Supplementary material 2

Introduction

The promoters of LdhL, a lactate dehydrogenase from L. sakei (Malleret et al. 1998), SlpA, a surface (S)-layer protein from L. acidophilus (Smit et al. 2001), and EmR, an erythromycin ribosomal methylase from L. fermentum (Fons et al. 1997) are regarded as three strong constitutive promoters in lactic acid bacteria. Studies on constitutive promoters to express proteins in L. fermentum strains have not yet been described. Therefore, the develepment of an efficient expression system for L. fermentum is highly desirable. The aim of this study was to identify strong constitutive promoters by comparing the production of a reporter protein-green fluorescent protein (GFP) driven by different promoters in L. fermentum.

Materials and Methods

Bacterial strains and plasmids are shown in Table 1, and primer sequences in Table 2. The gfp gene was amplified using the primers 123-1 and 123-2, while the promoter region of ldhL was amplified by the primers 123-3 and 123-4. After purification, these two fragments were ligated and amplified to generate a PldhL--gfp fragment by overlap extension PCR using primers 123-1 and 123-4. The resulting amplicon was digested by EcoR I and Kpn I, followed by insertion into pLEM415 to make the construct pLK123. pLK123 was then transformed into L. fermentum by electroporation. The pLK124 and pLK125 constructs were obtained using similar methods.

The L. fermentum strains (P415, P123, P124 and P125) were grown in MRS broth (containing 10 μg/ml erythromycin) at 37 °C anaerobically. To acquire fluorescent activity, the cultures were incubated with aeration (at 180 rpm) at 30 °C for 3 h. After centrifugation, the cell pellet was resuspended with phosphate-buffered saline (PBS, pH 7.0). A 10 μl bacterial suspension was then transferred and dried to attach onto Poly Prep slides (Sigma), followed by visualization under a fluorescence microscope (Olympus model BX51). Fluorescence measurements were performed as described by Siegele and Hu (1997). Briefly, the OD600 of the bacteria suspension was adjusted to < 0.3 in a volume of >3 ml. The 3 ml samples were transferred to a quartz cuvette, and fluorescence was measured in an fluorescence spectrophotometer (960, SHANGHAI PRECESION & SCIENTIFIC INSTRUMENT CO., LTD, Shanghai, China). Fluorescent intensity = fluorescence/OD600. The total protein extracts of bacteria concentration were measured by Bradford assay (Applygen Technologies Inc., Beijing, China). Protein extracts (5 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (10% acrylamide) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) for immunodetection. Target proteins were detected using rabbit anti-GFP (1:1000, Cell Signaling Technology, Boston, MA, USA), as primary antibodies, and HRP-conjugated anti-rabbit IgG, (1:10000) as a secondary antibody. Specifically bound peroxidase was detected by Western blot detection reagent (AmershamTM ECLTM Prime Western Blotting Detection Reagent, GE Healthcare, UK Limited Little Chalfont Buckinghamshire, UK) and then exposed to x-ray (GE Healthcare, UK) for 5-60 s.

Results

Three plasmids expressing GFP under the control of PldhL-gfp, PslpA-gfp and PemR-gfp respectively were generated into the backbone of the shuttle vector pEM415 in L. fermentum. The expression levels of GFP were assessed using fluorescence microscope, Western blot and fluorescence spectrophotometer, respectively.

The expression of GFP driven by all the three promoters was clearly observed under fluorescence microscope while GFP expression was undetectable in L. fermentum P415 (empty vector control) (Fig. A-D) . Based on the fluorescent intensities detected and Western blot analysis, the PldhL promoter appears to be the most active in L. fermentum (P < 0.05). The expression efficiencies of these chosen promoters in L. fermentum could be ranked as PldhL > PslpA > PemR. (Fig. E and F).

Table 1 Characteristics and sources of bacterial strains and plasmids used in this study

Characteristics / Source
Strains
L. fermentum I5007 / Wild type / Our lab
L. fermentum P123 / I5007 harboring pLK123 / This work
L. fermentum P124 / I5007 harboring pLK124 / This work
L. fermentum P125 / I5007 harboring pLK125 / This work
L. fermentum P415 / I5007 harboring pLEM415 / This work
E. coli DH5α / TIANGEN, Beijing, China
Plasmids
pLEM415 / AmpR, EmR, shuttle vector
pLK123 / The shutter plasmid carrying the P ldhL- gfp / This work
pLK124 / The shutter plasmid carrying the PslpA- gfp / This work
pLK125 / The shutter plasmid carrying the PemR- gfp / This work

Table 2 The primers used in the study

Primers / Sequence (5'-3') / Outcome
P123-1 / CGGAATTCGCCCTTTCCGGACAGAAGAT
P123-2 / CGTCATTATAATGGCTAGCAGTAAAGGAGA / Construction of pLK123
P123-3 / TGCTAGCCATTATAATGACGTCCTTTCTGT
P123-4 / GGGGTACCACAATGTTGGTCAAGGACTG
P124-1 / CGGAATTCGCCCTTTCCGGACAGAAGAT
P124-2 / AAAAGACCATATGGCTAGCAGTAAAGGAGA / Construction of pLK124
P124-3 / TGCTAGCCATATGGTCTTTTCCTCCTTGAA
P124-4 / GGGGTACCACAATGTTGGTCAAGGACTG
P125-1 / CGGAATTCGCCCTTTCCGGACAGAAGAT
P125-2 / GGAGAGATTACATGGCTAGCAGTAAAGGAG / Construction of pLK125
P125-3 / GCTAGCCATGTAATCTCTCCTGAAGTGATT
P125-4 / GGGGTACCACAATGTTGGTCAAGGACTG

GAATTC, EcoRⅠ; GGTACC, KpnⅠ

Fig. A-D: Four different strains of L. fermentum (P415, P123, P124 and P125) under fluorescence microscope (× 400)

Fig. E: Western blot analysis of cell lysates from different strains

Fig. F: Fluorescent intensity of different strains

References

Fons M, Hégé T, Ladiré M, Raibaud P, Ducluzeau R, Maguin E (1997) Isolation and characterization of a plasmid from Lactobacillus fermentum conferring erythromycin resistance. Plasmid 37(3):199-203

Malleret C, Lauret R, Ehrlich SD, Morel-Deville F, Zagorec M (1998) Disruption of the sole ldhL gene in Lactobacillus sakei prevents the production of both L- and D-lactate. Microbiology 144 ( Pt 12)3327-3333

Siegele DA, Hu JC (1997) Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proc Natl Acad Sci U S A 94(15):8168-8172

Smit E, Oling F, Demel R, Martinez B, Pouwels PH (2001) The S-layer protein of Lactobacillus acidophilus ATCC 4356: Identification and characterisation of domains responsible for S-protein assembly and cell wall binding. J Mol Biol 305(2):245-257

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