Antisense RNA Targeting of Primase Interferes with Bacteriophage Replication inStreptococcus thermophilus

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Abstract
The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved inStreptococcus thermophilusbacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) fromS. thermophilusphage κ3 provided significant protection from κ3 and two other Sfi21-type phages. Expression ofpri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of κ3 to 3 × 10−3and 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 × 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 ofpri3.10-ASresulted 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 inS. thermophilus.