Vásquez Alejandra, Molin Göran, Pettersson Bertil, Antonsson Martin, Ahrné Siv
Division of Food Technology/Lab of Food Hygiene, Lund University, Lund, Sweden.
Syst Appl Microbiol. 2005 Jul;28(5):430-41. doi: 10.1016/j.syapm.2005.02.011.
The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.
对干酪乳杆菌/副干酪乳杆菌以及相关物种玉米乳杆菌和鼠李糖乳杆菌的16S rRNA基因序列差异进行了研究。通过对总染色体DNA进行限制性内切酶分析（REA）以及对PCR扩增的16S rRNA基因片段进行时间温度梯度凝胶电泳（TTGE），对37株主要来源于人类或奶酪的菌株进行了分组。REA证实所有菌株在基因组上都是独特的，并区分出三个主要聚类，一个鼠李糖乳杆菌聚类和两个包含副干酪乳杆菌菌株的聚类。TTGE得到的分组除了一个例外与REA聚类相对应。在TTGE聚类中，所有副干酪乳杆菌菌株形成一个总体组，有一条共同的TTGE条带，并且由于四个亚组中存在不止一条TTGE条带，该组又被细分为五个亚组。通过对显示这种现象的菌株的16S rRNA基因进行扩增和克隆来研究多条TTGE条带的出现情况，从而对每个菌株的12个克隆进行测序，几乎在所有情况下都证明了多态性。对显示序列变异的克隆进行TTGE以及通过菌株的核糖体分型揭示的16S rDNA测序，证实了16S rRNA基因内存在多态性。来自单个克隆的扩增DNA的迁移特征与该克隆所源自菌株的TTGE图谱中的特定条带相对应。用16S rDNA探针进行Southern印迹杂交表明干酪乳杆菌/副干酪乳杆菌中存在至少五个16S rRNA基因。在该复合体成员的16S rRNA基因片段中观察到比以前报道的更高程度的可变位置。干酪乳杆菌（CCUG 21451T）和玉米乳杆菌（CCUG 35515T）的16S rRNA基因拷贝之间的序列比较表明，这两个物种在一些拷贝中共享几乎相同的序列，而其他拷贝则差异更大。我们的结果为在干酪乳杆菌/副干酪乳杆菌复合体中难以明确分类提供了一种解释。
