UBC Theses and Dissertations
Observations on the control of biosynthesis of valine and isoleucine in Escherichia coli Stapleton, Joyce Alice
Previous work in many laboratories has established that the addition of L-valine to a culture of Escherichia coli K-12 growing exponentially on minimal medium causes inhibition resulting in persistent linear growth. The inhibition can be removed by addition of isoleucine. Growth of other strains of E. coli e.g. E. coli B and two mutants of K-12 (E. coli AB1020 and AB1005) was not affected by addition of valine. E. coli LL5, another mutant of K-12, showed no inhibition of growth by valine added in concentrations up to 1 x 10ˉ⁴M. However, the addition of 1 x 10ˉ³M valine caused a decrease in the logarithmic growth rate, and 1 x 10ˉ²M valine caused persistent linear growth. The inhibition of growth by valine in LL5 was antagonized by L-isoleucine (as is the case with wild-type E. coli K-12), but not by α-ketobutyrate, threonine or pyruvate. Kinetic studies showed that the maximal inhibition of acetohydroxy acid synthetase (AHAS) by 1.5 x 10ˉ³ M valine was 84% for E. coli K-12, 55% for E. coli B, 75% for E. coli AB1005, 77% for E. coli AB1020 and only 20% for E. coli LL5. It is proposed that the persistence of linear growth of K-12 in valine-containing medium is the result of incomplete (84%) feed-back inhibition of AHAS. Analysis of the data for valine inhibition of AHAS was carried out by the method of Levitsky and Koshland (1969) using Hill Plots. E. coli strains K-12, AB1005 and AB10 20 showed "positive cooperativity" between inhibitor (valine) binding sites at low valine concentrations, and "negative cooperativity" between inhibitor binding sites at high valine concentrations. The AHAS of E. coli strains E and LL5, however, showed only negative cooperativity between binding sites for inhibitor, which could be the mechanism for incomplete inhibition of AHAS by valine. Preliminary kinetic analyses using Michaelis-Menten plots were carried out with strains K-12 and LL5. The levels of threonine deaminase (TD) and AHAS were also examined in four of the E. coli strains, K-12, B, AB1020 and LL5. AHAS was repressed and TD derepressed in E. coli K-12 grown (linearly) with 10ˉ³M valine. In E. coli strains B and AB1020, growth with 10ˉ³M valine had no effect on the levels of AHAS or (derepressed) TD. In E. coli strain LL5, growth with 10ˉ³M valine did not change the AHAS level but caused significant derepression of TD. Sensitivity of growth to valine has been correlated with three properties of the organism: 1) Feed-back sensitivity of acetohydroxy acid synthetase, to L-valine; 2) The level of acetohydroxy acid synthetase in the cell; 3) The level of biosynthetic threonine deaminase - the regulatory enzyme for isoleucine biosynthesis.