A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum (original) (raw)
Related papers
Use of bacteriocin promoters for gene expression in Lactobacillus plantarum C11
Journal of Applied Microbiology, 2004
Aims: To exploit promoters involved in production of the bacteriocin sakacin P for regulated overexpression of genes in Lactobacillus plantarum C11. Methods and Results: Production of sakacin P by Lact. sakei LTH673 is controlled by a peptide-based quorum sensing system that drives strong, regulated promoters. One of these promoters (P orfX ) was used to establish regulated overexpression of genes encoding chloramphenicol acetyltransferase from Bacillus pumilus, aminopeptidase N from Lactococcus lactis or chitinase B from Serratia marcescens in Lact. plantarum C11, a strain that naturally possesses the regulatory machinery that is necessary for promoter activation. The expression levels obtained were highly dependent on which gene was used and on how the promoter was coupled to this gene. The highest expression levels (14% of total cellular protein) were obtained with the aminopeptidase N gene translationally fused to the regulated promoter. Conclusions: Sakacin promoters permit regulated expression of a variety of genes in Lact. plantarum C11. Significance and Impact of the Study: This study shows the usefulness of regulated bacteriocin promoters for developing new gene expression systems for lactic acid bacteria, in particular lactobacilli.
Construction of vectors for inducible and constitutive gene expression in Lactobacillus
Microbial Biotechnology, 2011
Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A b-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli. Fig. 2. Nucleotide sequence of select intergenic regions. Native promoter regions for (A) FOS, (B) lac and (C) tre operons and (D) pgm. The consensus catabolite response element (cre) sequence WTGNAANCGNWNNCW (Barrangou et al., 2003) was used to identify cre-like sequences in the promoter regions. Putative -35 and -10 regions are in bold; putative cre-like sequences are underlined; putative ribosome binding sites (RBS) are boxed; and putative translation start sites are in shaded text. 359 T. Duong et al.
A transcriptional fusion vector, designated pNZ272, based on the promoterless ,-glucuronidase gene (gus4) ofEscherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage 4SK11G. The three resulting plasmids showed 0-glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific B-glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the * Corresponding author. Mailing address: Department of Biophysical Chemistry, NIZO, Kernhemseweg 2, 6718 ZB Ede, The Netherlands. Phone: 31-8380-59558. Fax: 31-8380-50400. Electronic mail address: NIZO@CAOS.CAOS.KUN. a reporter gene, whose expression should be easily detectable by using a chromogenic substrate. (10). Lactococcus lactis was grown in M17 broth (Difco Laboratories) (41) supplemented with 0.5% glucose (GM17). LAB were grown at 30°C (Lactococcus and Leuconostoc spp.) or 37°C (Lactobacillus spp.). Chloramphenicol was used at a concentration of 10 ,Lg/ml for LAB and 25 ,ug/ml for E. coli. Histochemical screening for gusA-positive clones was performed with 5-bromo-4-chloro-3-indolyl-f-D-glucuronide (X-Gluc) (Research Organics Inc., Cleveland, Ohio) at a final concentration of 0.5 mM.
Microbiology, 2005
Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using b-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50 % of total cellular protein.
Applied and environmental microbiology, 1994
A transcriptional fusion vector, designated pNZ272, based on the promoterless beta-glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed beta-glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a s...
Fems Microbiology Letters, 2010
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 TM fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 Â 10 À7 g of fluorescent EGFP (mL OD stationary culture ) À1 . Under the same conditions, the ldhL promoter produced 2.66 Â 10 À7 g of fluorescent EGFP (mL OD stationary culture ) À1 . 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. Ã Underlined base pairs in primers indicate introduced restriction sites; bold base pairs shown in primers indicate the RBS. Ap r , Ampicillin resistant; Em r , erythromycin resistant; Tet r , tetracycline resistant. FEMS Microbiol Lett 308 (2010) 8-15 c
Applied Microbiology and Biotechnology, 1999
A cassette based on the expression signals of the Lactobacillus brevis surface (S)-layer protein gene (slpA) was constructed. The low-copy-number vector pKTH2095, derived from pGK12, was used as the cloning vector. The eciency of slpA promoters in intracellular protein production was studied using three reporter genes, b-glucuronidase (gusA), luciferase (luc) and aminopeptidase N (pepN) in three dierent lactic acid bacteria hosts: Lactococcus lactis, Lactobacillus plantarum and Lactobacillus gasseri. The S-layer promoters were recognized in each strain and especially L. lactis and Lb. plantarum exhibited high levels of transcripts. The production kinetics of reporter proteins was studied as a function of growth. The GusA, Luc and PepN activities varied considerably among the lactic acid bacterial strains studied. The highest levels of b-glucuronidase and luciferase activity were obtained in L. lactis. The level of GusA obtained in L. lactis corresponded to over 15% of the total cellular proteins. The highest level of aminopeptidase N activity was achieved in Lb. plantarum where PepN corresponded up to 28% of the total cellular proteins at the late exponential phase of growth. This level of PepN activity is 30-fold higher than that in Lb. helveticus, which is the species from which the pepN gene originates.
Integrative Food-Grade Expression System Based on the Lactose Regulon of Lactobacillus casei
Applied and Environmental Microbiology, 2000
The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3 end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, -glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.