Expression and site-directed mutagenesis of the lactococcal abortive phage infection protein AbiK.
Abortive infection mechanisms of Lactococcus lactis form a heterogeneous group of phage resistance systems that act after early phage gene expression. One of these systems, AbiK, aborts infection of the three most prevalent lactococcal phage groups of the dairy industry. In this study, it is demonstrated that the antiphage activity depends on the level of expression of the abiK gene and on the presence of a reverse transcriptase (RT) motif in AbiK. The abiK gene was shown to be part of an operon that includes two additional open reading frames, with one of these encoding a phage-related transcriptional repressor named Orf4. Expression of AbiK is driven by two promoters, PabiK and Porf3, the latter being repressed by Orf4 in vivo. Binding of the purified Orf4 to the Porf3 promoter was demonstrated in vitro by gel retardation assays. The N-terminal half of the deduced AbiK protein possesses an RT motif that was modified by site-directed mutagenesis. Conservative mutations in key positions resulted in the complete loss of the resistance phenotype. These data suggest that an RT activity might be involved in the phage resistance activity of AbiK. A model for the mode of action of AbiK is proposed.
Fortier
Louis-Charles
LC
Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, and Groupe de Recherche en Ecologie Buccale, Université Laval, Québec, Canada G1K 7P4.
Bouchard
Julie D
JD
Moineau
Sylvain
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001435:Bacteriophages / Q000254:growth & development*; D007763:Lac Operon; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics* / Q000821:virology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames / Q000235:genetics; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
C106520:AbiK protein, Lactococcus lactis; D001426:Bacterial Proteins; D012194:RNA-Directed DNA Polymerase