title: Comparison of expression vectors in Lactobacillus reuteri strains.
abstract: The synthesis of heterologous proteins in lactobacilli is strongly influenced by the promoter selected for the expression. In addition, the activity of the promoters themselves may vary among different bacterial hosts. Three different promoters were investigated for their capability to drive enhanced green fluorescent protein (EGFP) expression in Lactococcus lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016(T) and in five L. reuteri strains isolated from chicken crops. The promoters of the Lactobacillus acidophilus surface layer protein gene (slp), L. acidophilus lactate dehydrogenase gene (ldhL) and enterococcal rRNA adenine N-6-methyltransferase gene (ermB) were fused to the coding sequence of EGFP and inserted into the backbone of the pTRKH3 shuttle vector (pTRKH3-slpGFP, pTRKH3-ldhGFP, pTRKH3-ermGFP). Besides conventional analytical methods, a new quick fluorimetric approach was set up to quantify the EGFP fluorescence in transformed clones using the Qubit() fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Under the same conditions, the ldhL promoter produced 2.66 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016(T) and in L. reuteri isolates.
authors.affiliation: Centro Ricerche Biotecnologiche, Istituto di Microbiologia - Università Cattolica del Sacro Cuore, Cremona, Italy. michela.lizier@unicatt.it
authors.forename: Michela
authors.initials: M
authors.lastname: Lizier
authors.forename: Pier G
authors.initials: PG
authors.lastname: Sarra
authors.forename: Roberto
authors.initials: R
authors.lastname: Cauda
authors.forename: Franco
authors.initials: F
authors.lastname: Lucchini
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002645:Chickens; D024881:Drug Resistance, Bacterial; D004917:Erythromycin / Q000494:pharmacology; D005243:Feces / Q000382:microbiology; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D007779:Lactobacillus acidophilus / Q000235:genetics; D052200:Limosilactobacillus reuteri / Q000235:genetics* / Q000302:isolation & purification; D013294:Lactococcus lactis / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012641:Selection, Genetic; D012359:tRNA Methyltransferases / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004917:Erythromycin; D012359:tRNA Methyltransferases
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