Promotor analysis and transcriptional regulation of Lactobacillus pentosus genes involved in xylose catabolism
1994
Lokman, B.C. | Leer, R.J. | Sorge, R. van | Pouwels, P.H. (TNO Nutrition and Food Research, Rijswijk (Netherlands). Dept. of Molecular Genetics and Genetechnology)
The xyl genes in Lactobacillus pentosus are induced by xylose and repressed by glucose, ribose, and arabinose. Northern blot analysis showed that regulation is mediated at the transcriptional level. Under inducing conditions, two xylA transcripts were detected, a major transcript of 1.5 kb and a minor transcript of 3 kb. The 3 kb transcript also comprises sequences from xylB, suggesting that xylA and xylB are transcribed together. A 1.2 kb xylR transcript was found under inducing and non-inducing conditions. In the presence of xylose, a second xylR transcript (over 7 kb) was detected, which includes sequences from two upstream genes, xylQ and xylP. The transcription start sites for xylA and xylR were mapped by primer extension and S1 nuclease experiments at 42 and 83 nucleotides, respectively upstream of the translation start sites. Induction by xylose of the chloramphenicol acetyltransferase (CAT) gene under control of the xylA promoter, on a multicopy plasmid, was 60 to 80-fold, but only 3 to 10-fold in the presence of glucose and xylose. Expression of CAT under control of the xylR promoter was constitutive at a level tenfold less than that observed under control of the xylA promoter. Sequence analysis suggests the presence of two operator-like elements, one overlapping with the promoter -35 region of xylA and controlling the expression of xylA by binding factors involved in catabolite repression, and a second operator downstream of the promoter -10 region of xylA, which may bind the product of xylR, the repressor. Titration experiments with multiple copies of these elements showed that, under inducing conditions, expression of xylA in wild-type L. pentosus is suboptimal.
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