Angell S, Schwarz E, Bibb M J
John Innes Institute, John Innes Centre, Norwich, UK.
Mol Microbiol. 1992 Oct;6(19):2833-44. doi: 10.1111/j.1365-2958.1992.tb01463.x.
Mutants (glk) of Streptomyces coelicolor A3(2) that are resistant to the non-utilizable glucose analogue 2-deoxyglucose are deficient in glucose kinase activity, defective in glucose repression, and usually unable to utilize glucose. A 2.9 kb BclI fragment, previously shown to restore a wild-type phenotype to a glk deletion mutant that lacks the entire segment, contains two complete open reading frames that would encode proteins of 20.1 kDa (ORF2) and 33.1 kDa (ORF3). ORF3 is transcribed from its own promoter, and also from a promoter that initiates transcription upstream of ORF2. A derivative of the temperate phage phi C31 containing ORF3 alone restored a wild-type phenotype when used to lysogenize the deletion mutant. The product of ORF3 is homologous to members of a family of repressor proteins encoded by xylR in Bacillus subtilis and Lactobacillus pentosus, and by nagC in Escherichia coli. Although this might suggest that ORF3 encodes a positive activator for glucose kinase, rather than the enzyme itself, ORF3 restored the ability to metabolize glucose to an E. coli glk mutant, and activity gels of cell extracts of E. coli containing ORF3 cloned in the pT7-7 expression vector demonstrated that the ORF3 product has glucose kinase activity.
天蓝色链霉菌A3(2)对不可利用的葡萄糖类似物2-脱氧葡萄糖具有抗性的突变体（glk）缺乏葡萄糖激酶活性，存在葡萄糖阻遏缺陷，并且通常无法利用葡萄糖。一个2.9 kb的BclI片段，先前已证明可使缺乏整个片段的glk缺失突变体恢复野生型表型，该片段包含两个完整的开放阅读框，分别编码20.1 kDa（ORF2）和33.1 kDa（ORF3）的蛋白质。ORF3从其自身的启动子转录，也从一个在ORF2上游启动转录的启动子转录。仅含有ORF3的温和噬菌体phi C31的衍生物用于溶源化缺失突变体时可恢复野生型表型。ORF3的产物与枯草芽孢杆菌和戊糖乳杆菌中由xylR编码以及大肠杆菌中由nagC编码的阻遏蛋白家族成员同源。尽管这可能表明ORF3编码的是葡萄糖激酶的正激活剂而非酶本身，但ORF3恢复了大肠杆菌glk突变体代谢葡萄糖的能力，并且在pT7-7表达载体中克隆了ORF3的大肠杆菌细胞提取物的活性凝胶显示ORF3产物具有葡萄糖激酶活性。
