Introduction

Lactic acid bacteria (LAB) are food-grade microorganisms widely used in the food and animal industries. Lactococcus lactis is one species of LAB that has been used as a component of starter culture for fermented dairy products such as cheese and fermented milk [23]. Many studies have shown that L. lactis can be genetically engineered [13, 15, 26]. Recent researches in L. lactis have focused on the development of oral vaccines [2, 21, 26]. Generally, many genetic elements are required to develop a recombinant oral vaccine using LAB [12]. Among these elements, promoters are important and essential for foreign antigen expression. However, finding a promoter that fulfills all needed requirements is not easy. Therefore, screening and selecting of promoters is an important and on-going avenue of research.

Despite many efforts to screen promoters, the conventionally used strategies were very similar in terms of construction of promoter library and use of reporter genes such as lacZ, lacG, gusA, cat, gfp, and luxAB [3]. Construction of promoter library with high diversity requires a lot of labor, cost, and time. To avoid these problems and to screen and select novel promoters, we designed a new strategy using microarray data. In this study, we cloned and characterized a novel tuf promoter (Ptuf) of L. lactis IL1403 which was selected through analysis of previously published microarray data.

Materials and Methods

Bacterial Strains and Plasmids

The bacterial strains and plasmids used in this work are listed in Table 1. Escherichia coli Top10F’ was used as a host bacterium for construction of pGEM-T easy-derived vectors and BmpB expression. L. lactis IL1403 was used as a host bacterium for construction of pIL252-derived vectors and BmpB expression.

Table 1 Bacterial strains and plasmids
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Culture Media, Bacterial Transformation, and DNA Isolations

E. coli, L. lactis, and Lactobacillus strains were cultivated in Luria-Bertani broth, M17 broth containing 0.5% (w/v) glucose (called M17G), and MRS broth, respectively. The used antibiotics were ampicillin (100 μg/ml) for E. coli and erythromycin (5 μg/ml) for LAB. All bacterial species were transformed by electroporation as described previously [1] and according to manufacturer’s instructions. Plasmid DNA was isolated from E. coli with the Plasmid Purification Mini Kit (Nucleogen, South Korea) according to manufacturer’s instructions. Plasmid DNA from L. lactis and Lactobacillus strains was isolated as described previously [16].

Analysis of Microarray Data for IL1403 and Statistical Analysis

We collected two sets of microarray data on IL1403 from the Gene Expression Omnibus (GEO) database of National Center for Biotechnology Information (NCBI) website (Series GSE5761 and GSE4872). These sets contain exponential phase-specific gene expression profiles of IL1403 cultivated in either a chemically defined medium (CDM) (Sample GSM134562, GSM134563 and GSM134564 within Series GSE5761) [17] or a CDM with ten-fold reduced concentrations of isoleucine, leucine, and valine (Sample GSM109311, GSM109314, GSM109315 within Series GSE,4872). Sorting of microarray data and linear regression was carried out using Microsoft Excel or R [22].

Construction of Plasmid Vectors

pIL.CatT was constructed by subcloning a 971-bp fragment containing the RBS region, cat, the transcriptional terminator for pepN of IL1403, an N-terminal SalI site, and a C-terminal EcoRI site into the SalI and EcoRI sites of pIL252. A schematic diagram for the construction of expression vectors is shown in Fig. 1. Four promoters were cloned by PCR-amplification (Table 2) with IL1403 genomic DNA as a template using the i-MAXTM II DNA polymerase (iNtRON BIOTECHNOLOGY, South Korea). The amplicons and pET.MbmpB [9] were cut with NdeI, and the resulting fragments were ligated. The ligated DNA was used as a template for PCR-amplification using four sets of primers (Table 2 and Fig. 1). Each amplicon was, respectively, subcloned into pGEM-T easy and pIL.CatT to generate E. coli expression vectors (pT.Ptuf.MbF, pT.Ppfk.MbF, and pT.Pusp.MbF) and LAB expression vectors (pILPdnaJ.Mb, pILPtuf.Mb, pILPpfk.Mb, and pILPusp.Mb).

Fig. 1
figure 1

Schematic diagram for the construction of BmpB expression vectors. TpepN transcription terminator of the IL1403 pepN gene; ermAM erythromycin resistance gene; Amp R ampicillin resistance gene; cat chloramphenicol resistance gene; repD replication protein D; repE replication protein E

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Table 2 Primers for cloning and sequencing
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Analysis of Nucleotide Sequences

DNA sequencing was carried out using Applied Biosystems 3730xl by the National Instrumental Center for Environmental Management (NICEM, Seoul National University, South Korea).

