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Control of expression of Lla I restriction in Lactococcus lactis
Author(s) -
O'sullivan Daniel J.,
Klaenhammer T. R.
Publication year - 1998
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1998.00748.x
Subject(s) - biology , operon , lactococcus lactis , ribosomal binding site , plasmid , genetics , microbiology and biotechnology , gene , start codon , primer extension , restriction site , lac operon , open reading frame , escherichia coli , restriction enzyme , messenger rna , peptide sequence , translation (biology) , bacteria , lactic acid
The plasmid encoded Lla I R/M system from Lactococcus lactis ssp. lactis consists of a bidomain methylase, with close evolutionary ties to type IIS methylases, and a trisubunit restriction complex. Both the methylase and restriction subunits are encoded on a polycistronic 6.9 kb operon. In this study, the 5′ end of the llaI 6.9 kb transcript was determined by primer extension analysis to be 254 bp upstream from the first R/M gene on the operon, llaIM . Deletion of this promoter region abolished Lla I restriction in L. lactis . Analysis of the intervening sequence revealed a 72‐amino‐acid open reading frame, designated llaIC , with a conserved ribosome binding site and helix–turn–helix domain. Overexpression of llaIC in Escherichia coli with a T7 expression vector produced the predicted protein of 8.2 kDa. Mutation and in trans complementation analyses indicated that C· Lla I positively enhanced Lla I restriction activity in vivo . Northern analysis and transcriptional fusions of the llaI promoter to a lacZ reporter gene indicated that C· Lla I did not enhance transcription of the llaI operon. Databank searches with the deduced protein sequence for llaIC revealed significant homologies to the E. coli Rop regulatory and mRNA stabilizer protein. Investigation of the effect of C· Lla I on enhancement of Lla I restriction in L. lactis revealed that growth at elevated temperatures (40°C) completely abolished any enhancement of restriction activity. These data 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.