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.
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