Characterization of the lactococcal group II intron target site in its native host.
The Lactococcus lactis group II intron (Ll.ltrB) retrohomes into the ltrB gene at high efficiency. To date, the critical DNA bases recognized in vivo by the Ll.ltrB ribonucleoprotein (RNP) have been exclusively elucidated in Escherichia coli. However, recent evidence indicates host-dependant differences in Ll.ltrB mobility, raising the possibility of limitations of the current model for RNP-homing site recognition in the native L. lactis host. In this work, intron retargeting experiments in L. lactis have demonstrated that adherence to specific target site critical bases is not sufficient to predict success or failure of chromosomal invasion, as in E. coli. Accordingly, a quantitative real-time PCR (QPCR) assay was developed to test target site nucleotides previously demonstrated as critical for homing in E. coli, for relevance in its native host. This two-plasmid QPCR homing assay is highly sensitive and, unlike previous E. coli-based assays, resolves differential homing efficiencies in the absence of selection. As in E. coli, deviation from wild type at target site positions -23, -21, -20, -19, and +5 resulted in lower homing efficiencies in L. lactis. Furthermore, the same trends are observed when assaying select variants in Enterococcus faecalis. Our results suggest that these target site positions are critical in both E. coli and L. lactis.
Sela
David A
DA
University of California at Davis, Department of Viticulture and Enology, One Shields Ave., Davis, CA 95616, USA.
Rawsthorne
Helen
H
Mills
David A
DA
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013293:Enterococcus faecalis / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics*; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C099786:LtrB protein, Lactococcus lactis