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.

1

Three different strains of Lactobacillus reuteri isolated from chicken crop transformed with pTRKH3-ermGFP. Upper panels show fluorescence following DNA-intercalating bisbenzimide staining. Lower panels show GFP fluorescence. (a) Strain H09, transformed. (b) Strain N10, transformed. (c) Strain N09, transformed. (d) Strain N09, wild type.

2

Influence of culture conditions on erm-GFP expression in Lactobacillus reuteri. (a) Western blot analysis of cell lysates and cell-free medium from L. reuteri DSM 20016^T/pTRKH3-ermGFP grown in different culture conditions. Cells were grown in several combinations of the following parameters: MRS broth (–) or MRS broth buffered with KHPO₄ (B), 30°C (30°) or 37°C (37°), anaerobiosis (–) or aeration (O₂) (see Materials and methods). (b) GFP content in lag-phase cell lysates of L. reuteri I09/pTRKH3-ermGFP was assessed after culturing in different conditions. Cells were grown in several combinations of the following parameters: 30°C (30°) or 37°C (37°), anaerobiosis (–) or aeration (O₂), MRS broth (–) or MRS broth buffered with KHPO₄ (Buff.). The GFP content is referred to the same amount of cells (measured as OD_{600 nm}). w.t., Wild type.

3

Western blot analysis of cell lysates and surnatants from Lactococcus lactis MG1363 bearing different vectors. M, Molecular weight marker; w.t., lysate, L. lactis MG1363, untransformed; L, lysate, ldhL promoter; E, lysate, ermB promoter; Es, surnatant, ermB promoter; S, lysate, slp promoter; Ss, surnatant, slp promoter; C+, 100 pg of purified 6xHis-EGFP. The lysate from cells transformed with the slp-vector shows a double band, corresponding to the presence of the SLP-leader peptide fused to the N-terminal of the reporter (upper band) and to the processed peptide (lower band). In the next lane, the sample containing the spent culture medium (Ss) shows only the secreted processed GFP. The concentration of GFP in such sample is higher than in the corresponding one (Es) obtained with the ‘stronger’ermB vector.

4

Activity of the three promoters in different species. w.t., Wild type; Erm, pTRKH3-ermGFP transformants; Ldh, pTRKH3-ldhGFP transformants; Slp, pTRKH3-slpGFP transformants. Fluorescent GFP content in lag-phase cell lysates was assessed for the three vectors in Lactococcus lactis MG1363 cultured in GM17 at 30°C, Lactobacillus reuteri DSM 20016^T and L. reuteri N09 both cultured in buffered MRS at 30°C. The fluorescence of each sample was compared with a purified 6xHis-EGFP standard by means of a Qubit^™ fluorometer. The fluorescent GFP content is reported to a similar amount of cells (measured as OD_{600 nm}) for each species.

5

Western blot analysis of cell lysates from the crop isolate N09 of Lactobacillus reuteri transformed with three different vectors and cultured in MRS at 37°C or in buffered MRS at 30°C. M, Molecular weight marker; S 37°C, slp-GFP vector, MRS at 37°C; L 37°C, ldh-GFP vector, MRS at 37°C; E 37°C, erm-GFP vector, MRS at 37°C; S 30°C+B, slp-GFP vector, buffered MRS at 30°C; L 30°C+B, ldh-GFP vector, buffered MRS at 30°C; E 30°C+B, erm-GFP vector, buffered MRS at 30°C; w.t., L. reuteri N09 untransformed; C+, 300 pg of purified 6xHis-EGFP.