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Applied and Environmental Microbiology, February 2002, p. 588-596, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.588-596.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Expression of Antisense RNA Targeted against Streptococcus thermophilus Bacteriophages{dagger}

Joseph M. Sturino and Todd R. Klaenhammer*

Departments of Food Science and Microbiology, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695-7624

Received 12 July 2001/ Accepted 8 November 2001

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 {kappa}3, 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 {kappa}3-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.

* Corresponding author. Mailing address: Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695-7624. Phone: (919) 515-2971. Fax: (919) 515-7124. E-mail:Klaenhammer{at}ncsu.edu.

{dagger} Paper number FSR01-25 of the Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695-7624.

Applied and Environmental Microbiology, February 2002, p. 588-596, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.588-596.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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