Control of expression of LlaI restriction in Lactococcus lactis

Molecular Microbiology
D J O'Sullivan, Todd R Klaenhammer

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 pr...Continue Reading

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Citations

Mar 13, 2008·Nucleic Acids Research·Iwona Mruk, Robert M Blumenthal
Aug 31, 2013·Nucleic Acids Research·Wil A M Loenen, Elisabeth A Raleigh
Sep 4, 2004·Applied and Environmental Microbiology·Jonathan O'DriscollDouwe Van Sinderen
Sep 2, 2005·Molecular Microbiology·Jonathan O'DriscollDouwe van Sinderen

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