Tsai Yu-Kuo, Chen Hung-Wen, Lo Ta-Chun, Lin Thy-Hou
Prof. Thy-Hou Lin laboratory, Institute of Molecular Medicine and Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan, ROC.
Microbiology (Reading). 2009 Mar;155(Pt 3):751-760. doi: 10.1099/mic.0.021907-0.
Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac(+) clones could be obtained. A gene cluster (lacTEGF-galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac(-)) and its Lac(+) revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac-gal gene clusters was different. The protein sequence identity between the lac-gal gene cluster and those reported previously for some L. casei (Lac(+)) strains was high; namely, 96-100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac(+) revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac(+) revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac(-) to Lac(+) for L. casei ATCC 27139.
乳糖代谢是干酪乳杆菌菌株中一种可变的表型。在本研究中，我们发现干酪乳杆菌ATCC 27139无法利用乳糖。然而，当以乳糖作为唯一碳源时，可以获得自发的Lac(+)克隆。对参与干酪乳杆菌ATCC 27139（Lac(-)）及其Lac(+)回复突变株（命名为R1株）中乳糖和半乳糖代谢的一个基因簇（lacTEGF-galKETRM）进行了测序和特征分析。我们发现，两个乳糖 - 半乳糖基因簇中，只有位于lacTEGF启动子（lacTp）中的一个核苷酸不同。该乳糖 - 半乳糖基因簇与先前报道的一些干酪乳杆菌（Lac(+)）菌株的蛋白质序列同一性很高；即，发现同一性为96 - 100%，且未发现提前终止密码子。在干酪乳杆菌ATCC 27139的其他41个独立分离的Lac(+)回复突变株中，也检测到位于lacTp启动子区域内的一个单点突变。根据检测到的点突变位置，回复突变株可分为六类。对lacTp转录进行的引物延伸实验表明，这六类Lac(+)回复突变株的lacTp启动子具有功能，而干酪乳杆菌ATCC 27139的lacTp启动子则没有功能。Northern印迹实验进一步证实，R1株的lacTEGF操纵子被乳糖诱导但被葡萄糖抑制，而干酪乳杆菌ATCC 27139从未检测到印迹信号。这些结果表明，lacTp启动子中的一个单点突变能够恢复一个功能完全的lacTEGF操纵子的转录，并导致干酪乳杆菌ATCC 27139的表型从Lac(-)转变为Lac(+)。
