 1 and 2 contained preparations restricted with PstI and lanes 3 and 4 contained preparations restricted with EcoRV.