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Applied and Environmental Microbiology, March 2004, p. 1735-1743, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1735-1743.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Antisense RNA Targeting of Primase Interferes with Bacteriophage Replication in Streptococcus thermophilus

Joseph M. Sturino and Todd R. Klaenhammer*

Genomic Sciences Program, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695-7624

Received 3 June 2003/ Accepted 21 November 2003

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) from S. thermophilus phage {kappa}3 provided significant protection from {kappa}3 and two other Sfi21-type phages. Expression of pri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of {kappa}3 to 3 x 10-3 and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 x 10-3. The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5' region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection in S. thermophilus.

* 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.

Applied and Environmental Microbiology, March 2004, p. 1735-1743, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1735-1743.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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