Wagner E, Marcandier S, Egeter O, Deutscher J, Götz F, Brückner R
Mikrobielle Genetik, Universität Tübingen, Germany.
J Bacteriol. 1995 Nov;177(21):6144-52. doi: 10.1128/jb.177.21.6144-6152.1995.
By transposon Tn917 mutagenesis, 16 mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild-type strain. The transposons were found to reside in three adjacent locations in the genome of S. xylosus. The nucleotide sequence of the chromosomal fragment affected by the Tn917 insertions yielded an open reading frame encoding a protein with a size of 328 amino acids with a high level of similarity to glucose kinase from Streptomyces coelicolor. Weaker similarity was also found to bacterial fructokinases and xylose repressors of gram-positive bacteria. The gene was designated glkA. Immediately downstream of glkA, two open reading frames were present whose deduced gene products showed no obvious similarity to known proteins. Measurements of catabolic enzyme activities in the mutant strains grown in the presence or absence of sugars established the pleiotropic nature of the mutations. Besides beta-galactosidase activity, which had been used to detect the mutants, six other tested enzymes were partially relieved from repression by glucose. Reduction of fructose-mediated catabolite repression was observed for some of the enzyme activities. Glucose transport and ATP-dependent phosphorylation of HPr, the phosphocarrier of the phosphoenolpyruvate:carbohydrate phosphotransferase system involved in catabolite repression in gram-positive bacteria, were not affected. The cloned glkA gene fully restored catabolite repression in the mutant strains in trans. Loss of GlkA function is thus responsible for the partial relief from catabolite repression. Glucose kinase activity in the mutants reached about 75% of the wild-type level, indicating the presence of another enzyme in S. xylosus. However, the cloned gene complemented an Escherichia coli strain in glucose kinase. Therefore, the glkA gene encodes a glucose kinase that participates in catabolite repression in S. xylosus.
通过转座子Tn917诱变，分离出16株木糖葡萄球菌突变体，这些突变体在葡萄糖存在下的β-半乳糖苷酶活性水平高于野生型菌株。发现转座子位于木糖葡萄球菌基因组的三个相邻位置。受Tn917插入影响的染色体片段的核苷酸序列产生了一个开放阅读框，编码一种大小为328个氨基酸的蛋白质，与天蓝色链霉菌的葡萄糖激酶具有高度相似性。还发现与革兰氏阳性菌的细菌果糖激酶和木糖阻遏物有较弱的相似性。该基因被命名为glkA。在glkA的紧下游，存在两个开放阅读框，其推导的基因产物与已知蛋白质没有明显的相似性。对在有或无糖存在下生长的突变菌株的分解代谢酶活性进行测量，确定了这些突变的多效性。除了用于检测突变体的β-半乳糖苷酶活性外，其他六种测试酶也部分解除了葡萄糖的阻遏作用。观察到某些酶活性的果糖介导的分解代谢阻遏作用降低。葡萄糖转运以及磷酸烯醇丙酮酸：碳水化合物磷酸转移酶系统的磷酸载体HPr的ATP依赖性磷酸化不受影响，该系统参与革兰氏阳性菌的分解代谢阻遏。克隆的glkA基因在反式中完全恢复了突变菌株中的分解代谢阻遏作用。因此，GlkA功能的丧失是分解代谢阻遏部分解除的原因。突变体中的葡萄糖激酶活性达到野生型水平的约75%，表明木糖葡萄球菌中存在另一种酶。然而，克隆的基因补充了大肠杆菌菌株中的葡萄糖激酶。因此，glkA基因编码一种参与木糖葡萄球菌分解代谢阻遏的葡萄糖激酶。
