Gärtner D, Geissendörfer M, Hillen W
Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Federal Republic of Germany.
J Bacteriol. 1988 Jul;170(7):3102-9. doi: 10.1128/jb.170.7.3102-3109.1988.
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.
木糖异构酶在枯草芽孢杆菌W23、168和BR151菌株中表达受到抑制，在木糖存在时可被诱导。在168和BR151菌株中，该表达也受到葡萄糖抑制，不过在W23菌株中未观察到这种效应。在多拷贝质粒上构建了一个木糖 - 氯霉素乙酰转移酶融合基因，源自W23的一个366碱基对（bp）DNA片段上的木糖启动子指导氯霉素抗性的表达。在这种多拷贝情况下，表达的调控不是很明显。木糖启动子是转录起始的强信号。通过核酸酶S1图谱分析鉴定了木糖mRNA的5'序列。该启动子由 -10序列TAAGAT、-35序列TTGAAA（间隔17 bp）以及一个上游多聚腺苷酸区段（17 bp中有14个A）组成。为了研究调控机制，构建了一个木糖 - lacZ融合基因，并作为单拷贝整合到枯草芽孢杆菌168的淀粉酶基因中。该菌株在木糖存在时于X - Gal（5 - 溴 - 4 - 氯 - 3 - 吲哚基 - β - D - 半乳糖苷）指示平板上生长呈蓝色，在葡萄糖存在时呈白色。定量分析表明，木糖对β - 半乳糖苷酶的诱导作用为100倍。在木糖加葡萄糖存在时，指示基因的表达被抑制到完全诱导水平的30%。当在多拷贝质粒上反式提供366 - bp木糖DNA片段时，该菌株获得了最大lacZ表达的约25%至60%。这一结果表明，在没有木糖时的抑制作用是由一种可溶性因子反式介导的，该因子在枯草芽孢杆菌168中低水平表达。木糖效应依赖于负调控。通过斑点印迹分析对mRNA量的估计明确表明，木糖诱导发生在转录水平。针对366 - bp木糖调控DNA的核苷酸序列讨论了可能的分子机制。
