CONTROL OF EXPRESSION OF LLAI RESTRICTION IN LACTOCOCCUS-LACTIS

The plasmid encoded Llal R/M system from Lactococcus lactis ssp, lacti s consists of a bidomain methylase, with close evolutionary ties to ty pe IIS methylases, and a trisubunit restriction complex, Both the meth ylase and restriction subunits are encoded on a polycistronic 6.9 kb o peron, In this study, the 5' end of the llal 6.9 kb transcript was det ermined by primer extension analysis to be 254 bp upstream from the fi rst R/M gene on the operon, llalM, Deletion of this promoter region ab olished Llal restriction in L. lactis. Analysis of the intervening seq uence revealed a 72-amino-acid open reading frame, designated llalC, w ith a conserved ribosome binding site and helix-turn-helix domain, Ove rexpression of llalC in Escherichia coil with a T7 expression vector p roduced the predicted protein of 8.2 kDa. Mutation and in trans comple mentation analyses indicated that C.Llal positively enhanced Llal rest riction activity in vivo. Northern analysis and transcriptional fusion s of the llal promoter to a lacZ reporter gene indicated that C.Llal d id not enhance transcription of the Ilal operon, Databank searches wit h the deduced protein sequence for IlalC revealed significant homologi es to the E. coil Rop regulatory and mRNA stabilizer protein, Investig ation of the effect of C.Llal on enhancement of Llal restriction in L. lactis revealed that growth at elevated temperatures (40 degrees C) c ompletely abolished any enhancement of restriction activity, These dat a provide molecular evidence for a mechanism on how the expression of a restriction system in a prokaryote can be drastically reduced during elevated growth temperatures, by a small regulatory protein.