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GENETIC CONTROL OF BIOSYNTHESIS IN BACILLUS SUBTILIS

枯草芽孢杆菌生物合成的遗传控制

基本信息

批准号：
3282515
负责人：
STANLEY A ZAHLER
金额：
$ 8.65万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-04-01 至 1989-09-30
项目状态：
已结题

项目摘要

The genetic controls that operate in biosynthetic pathways in Gram-positive bacteria have not previously been amenable to analysis. To a considerable extent, the difficulties that have hindered such studies have now been overcome. We have methods for studying cis-trans relationships between alleles, for determining dominance and recessiveness, for inserting transposons into and near to genes, and for cloning genes on a variety of multicopy vectors. We now plan to use these new techniques to study the synthesis of the branched-chain amino acids isoleucine, valine and leucine by Bacillus subtilis, using a combination of genetics, biochemistry and molecular biology. In the early stages of the work we will study the controls involved in the expression of the ilvBC-leu operon, encoding the second and third enzymes of the isoleucine-valine pathway and the four cistrons responsible for the first three steps of the leucine pathway. This operon differs from known biosynthetic operons in Escherichia coli and other bacteria, since it combines genes for enzymes of two separate (but related) pathways. We will clone and sequence the region near the beginning of the ilvB gene to learn if the region contains recognizable sequences suitable for transcription controls, and if it contains binding sites for trans-acting control proteins that are involved in the expression of the operon. We will also examine the region between the ilvC and leuA cistrons, to learn if B. subtilis has a way to control the leucine pathway without affecting the isoleucine-valine pathway. Other genes of the pathways will also be studied. The ilvA gene (threonine deaminase) has already been examined by us. We have shown that the production of that enzyme is constitutive in B. subtilis strain 168, and under isoleucine control in other strains, e.g., strain W23. We will determine the differences between the genes from the two strains to learn how ilvA is controlled in strain W23. Later we will examine the ilvD gene (dihydroxyacid dehydratase) to learn if it is coordinately controlled with ilvA. It is our hope that we will uncover new methods of byosynthetic control used by Gram-positive bacteria and not by Gram-negative ones.

革兰氏阳性菌生物合成途径中的遗传控制在此之前，细菌还不能进行分析。到了相当大的在很大程度上，阻碍这类研究的困难现在已经克服困难。我们有研究顺反关系的方法决定显性和隐性的等位基因转座子进入和接近基因，以及在各种不同的多拷贝载体。我们现在计划使用这些新技术来研究支链氨基酸异亮氨酸、缬氨酸和亮氨酸的合成由枯草芽孢杆菌利用遗传学、生物化学和分子生物学。在工作的早期阶段，我们将研究参与ilvBC-leu操纵子表达的调控，编码异亮氨酸-缬氨酸途径的第二和第三酶以及四种负责亮氨酸途径前三步的顺反子。该操纵子不同于已知的大肠杆菌生物合成操纵子和其他细菌，因为它结合了两个独立的酶的基因(但相关)路径。我们将克隆该区域并对其进行测序开始学习ilvB基因的区域是否包含可识别的适合转录控制的序列，如果它包含结合参与表达的反式作用控制蛋白的位置这部歌剧。我们还将研究ILVC和LeuA之间的区域顺反子，以了解枯草杆菌是否有办法控制亮氨酸途径而不影响异亮氨酸-缬氨酸途径。这些途径的其他基因也将被研究。Ilva基因(苏氨酸脱氨酶)已经由我们检查过了。我们已经证明了该酶的产生在枯草杆菌168菌株中是构成的，并且在异亮氨酸控制下的其他菌株，例如菌株W23。我们会确定两个菌株的基因之间的差异以了解伊尔瓦是如何在W23菌株中被控制的。稍后我们将检测ilvd基因。 (二羟基酸脱水酶)，以了解它是否与伊尔瓦。我们希望我们能发现合成的新方法。由革兰氏阳性菌使用的对照，而不是由革兰氏阴性菌使用的对照。