Luchansky J B, Tennant M C, Klaenhammer T R
Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695-7624.
J Dairy Sci. 1991 Oct;74(10):3293-302. doi: 10.3168/jds.S0022-0302(91)78515-9.
Lactobacillus strain ADH is a bile-resistant, bacteriocin-producing human isolate that was phenotypically classified within the Lactobacillus acidophilus group. Total DNA and phage DNA extracted from strain ADH were separately digested with BclI and ligated with BclI-digested pGK12. Following electroporation of these ligation mixtures directly into strain ADH, electrotransformants were recovered at frequencies of 1.5 x 10(3) and 2.0 x 10(4)/micrograms of pGK12 for preparations of pGK12::phage DNA and pGK12::total DNA, respectively. Among the electrotransformants screened, 6 and 22% contained passenger DNA of either phage DNA or chromosomal origin, respectively, as determined by restriction-enzyme analyses and hybridization assays. Derivatives of pGK12 containing passenger DNA of chromosomal (pTRK120) or phage (pTRK121) origin and pTRK15 (native cryptic plasmid) were evaluated for use as species-specific probes. The strain ADH-derived probes hybridized primarily to members of the B-1 and B-2 lactobacilli homology groups and demonstrated strain-specific polymorphisms within these groups. Identical hybridization patterns were established for strain ADH and Lactobacillus gasseri VPI 6033 (ATCC 19992). Identification of DNA probes and establishment of a host-vector cloning system have facilitated our efforts to characterize the Lactobacillus chromosome and to distinguish between closely related species thought to be important inhabitants of the gastrointestinal tract.
乳酸杆菌菌株ADH是一种耐胆汁、能产生细菌素的人体分离菌株，在表型上被归类于嗜酸乳杆菌组。从菌株ADH中提取的总DNA和噬菌体DNA分别用BclI消化，并与经BclI消化的pGK12连接。将这些连接混合物直接电穿孔导入菌株ADH后，对于pGK12::噬菌体DNA和pGK12::总DNA的制备，电转化子的回收频率分别为每微克pGK12 1.5×10³和2.0×10⁴个。在筛选出的电转化子中，通过限制性酶切分析和杂交试验确定，分别有6%和22%含有噬菌体DNA或染色体来源的外源DNA。对含有染色体（pTRK120）或噬菌体（pTRK121）来源的外源DNA的pGK12衍生物以及pTRK15（天然隐蔽质粒）作为种特异性探针的用途进行了评估。源自菌株ADH的探针主要与B-1和B-2乳杆菌同源组的成员杂交，并在这些组内显示出菌株特异性多态性。菌株ADH和加氏乳杆菌VPI 6033（ATCC 19992）建立了相同的杂交模式。DNA探针的鉴定和宿主-载体克隆系统的建立有助于我们对乳杆菌染色体进行表征，并区分被认为是胃肠道重要定居菌的密切相关物种。
