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 3 provided significant protection from 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 3 to 3 ؋ 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 ؋ 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.Strains of lactic acid bacteria are used in starter cultures or culture adjuncts during the manufacture of a variety of fermented dairy products. Phage contamination during product manufacture can result in significant loss of starter culture activity and remains the leading cause of failed batch fermentations. These losses are particularly severe when highly specialized strains, which are themselves a valuable product of scientific discovery and product development, become susceptible to phage attack. In this case, costs committed for strain development will not be recovered if the expected lifetime of a new, highly specialized strain is diminished by the appearance of lytic phages capable of attacking it. The crux of the problem is that the dairy environment and fermentation substrate provide a continuous reservoir for the influx of new virulent phages (7, 25), while existing phages adapt by mutation and recombination (4, 13). Together, these events enable the appearance of subpopulations of phages capable of subverting previously resistant cultures and necessitate the development of strains of lactic acid bacteria with enhanced phage resistance properties.Novel and more efficacious phage defense strategies continue to be developed, including the expression of antisense RNA targeted against phage-encoded transcripts. These antisense RNAs have been constitutively expressed by starter strains (reviewed in references 2, 24, 32, and 34) or triggered in response to phage infection through the use of phage-encoded promoters and/or origins of replication (32, 34). Regardless of the delivery strategy employed, antisense RNAs act to interfere with phage development by pr...