Blaiotta Giuseppe, Fusco Vincenzina, Ercolini Danilo, Aponte Maria, Pepe Olimpia, Villani Francesco
Department of Food Science, School of Agriculture, The University of Naples Federico II, via Università 100, 80055 Portici, Italy.
Appl Environ Microbiol. 2008 Jan;74(1):208-15. doi: 10.1128/AEM.01711-07. Epub 2007 Nov 9.
A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.
获得了显示116个部分（499bp）嗜热栖热放线菌hsp60（groEL，编码60kDa热休克蛋白）核苷酸序列多样性的系统发育树，并将其与先前描述的16S rRNA和tuf基因序列的系统发育树进行比较。本研究产生的树的拓扑结构显示出与先前报道的嗜热栖热放线菌物种分布相似但不完全相同。然而，根据最新的系统研究，在所分析的序列中检测到/鉴定出43个单物种簇的明显分化。hsp60核苷酸序列的变异性略高于16S rRNA序列，这为设计或开发分子检测方法提供了更好的机会，从而能够鉴定和区分远缘或非常近缘的嗜热栖热放线菌物种。因此，我们的结果表明，hsp60可被视为推断嗜热栖热放线菌属成员分类学和系统发育的优秀分子标记，并且所选择的引物可用于简单的PCR程序，从而直接对hsp60片段进行测序。此外在本研究中，我们对所有499bp的hsp60部分序列进行了计算机辅助限制性内切酶分析，结果表明，通过使用AluI和TacI两种内切酶（在单独反应中）获得的PCR限制性片段长度多态性（RFLP）模式，除了植物乳杆菌/戊糖乳杆菌这一对菌株外，能够鉴定和区分所有43种所考虑的嗜热栖热放线菌物种。然而，通过用Sau3AI或MseI进一步分析，可以区分后一种菌株。hsp60 PCR-RFLP方法有效地应用于鉴定和区分从奶酪和干发酵香肠中分离出的总共110株野生嗜热栖热放线菌菌株（包括密切相关的物种，如干酪乳杆菌和鼠李糖乳杆菌或植物乳杆菌和戊糖乳杆菌）。
