title: Quantitative transcript analysis of the inducible expression system pSIP: comparison of the overexpression of Lactobacillus spp. β-galactosidases in Lactobacillus plantarum.
abstract: BACKGROUND
  Two sets of overlapping genes, lacLMReu and lacLMAci, encoding heterodimeric β-galactosidases from Lactobacillus reuteri and Lactobacillus acidophilus, respectively, have previously been cloned and expressed using the pSIP vector system and Lactobacillus plantarum WCSF1 as host. Despite the high similarity between these lacLM genes and the use of identical cloning and expression strategies, strains harboring lacLMReu produced about twenty-fold more β-galactosidase than strains containing lacLMAci.
  
  
  RESULTS
  In this study, the plasmid copy numbers (PCN) of expression vectors pEH9R (lacLMReu) and pEH9A (lacLMAci) as well as the transcription levels of both lacLM genes were compared using quantitative PCR methods. Analyses of parallel fermentations of L. plantarum harboring either pEH9R or pEH9A showed that the expression plasmids were present in similar copy numbers. However, transcript levels of lacLM from L. reuteri (pEH9R) were up to 18 times higher than those of lacLM from L. acidophilus (pEH9A). As a control, it was shown that the expression levels of regulatory genes involved in pheromone-induced promoter activation were similar in both strains.
  
  
  CONCLUSIONS
  The use of identical expression strategies for highly similar genes led to very different mRNA levels. The data indicate that this difference is primarily caused by translational effects that are likely to affect both mRNA synthesis rates and mRNA stability. These translational effects thus seem to be a dominant determinant for the success of gene expression efforts in lactobacilli.
authors.affiliation: Food Biotechnology Lab, Department of Food Sciences and Technology, University of Natural Resources and Life Sciences Vienna, Austria.
authors.forename: Tien-Thanh
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
authors.forename: Thomas
authors.initials: T
authors.lastname: Maischberger
authors.forename: Philipp
authors.initials: P
authors.lastname: Schmelzer
authors.forename: Geir
authors.initials: G
authors.lastname: Mathiesen
authors.forename: Vincent Gh
authors.initials: VG
authors.lastname: Eijsink
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
mesh_terms: D005285:Fermentation; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D007779:Lactobacillus acidophilus / Q000201:enzymology*; D048191:Lactiplantibacillus plantarum / Q000378:metabolism*; D052200:Limosilactobacillus reuteri / Q000201:enzymology*; D010675:Pheromones / Q000494:pharmacology; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D010675:Pheromones; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001616:beta-Galactosidase