NDLI: An Explosive Antisense RNA Strategy for Inhibition of a Lactococcal Bacteriophage†

Content Provider	PubMed Central
Author	Walker, Shirley A. Klaenhammer, Todd R.
Copyright Year	2000
Abstract	The coding regions of six putative open reading frames (ORFs) identified near the phage φ31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage φ31 were used to develop antisense constructs to inhibit the proliferation of phage φ31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (TT7) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage φ31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage φ31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage φ31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::TT7 resulted in a threefold decrease in the EOP of phage φ31 (EOP = 0.11 ± 0.03 [mean ± standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage φ31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.
Starting Page	310
File Format	PDF
ISSN	10985336
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	1
Volume Number	66
Language	English
Publisher	American Society for Microbiology
Publisher Date	2000-01-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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