@article{walker_klaenhammer_2001, title={Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways}, volume={67}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.67.1.251-259.2001}, abstractNote={ABSTRACT A novel system that leaks β-galactosidase (β-gal) without a requirement for secretion or export signals was developed in Lactococcus 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 (P 15A10 ) encoding the transcriptional activator. When a high-copy-number P 15A10 :: lacZ.st fusion 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 P 15A10 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. }, number={1}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Walker, SA and Klaenhammer, TR}, year={2001}, month={Jan}, pages={251–259} } @article{walker_klaenhammer_2000, title={An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage}, volume={66}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.66.1.310-319.2000}, abstractNote={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 (T T7 ) 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 ori31 and P6::anti-ORF 4H::T T7 resulted in a threefold decrease in the EOP of phage φ31 (EOP = 0.11 ± 0.03 [mean ± standard deviation]) compared to the presence of ori31 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 ori31 compared 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 ori31 plus 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. }, number={1}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Walker, SA and Klaenhammer, TR}, year={2000}, month={Jan}, pages={310–319} } @misc{klaenhammer_conkling_o'sullivan_djordjevic_walker_taylor_1998, title={Bacteriophage-triggered cell suicide systems and fermentation methods employing the same}, volume={5,792,625}, number={1998 Aug. 11}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Klaenhammer, T. R. and Conkling, M. A. and O'Sullivan, D. and Djordjevic, G. and Walker, S. A. and Taylor, C. G.}, year={1998} } @article{walker_dombroski_klaenhammer_1998, title={Common elements regulating gene expression in temperate and lytic bacteriophages of Lactococcus species}, volume={64}, number={3}, journal={Applied and Environmental Microbiology}, author={Walker, S. A. and Dombroski, C. S. and Klaenhammer, T. R.}, year={1998}, pages={1147–1152} } @article{walker_klaenhammer_1998, title={Molecular characterization of a phage-inducible middle promoter and its transcriptional activator from the lactococcal bacteriophage phi 31}, volume={180}, number={4}, journal={Journal of Bacteriology}, author={Walker, S. A. and Klaenhammer, T. R.}, year={1998}, pages={921–931} } @article{djordjevic_osullivan_walker_conkling_klaenhammer_1997, title={A triggered-suicide system designed as a defense against bacteriophages}, volume={179}, ISSN={["0021-9193"]}, DOI={10.1128/jb.179.21.6741-6748.1997}, abstractNote={A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.}, number={21}, journal={JOURNAL OF BACTERIOLOGY}, author={Djordjevic, GM and OSullivan, DJ and Walker, SA and Conkling, MA and Klaenhammer, TR}, year={1997}, month={Nov}, pages={6741–6748} } @misc{vandenbergh_walker_kunkar_1997, title={Method of making a lactococcal bacteriocin}, volume={5,702,923}, number={1997 Dec. 30}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Vandenbergh, P. A. and Walker, S. A. and Kunkar, B. S.}, year={1997} }