LeBlanc D J, Lee L N, Abu-Al-Jaibat A R, Sreenivasan P K, Fives-Taylor P M
Department of Microbiology, University of Texas Health Science Center at San Antonio.
Oral Microbiol Immunol. 1993 Apr;8(2):94-9. doi: 10.1111/j.1399-302x.1993.tb00552.x.
A collection of 39 isolates of Actinobacillus actinomycetemcomitans, obtained from laboratories located in 5 different geographical regions of the United States, was examined for the presence of plasmid DNA. Only 2 of the strains examined, designated VT736 and VT745, harbored detectable plasmids. Strain VT736 contained a 1.9 kb plasmid species (pVT736-1) and a larger ( > 30 kb) species (pVT736-2). Both plasmids were detected in the covalently closed circular DNA fraction of dye buoyant density gradients. However, only the smaller plasmid was observed in agarose gels containing plasmid-enriched cell lysates prepared by a rapid screening procedure. Strain VT745 contained a single, 24 kb, plasmid (pVT745) that was observed consistently in plasmid-enriched lysates, as well as in the plasmid band of dye buoyant density gradients. A restriction endonuclease map of pVT736-1 was constructed. The plasmid contained one site each for the enzymes HincII, KpnI and XhoI, located 600 to 700 bp from each other on the pVT736-1 map. HincII-digested pVT736-1 DNA could not be cloned in Escherichia coli. However, intact pVT736-1 digested with KpnI or XhoI could be cloned in E. coli on pUC19 or pGEM7Zf(-), respectively. KpnI-digested pVT736-1 was cloned in both orientations on pUC19, but XhoI-digested pVT736-1 was clonable in only one orientation on pGEM7Zf(-). Each of the 3 types of chimeric plasmid constructs provided a potential A. actinomycetemcomitans/E. coli shuttle plasmid for the development of a genetic transfer system in A. actinomycetemcomitans.
从美国5个不同地理区域的实验室获得了39株伴放线放线杆菌分离株，检测其是否存在质粒DNA。在所检测的菌株中，只有2株（命名为VT736和VT745）含有可检测到的质粒。菌株VT736含有一个1.9 kb的质粒（pVT736 - 1）和一个更大的（> 30 kb）质粒（pVT736 - 2）。两种质粒都在染料浮力密度梯度的共价闭合环状DNA组分中检测到。然而，在通过快速筛选程序制备的富含质粒的细胞裂解物的琼脂糖凝胶中，仅观察到较小的质粒。菌株VT745含有一个单一的24 kb质粒（pVT745），在富含质粒的裂解物以及染料浮力密度梯度的质粒条带中均能始终观察到。构建了pVT736 - 1的限制性内切酶图谱。该质粒在pVT736 - 1图谱上分别含有HincII、KpnI和XhoI酶的一个位点，彼此相距600至700 bp。用HincII消化的pVT736 - 1 DNA不能在大肠杆菌中克隆。然而，用KpnI或XhoI消化的完整pVT736 - 1可分别在pUC19或pGEM7Zf(-)上克隆到大肠杆菌中。用KpnI消化的pVT736 - 1以两种方向克隆到pUC19上，但用XhoI消化的pVT736 - 1仅能以一种方向克隆到pGEM7Zf(-)上。这3种嵌合质粒构建体中的每一种都为开发伴放线放线杆菌的基因转移系统提供了一种潜在的伴放线放线杆菌/大肠杆菌穿梭质粒。
