A biochemical and genetic study of isoleucine and valine biosynthesis in {\it Escherichia coli\/} and {\it Serratia marcescens}

Ming-Fong Lu, Purdue University

Abstract

A plasmid was constructed that carried the ilvG and ilvM genes and the associated promoter and leader regions derived from the K-12 strain of Escherichia coli. The ilvG gene contained a +1 frameshift mutation that enabled the plasmid to specify acetohydroxy acid synthase II. The plasmid was modified by deletions into the terminus of and within the ilvM gene and insertions into the ilvM gene. The effects of these modifications on the phenotypes of the plasmids were examined in a host strain that lacked all three isozymes of acetohydroxy acid synthase. Most of the ilvM mutant plasmids so obtained permitted growth of the host strain in the absence of isoleucine but not in the absence of valine. Growth in the presence of valine, however, was very slow. The enzyme activity of acetohydroxy acid synthase obtained in this study was less than 10% of the wild type activity for the various ilvM mutations. The ilvM and ilvG genes can be expressed independently of each other.^ A deletion mutation covering the Serratia marcescens terminator region of the ilv leader and amino terminus of the ilvG gene was shown to have only a limited escape from repression but very little if any control by amino acid limitation. Transaminase assays showed that the rho mutation could neither improve the low level control observed in this strain, nor could it lead to a markedly higher escape from repression. Nucleotide sequence analysis of S. marcescens indicated a low homology in the promoter region yet the attenuator region was highly conserved. The sequences of the ilvG and ilvM genes also showed moderately conserved nucleotide and deduced amino acid sequences. However a reading frame difference near the end of the ilvM region in S. marcescens from that in E. coli makes translation stop 6 nucleotides upstream instead of 19 nucleotides upstream of the ilvE gene as in E. coli K-12. This could possibly couple the translation of ilvM and ilvE to each other. The implication of this phenomenon is further discussed. (Abstract shortened with permission of author.) ^

Degree

Ph.D.

Advisors

Major Professor: H. E. Umbarger, Purdue University.

Subject Area

Biology, Molecular|Biology, Microbiology|Chemistry, Biochemistry

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