Expression of the Bacillus subtilis xyl operon is repressed at the level of transcription and is induced by xylose

Abstract

Expression of xylose isomerase was repressed in Bacillus subtilis strains W23, 168, and BR151 and could be induced in the presence of xylose. The expression was also glucose repressed in strains 168 and BR151, although this effect was not observed with W23. A xyl-cat fusion gene was constructed on a multicopy plasmid, from which the xyl promoter located on a 366-base-pair (bp) DNA fragment derived from W23 directed the expression of chloramphenicol resistance. The regulation of expression was not very pronounced in this multicopy situation. The xyl promoter is a strong signal for transcription initiation. The 5' sequence of the xyl mRNA was identified by nuclease S1 mapping. The promoter consisted of the -10 sequence TAAGAT, the -35 sequence TTGAAA spaced by 17 bp, and an upstream poly(A) block with 14 As out of 17 bp. To study the regulation, a xyl-lacZ fusion gene was constructed and integrated as a single copy into the amygene of B. subtilis 168. This strain grows blue on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) indicator plates in the presence of xylose and white in the presence of glucose. Quantitatively, the induction of beta-galactosidase by xylose was 100-fold. In the presence of xylose plus glucose, the expression of the indicator gene was repressed to 30% of the fully induced level. About 25 to 60% of the maximal lacZ expression was obtained with this strain when the 366-bp xyl DNA fragment was provided in trans on a multicopy plasmid. This result indicates that repression in the absence of xylose is mediated in trans by a soluble factor which is expressed at a low level in B. subtilis 168. The xylose effect depended on negative regulation. The estimations of mRNA amounts by dot blot analysis showed unambiguously that the induction by xylose occurs at the level of transcription. The possible molecular mechanisms are discussed with respect to the nucleotide sequence of the 366-bp xyl regulatory DNA.