Research Group in Oral Health and Systemic Disease, University of Louisville School of Dentistry, 501 S. Preston Street, Louisville, KY 40202, USA.
Plasmid. 2013 May;69(3):211-22. doi: 10.1016/j.plasmid.2013.01.002. Epub 2013 Jan 23.
To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria.
为了阐明基因的假定功能，需要有效的遗传特征工具来促进其在细菌染色体上的失活、缺失或修饰。在本研究中，确定了大肠杆菌/伴放线放线杆菌穿梭载体 pYGK 的核苷酸序列，使我们能够重新设计和构建一种新的穿梭克隆载体 pJT4，以及无启动子的 lacZ 转录/翻译融合质粒 pJT3 和 pJT5。质粒 pJT4 和 pJT5 含有维持穿梭载体复制所需的复制起点。此外，构建了一种新的自杀载体 pJT1，用于产生口腔病原体 A. actinomycetemcomitans 中基因的无痕和无标记缺失突变。质粒 pJT1 是一种基于 pUC 的自杀载体，对蔗糖敏感性具有反向选择。该载体在基因缺失后不会在染色体上留下抗生素标记或痕迹，因此提供了在同一遗传背景下组合多个突变的选择。pJT1 的有效性通过构建 A. actinomycetemcomitans 的 qseB 单缺失（ΔqseB）突变体和 lsrRK 双缺失突变体（ΔlsrRK）得到了证明。这些新载体可为 A. actinomycetemcomitans 及其他 HACEK（嗜血杆菌属、放线共生放线杆菌、心杆菌属、侵蚀艾肯菌和金氏金杆菌）革兰氏阴性菌组的遗传研究提供替代方案。
