Analysis of PurR from Lactococcus lactis
Special Symposium - Innovations in Food Technology (LMC Congress)
Innovations in Food Technology - Poster Session (Food - P2)
Keywords: purine, PurR, DNA binding, purification, Lactococcus
Purine nucleotides constitute an essential part of every cell as building blocks of nucleic acids and cofactors, for energizing intermediates in numerous cellular processes, and as carrier of chemical energy between biological reactions. In the Gram-positive lactic acid bacterium Lactococcus lactis, purines can be salvaged from the environment or synthesized de novo. The biosynthetic genes are under transcriptional regulation by the regulatory protein PurR, which responds in a feed-forward manner to the starting molecule for salvage and biosynthesis, 5-phosphoribosyl-1-pyrophosphate (PRPP). Lactococcal PurR is homologous to the PurR protein in the Gram-positive model organism Bacillus subtilis. Although PurR works as a repressor in B. subtilis, the homolog in Lactococcus lactis has been proven to act as an activator. Lactococcal PurR activates promoters with a conserved distance between the minus ten region and a "PurBox" motif with a consensus sequence of AWWWCCGAACWWT. Here, lactococcal PurR was expressed heterologously in Escherichia coli and purified. The stoichiometry and DNA binding activity were examined, as was the effect of the inducer PRPP on DNA binding. While PRPP causes the B. subtilis repressor to relief DNA binding, the effect of PRPP on the lactococcal activator was found to be far smaller. Additionally, the in vitro footprint (DNase I protection) was used to confirm the PurBox motif as a true binding site of PurR. The in vitro results are discussed together with the genetic evidence, and a combined model for PurR mediated regulation is presented.
Presented Thursday 20, 13:30 to 14:40, in session Innovations in Food Technology - Poster Session (Food - P2).
