title: Control of expression of LlaI restriction in Lactococcus lactis.
abstract: The plasmid encoded LlaI 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 llal 6.9 kb transcript was determined by primer extension analysis to be 254 bp upstream from the first R/M gene on the operon, llalM. Deletion of this promoter region abolished LlaI restriction in L. lactis. Analysis of the intervening sequence revealed a 72-amino-acid open reading frame, designated llalC, with a conserved ribosome binding site and helix-turn-helix domain. Overexpression of llalC 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-LlaI positively enhanced LlaI restriction activity in vivo. Northern analysis and transcriptional fusions of the llal promoter to a lacZ reporter gene indicated that C x LlaI did not enhance transcription of the llal operon. Databank searches with the deduced protein sequence for llalC revealed significant homologies to the E. coli Rop regulatory and mRNA stabilizer protein. Investigation of the effect of C x LlaI on enhancement of LlaI restriction in L. lactis revealed that growth at elevated temperatures (40 degrees 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.
authors.affiliation: Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695-7624, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: O'Sullivan
authors.forename: T R
authors.initials: TR
authors.lastname: Klaenhammer
mesh_terms: D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D007763:Lac Operon; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D009876:Operon; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification; D012323:RNA Processing, Post-Transcriptional; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C112323:LlaIC protein, Lactococcus lactis; D012313:RNA; C069779:DNA modification methylase LlaI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)