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Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac^– to Lac⁺

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

Correspondence
Thy-Hou Lin
thlin{at}life.nthu.edu.tw

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.

The GenBank accession numbers for the sequences of the 16S rDNA and lac–gal gene cluster for L. casei ATCC 27139 are EU670679 and EU670680, respectively.

Two supplementary figures, showing the lactose and galactose metabolism pathways that have been identified in lactic acid bacteria and a comparison of promoter region and regulatory genetic elements of the lacTEGF operon in L. casei strains, and four supplementary tables, of API 50 CH fermentation profiles of L. casei ATCC 27139, potential RBSs and start codons, comparisons of the start and stop codons of the lacTEGF and galKETRM operons, and estimates of the homology of the lac and gal operons in L. casei ATCC 27139 (Lac^–) and L. casei (Lac⁺) strains, are available with the online version of this paper.