Showing papers by "Todd R. Klaenhammer published in 1998"

Molecular Characterization of a Phage-Inducible Middle Promoter and Its Transcriptional Activator from the Lactococcal Bacteriophage φ31

Abstract: An inducible middle promoter from the lactococcal bacteriophage φ31 was isolated previously by shotgun cloning an 888-bp fragment (P 15A10 ) upstream of the β-galactosidase (β-Gal) gene ( lacZ.st ) from Streptococcus thermophilus (D. J. O’Sullivan, S. A. Walker, S. G. West, and T. R. Klaenhammer, Bio/Technology 14:82–87, 1996). The promoter showed low levels of constitutive β-Gal activity which could be induced two- to threefold over baseline levels after phage infection. During this study, the fragment was subcloned and characterized to identify a smaller, tightly regulated promoter fragment which allowed no β-Gal activity until after phage infection. This fragment, defined within nucleotides 566 to 888 (P 566–888 ; also called fragment 566–888), contained tandem, phage-inducible transcription start sites at nucleotides 703 and 744 (703/744 start sites). Consensus −10 regions were present upstream of both start sites, but no consensus −35 regions were identified for either start site. A transcriptional activator, encoded by an open reading frame (ORF2) upstream of the 703/744 start sites, was identified for P 566–888 . ORF2 activated P 566–888 when provided in trans in Escherichia coli . In addition, when combined with pTRK391 (P 15A10 :: lacZ.st ) in Lactococcus lactis NCK203, an antisense ORF2 construct was able to retard induction of the phage-inducible promoter as measured by β-Gal activity levels. Finally, gel shift assays showed that ORF2 was able to bind to promoter fragment 566–888. Deletion analysis of the region upstream from the tandem promoters identified a possible binding site for transcriptional activation of the phage promoters. The DNA-binding ability of ORF2 was eliminated upon deletion of part of this region, which lies centered approximately 35 bp upstream of start site 703. Deletion analysis and mutagenesis studies also elucidated a critical region downstream of the 703/744 start sites, where mutagenesis resulted in a two- to threefold increase in β-Gal activity. With these improvements, the level of expression achieved by an explosive-expression strategy was elevated from 3,000 to 11,000 β-Gal units within 120 min after induction.

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.

Inducible gene expression systems in Lactococcus lactis.

Abstract: Lactococcus lactis is industrially important microorganism used in many dairy fermentations. Numerous genes and gene expression signals from this organism have now been identified and characterized. Recently, several naturally occurring, inducible gene-expression systems have also been described in L. lactis. The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes. Novel gene-expression systems in Lactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.

Common elements regulating gene expression in temperate and lytic bacteriophages of Lactococcus species

Abstract: A phage-inducible middle promoter (P15A10) from the lytic, lactococcal bacteriophage φ31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (>95%) to the right cohesive ends of two temperate phages of the P335 species, φr1t and φLC3. Sequencing upstream and downstream of P15A10 showed that the high degree of homology between φ31 and φr1t continued beyond the phage promoter. With the exception of one extra open reading frame in φ31, the sequences were highly homologous (95 to 98%) between nucleotides 13448 and 16320 of the published φr1t sequence. By use of a β-galactosidase (β-Gal) gene under the control of a smaller, more tightly regulated region within the P15A10promoter, P566–888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage φr1t induced the P566–888 promoter, as determined from an increase in β-Gal activity. Hybridization of nine other lactococcal strains with32P-labeled P566–888 showed that theLactococcus lactis strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter. Mitomycin C induced the resident prophages in all these strains and concurrently induced the P566–888 promoter, as determined from an increase in β-Gal activity. DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of φr1t. In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P566–888 from the lytic phage φ31. These results point to a conserved mechanism in the regulation of gene expression between the lytic phage φ31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.

A method for mapping phage-inducible promoters for use in bacteriophage-triggered defense systems

Abstract: We have recently developed a novel bacteriophage-protection system for Lactococcus lactis, based on a two-component genetic ‘trap’. An inducible promoter from a lytic bacteriophage is used to activate a lethal gene after infection, killing the host cell and halting phage proliferation. To expand the potential use of this novel defense strategy, promoters specific to any particular phage of interest must be available for fusion to a universal death gene. A method to localize regulated promoters within the context of the total phage genome was evaluated. The ‘capping’ activity of the vaccinia virus guanylyltransferase was exploited to label newly synthesized mRNA extracted from infected cells at sequential time points over the course of a phage infection. The labeled mRNAs were then used as probes in Southern hybridization reactions to identify restriction fragments in the phage genome where new transcripts were initiated during progression of the phage lytic cycle. This method has been used successfully in our laboratory to map the general location of a number of inducible promoters on the genomes of bacteriophages attacking lactic acid bacteria. Once identified and cloned, small fragments encoding inducible promoters can be partially sequenced and primer extension reactions carried out on phage RNA, isolated over the course of an infection, to pinpoint the precise location of the promoter. In this study, we illustrate the use of the capping method to map the phage-inducible promoter on the genome of the lactococcal bacteriophage skl. This approach provides a rapid and efficient means to identify promoter regions on the genomes of relatively uncharacterized phages. These promoters can then be used in a variety of applications, including phage-triggered defenses and inducible gene expression systems.