Ying C W, Ordal G W
Department of Biochemistry, School of Chemical Sciences, University of Illinois, Urbana 61801.
J Bacteriol. 1989 Mar;171(3):1631-7. doi: 10.1128/jb.171.3.1631-1637.1989.
The cheF gene, which is involved in chemotaxis in Bacillus subtilis, has been cloned, expressed, and sequenced. This gene is contained in a 0.7-kilobase PstI DNA fragment that was isolated from a lambda Charon 4A B. subtilis chromosomal DNA library. This fragment was subcloned into the expression vector pSI-1 and shown to complement the cheF mutation both for chemotaxis and for methanol production in response to the addition of attractants. Plasmid-encoded DNA expression in B. subtilis maxicells indicated that a membrane-associated polypeptide of 20-kilodaltons was expressed from this 0.7-kilobase DNA. The nucleotide sequence of this DNA fragment was determined, and an open reading frame capable of encoding a putative 175-amino-acid protein (Mr 20,002) was identified. In an effort to understand the function of the cheF protein, the dosage of the cheF gene product was varied by altering the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) during growth. In the presence of high concentrations of IPTG, chemotaxis was inhibited and methanol production was impaired.
参与枯草芽孢杆菌趋化作用的cheF基因已被克隆、表达和测序。该基因包含在一个0.7千碱基的PstI DNA片段中，该片段是从λ噬菌体Charon 4A枯草芽孢杆菌染色体DNA文库中分离出来的。这个片段被亚克隆到表达载体pSI-1中，并显示出在添加引诱剂时，对cheF突变体的趋化作用和甲醇产生具有互补作用。在枯草芽孢杆菌大细胞中质粒编码的DNA表达表明，从这个0.7千碱基的DNA中表达出一种20千道尔顿的膜相关多肽。测定了该DNA片段的核苷酸序列，并鉴定出一个能够编码假定的175个氨基酸的蛋白质（分子量20,002）的开放阅读框。为了了解cheF蛋白的功能，在生长过程中通过改变IPTG（异丙基-β-D-硫代半乳糖苷）的浓度来改变cheF基因产物的剂量。在高浓度IPTG存在下，趋化作用受到抑制，甲醇产生受损。
