Sela David A, Rawsthorne Helen, Mills David A
University of California at Davis, Department of Viticulture and Enology, One Shields Ave., Davis, CA 95616, USA.
Plasmid. 2007 Sep;58(2):127-39. doi: 10.1016/j.plasmid.2007.02.003. Epub 2007 Apr 3.
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.
乳酸乳球菌II组内含子（Ll.ltrB）能高效地归巢到ltrB基因中。迄今为止，Ll.ltrB核糖核蛋白（RNP）在体内识别的关键DNA碱基仅在大肠杆菌中得到了阐明。然而，最近的证据表明Ll.ltrB的移动性存在宿主依赖性差异，这增加了当前RNP归巢位点识别模型在天然乳酸乳球菌宿主中存在局限性的可能性。在这项工作中，乳酸乳球菌中的内含子重靶向实验表明，与在大肠杆菌中一样，仅遵循特定靶位点关键碱基并不足以预测染色体入侵的成功或失败。因此，开发了一种定量实时PCR（QPCR）检测方法，以测试先前在大肠杆菌中被证明对归巢至关重要的靶位点核苷酸在其天然宿主中的相关性。这种双质粒QPCR归巢检测方法高度灵敏，与以前基于大肠杆菌的检测方法不同，它能在没有选择压力的情况下分辨出不同的归巢效率。与在大肠杆菌中一样，靶位点位置-23、-21、-20、-19和+5处与野生型的偏差导致乳酸乳球菌中的归巢效率降低。此外，在检测粪肠球菌中的选定变体时也观察到了相同的趋势。我们的结果表明，这些靶位点位置在大肠杆菌和乳酸乳球菌中都至关重要。
