Shirley A. Walker

An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage.

ABSTRACT The coding regions of six putative open reading frames (ORFs) identified near the phage φ31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage φ31 were used to develop antisense constructs to inhibit the proliferation of phage φ31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (TT7) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage φ31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage φ31 origin of replication,ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage φ31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31and P6::anti-ORF 4H::TT7 resulted in a threefold decrease in the EOP of phage φ31 (EOP = 0.11 ± 0.03 [mean ± standard deviation]) compared to the presence ofori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage φ31 showed that ori31plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.

Leaky Lactococcus Cultures That Externalize Enzymes and Antigens Independently of Culture Lysis and Secretion and Export Pathways

ABSTRACT A novel system that leaks β-galactosidase (β-gal) without a requirement for secretion or export signals was developed inLactococcus lactis by controlled expression of integrated phage holin and lysin cassettes. The late promoter of the lytic lactococcal bacteriophage φ31 is an 888-bp fragment (P15A10) encoding the transcriptional activator. When a high-copy-number P15A10::lacZ.stfusion was introduced into L. lactis strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant β-gal activity were detected in the supernatant (approximately 85% of the total β-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed that the phenotype resulted from expression of Tac31A from the P15A10 fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains. Despite the high levels of β-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy. Integration of the holin-lysin cassette of phage r1t, under the control of the phage φ31 late promoter, into the host genome of MG1363 yielded a similar “leaky” phenotype, indicating that holin and lysin might play a critical role in the release of β-gal into the medium. In addition to β-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system. Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell. These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals.

The Genetics of Phage Resistance in Lactococcus lactis

Because of the nature of the cheese fermentation process, bacteriophage problems continue to appear in the Cheddar cheese industry, resulting in slowed or failed fermentation tanks and substantial economic losses. Extensive research in this area has lead to a vast accumulation of genetic information on Lactococcus lactis, the mechanisms by which it protects itself from phage attack, the phages themselves, and the phage—host interactions. This body of information has not only yielded a greater understanding of the natural defense mechanisms of Lc. lactis, but has also allowed for the development of novel, genetically engineered defense strategies. This chapter will address important aspects of phage resistance in Lc. lactis, both natural and novel. The means by which phages overcome these mechanisms will also be highlighted to illustrate the dynamic nature of phage—host interactions in the cheese environment. In addition, the continuing need to elevate the platform of knowledge of these model phage—host relationships will be emphasized, in our efforts to stay one step ahead of the dynamic evolutionary and adaptive ability of bacteriophages in fermentation environments.