An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage

Lactococcus lactis is an industrially important member of the lactic acid bacteria used in the fermentation of many dairy products, including sour cream, buttermilk, and various cheeses, such as cheddar. Because of the nature of the cheese fermentation process, bacteriophage problems continue to appear in the dairy industry, resulting in slowed or failed fermentation tanks and substantial economic losses. Efforts to protect L. lactis from phage attack have led to the identification of several phage resistance mechanisms naturally harbored by this important microorganism. These mechanisms can be divided into four groups: blockage of phage adsorption, blockage of phage DNA injection, restriction/modification, and abortive mechanisms (for extensive reviews, see references 2, 9, 10, 18, 23, and 33). These phage defense mechanisms are often located on plasmids, many of which have been successfully transferred, separately or in combination, to other industrial strains (6, 7, 16, 20, 29, 42, 47) to provide barriers to phage proliferation. Despite advances in this arena, however, the appearance of new phages capable of overcoming these defenses continues to pose a challenge to starter culture manufacturers. In addition, the use of biotechnology to develop highly specialized strains for specific functions may exacerbate this problem, since repeated use can allow the accumulation of phages capable of attacking the strain, thereby limiting its long-term usefulness in industry (9, 23, 33). Recent advances in the molecular biology of L. lactis and its bacteriophages have opened the door to the development of novel, recombinant phage defense mechanisms to complement the mechanisms described above. Two interesting examples of novel phage defense mechanisms involve the alteration or replacement of chromosomal elements to inhibit phage proliferation. In one case, site-specific integration was used to inactivate sequences in the NCK203 chromosome, which contributed to the emergence of new phages insensitive to certain resistance mechanisms (14, 36). Inactivation prevented the evolution and subsequent proliferation of recombinant lytic phages. In the second case, a bacteriophage receptor (pip), encoded by the L. lactis chromosome and involved in sensitivity to phages of the c2 species, was replaced with a mutated version (17). The replacement, which left no nonlactococcal DNA or antibiotic markers, resulted in a strain resistant to infection by c2 phages. In addition, phage sequences have been successfully utilized in the development of phage defense mechanisms. For example, a suicide system consisting of a phage-specific promoter from the lytic bacteriophage φ31 (φ31P) (41, 52) linked to a lethal gene (the LlaI restriction cassette) (40) was described recently (13). In this system, the infection transcriptionally activates a phage-specific, inducible promoter to express a lethal gene, thereby killing the host and destroying the phage genome. Another example is phage-encoded resistance, which provides a phage origin of replication in trans to compete with and inhibit phage replication (22, 37). This phage resistance mechanism was demonstrated with two origins of replication isolated from the P335 phages, φ50 (per50) (22) and φ31 (per31) (37). When present on a low-copy-number vector, per50 and per31 result in explosive plasmid amplification, with a concomitant reduction in phage efficiency of plaquing (EOP) (0.42 and 0.3, respectively) and plaque size (22, 37). On a high-copy-number vector, both cause a significant decrease in EOP (22, 37). In addition to the novel phage defense systems described above, antisense technology offers another approach to exploit phage sequences in providing barriers to phage attack. Binding of antisense RNA to the target mRNA prevents translation, either by preventing ribosome loading or by destabilizing the mRNA and making it more susceptible to RNase attack (27). Naturally occurring antisense mechanisms have been described for several systems (for reviews, see references 27 and 46). Although results have been inconsistent, antisense strategies have also been used successfully to control gene expression in animals (28), plants (15), and bacteria (8). In fact, Hirashima et al. (24, 25) utilized antisense RNA directed against various regions of the maturation protein, coat protein, and replicase gene to develop a novel immune system against RNA coliphage SP proliferation. These authors found that a 30-nucleotide antisense RNA directed against the Shine-Dalgarno sequence and base pairs just upstream of the maturation protein was sufficient to almost completely inhibit this phage (25). Previous attempts to use antisense mechanisms to protect L. lactis from bacteriophage attack, however, have yielded variable results. Using the major coat protein gene, mcp, of φF4-1, Chung et al. (5) achieved a reduction in EOP of 0.5 to 0.77, depending on the extent of the mcp gene included in the antisense construct. In contrast, Moineau et al. (35) found that the major structural proteins of the P335 phage ul36 were produced in excess, making them poor targets for antisense technology. Kim et al. (30–32) cloned an entire open reading frame (ORF) (gp51C) of unknown function from φ7-9 in an antisense orientation. When the entire coding region was used, this antisense construct resulted in a reduction in the EOP of φ7-9 to 10−2. This same EOP reduction was observed upon infection with phages related to φ7-9, which also contained this ORF. In addition, resistance to phage φ7-9 was achieved by antisense RNA directed to two ORFs immediately downstream of gp51C (gp18C and gp24C), although the resistance was far less efficient (EOP, ∼0.45) than that achieved with gp51C (32). More recently, Polzin et al. (K. M. Polzin, L. J. Collins, M. W. Lubbers, and A. W. Jarvis, Abstr. 5th Symp. Lactic Acid Bacteria, abstr. F2, 1996) used an antisense strategy to target several ORFs from the early and late regions of the prolate phage c2. The ORFs included e5, a putative subunit of DNA polymerase; e12, a putative transcription factor; l7, a major tail protein; and l12, a terminase. No inhibition was observed at low or high copy numbers for any of the constructs, even though there was some evidence that the antisense constructs lowered the expression levels of some of the ORFs. Lastly, in our laboratory, an antisense strategy was used to target the gene encoding the transcriptional activator, tac31A, of the phage φ31 late promoter (52). Although the anti-tac31A construct was able to significantly reduce expression from the phage promoter, it was not able to inhibit proliferation of φ31. The variable and largely negative results with antisense technology are unfortunate, especially since the recent availability of several complete and partial bacteriophage genome sequences (3, 34, 45, 50) makes the design of antisense strategies a viable option. In the present study, we have used available sequence information from near the late promoter and the right cohesive end of phage φ31 (51) to develop antisense constructs in both a high-copy-number vector and a low-copy-number explosive vector containing ori31, the putative phage φ31 origin of replication. The constructs allowed us to compare the efficacy of providing high levels of antisense RNA constitutively or explosively during the appropriate time in the phage lytic cycle.