Abstract
A gene cluster containing nine ORFs involved in the metabolism of lactose and galactose in L. rhamnosus TCELL-1 was sequenced and characterized. The order of the ORFs was lacTEGF and galKETRM. Northern blotting experiments revealed that the gene cluster could be transcribed as one lacTEGF-galKETRM mRNA though there were three major transcripts (lacTEGF, galKETRM and galETRM) detected for the gene cluster. The transcription of the lac or gal operon was independently induced in the presence of lactose or galactose. Northern blotting and primer extension experiments found the presence of four putative promoters upstream from the ORFs lacT (lacTp), galK (galKp1 and galKp2) and galE (galEp). The measurements of enzymatic activities of GalK, GalE and GalT suggested that expression of the gal operon was subjected to a glucose repression and galactose activation mechanism. We suspect that the gal operon could be regulated by a dual regulation mechanism, namely glucose repression possibly mediated by CcpA (Catabolite control protein A) and galactose induction through GalR and its binding sites. Besides, the β-galactosidase activity could also be detected in L. rhamnosus TCELL-1. These results indicated that the galactose moiety of lactose in L. rhamnosus TCELL-1 could be metabolized by two alternative pathways (the Leloir and the tagatose 6-phosphate pathways) while galactose metabolism could be mediated by the Leloir pathway. This work provides important information of sugar metabolism in L. rhamnosus. 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, under the lactose selection, the spontaneous revertant colonies (Lac+) were 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 as strain R1) was sequenced and characterized. We found that only one nucleotide located in the lacTEGF promoter (lacTp) of these 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 to 100% identity was found and no premature stop codon was identified on it. A single point mutation occurred on the same lacTp promoter region was also detected for 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. Northern blotting and primer extension experiments for the activity of the lacTp promoter further found that the lacTp promoter of strain R1 was functional while that of L. casei ATCC 27139 was not. These results suggest that a single point mutation on the lacTp promoter was able to restore the transcription of fully functional lacTEGF operon and caused a phenotype switch from Lac- to Lac+ for L. casei ATCC 27139 (Lac-).