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Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages
Joseph M Sturino1, Todd R Klaenhammer
1Departments of Food Science and Microbiology, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695-7624, USA.
Abstract:
Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S. thermophilus bacteriophages and one pac-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage kappa3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the kappa3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10(-7) and 10(-8), beyond the resistance conferred by the PER element alone (less than 10(-6)). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages.
Insights
Antisense RNA targeting the hel3.1 gene effectively inhibited Streptococcus thermophilus bacteriophage proliferation. This novel approach reduced plaque formation and burst size, offering a new strategy for phage control.
Area of Science:
- Microbiology
- Molecular Biology
- Bacteriophage Research
Background:
- Streptococcus thermophilus bacteriophages pose challenges in industrial settings.
- The hel3.1 gene, part of the Sfi21-type DNA replication module, is crucial for many important S. thermophilus bacteriophages.
- Targeting essential viral genes offers a potential strategy for phage control.
Purpose of the Study:
- To investigate the efficacy of antisense RNA targeting the hel3.1 gene to inhibit S. thermophilus bacteriophage proliferation.
- To evaluate the impact of antisense hel3.1 RNA on bacteriophage efficiency of plaquing (EOP), plaque size, and burst size.
- To assess the protective effect of antisense hel3.1 RNA against bacteriophage infection in S. thermophilus.
Main Methods:
- Antisense RNA complementary to the hel3.1 gene was designed and expressed.
- Bacteriophage proliferation was assessed using efficiency of plaquing (EOP) assays.
- Growth curves were generated to evaluate phage inhibition under varying multiplicities of infection.
- Antisense RNA was expressed in cis using a phage-encoded resistance (PER) plasmid.
Main Results:
- Expression of antisense hel3.1 RNA reduced EOP by 40-70% and decreased plaque size for sensitive bacteriophages.
- Antisense hel3.1 RNA significantly reduced bacteriophage burst size.
- Antisense hel3.1 conferred protection against bacteriophage kappa3 infection, even at a high multiplicity of infection.
- Combined antisense RNA and PER plasmid expression achieved EOP reductions to 10(-7)-10(-8).
Conclusions:
- Antisense RNA targeting the hel3.1 gene is a viable strategy to impede S. thermophilus bacteriophage proliferation.
- This study demonstrates the first successful application of antisense RNA for inhibiting S. thermophilus bacteriophages.
- Antisense RNA, particularly with explosive delivery, presents a promising tool for phage control in industrial applications.