Bacterial Cell Harvest for Western Blot Assay

A single bacterial colony was inoculated into 3 ml of fresh broth medium and incubated at 30°C without agitation or at 37°C with agitation for 17 h. The resulting culture (30 μl) was subsequently inoculated into 3 ml of fresh broth medium and incubated until the final optical density at 600 nm (OD600) was about 2.3 (E. coli), 2.3 (L. lactis), 3.6 (L. plantarum), or 6.0 (L. reuteri). All cultures were centrifuged at 5,000g for 10 min at 4°C to harvest cells, and the cells were stored at −80°C until analysis.

Protein Extraction, SDS-PAGE, and Western Blot Assay

In order to extract protein from E. coli, the harvested cells were washed twice with PBS and resuspended in 200 μl of PBS containing 1% (w/v) SDS. After briefly vortexing, resuspended cells were incubated at 95°C for 5 min. Cell debris was removed from the cell extract by centrifugation at 14,000g for 10 min. The protein-containing supernatant was stored at −80°C until analysis. In order to extract protein from LAB, the harvested cells were washed twice with PBS and resuspended in 200 μl of PBS. Glass beads (212–300 μm, Sigma) were supplemented, and the cells were incubated on ice and shaken intermittently for 1 h by vortex. Cell debris was removed from the cell extract by centrifugation at 14,000g for 10 min. The protein-containing supernatant was stored at −80°C until analysis. SDS-PAGE was carried out with a 12% poly-acrylamide gel. Each well was loaded with 20 μg of E. coli protein or 40 μg of LAB protein in the same volume. The expressed BmpB was detected with polyclonal anti-BmpB mouse serum (1:4,000, Aprogen, South Korea) and anti-mouse IgG, HRP-linked antibody (1:20,000, Cell Signaling Technology, USA) as described previously [9].

Results

Screening and Selecting of a Promoter Through Analysis of Microarray Data

We collected and analyzed two sets of previously published microarray data on IL1403 (Series GSE5761 and GSE4872) to identify highly expressed genes in IL1403. All data were sorted by normalized signals (Table 3). The first set (Series GSE5761) of data was derived from IL1403 cells cultivated in CDM. The second data set (Series GSE4872) was derived from IL1403 cells cultivated in CDM with ten-fold reduced concentrations of isoleucine, leucine, and valine, as compared to CDM. The spot having the highest expression signal in each set was identified as the same gene, tuf (Table 3), which encodes the translation elongation factor Tu (EF-Tu). According to the annotation data for the IL1403 genome (NC_002662), we could infer that the tuf gene has its own promoter and does not share that promoter with any other genes. Therefore, in this study, we examined the novel Ptuf.

Table 3 List of spots with high level of expression signal in microarray data for IL1403a
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Sequence Analysis and Cloning of a Putative Promoter

To compare the activity of the Ptuf with that of other promoters, we selected three other known lactococcal promoters for the dnaJ, pfkA, and usp45 genes (PdnaJ, PpfkA, and Pusp45) of L. lactis subsp. cremoris MG1363 [11, 20, 24]. They were reported to have activity in MG1363, and their cognate IL1403 promoters were also expected to have activity in IL1403 but their activity was not examined in IL1403. Therefore, we used them not only as positive controls but also as samples in this study. The 180-bp region of putative Ptuf containing the −35 and −10 hexamers was cloned (Fig. 2). The putative promoters for the other three genes were also cloned according to previous reports. Sequences for each cognate MG1363 promoter are similar to those of IL1403, with similarities of 95% (Ptuf, 180 bp), 86% (PdnaJ, 333 bp), 99% (PpfkA, 140 bp), and 92% (Pusp45, 193 bp). Each cloned promoter was, respectively, fused to the promoterless and modified bmpB gene (Fig. 1), which was derived from Brachyspira hyodysenteriae, a major pathogen of swine dysentery. Since administration of BmpB was reported to protect pigs from this disease [10], we selected the bmpB for potential use of L. lactis expressing BmpB as an oral vaccine.

Fig. 2
figure 2

Nucleotide sequence of IL1403 Ptuf. Two Ptuf sequences derived from IL1403 and MG1363 were aligned using ClustalW. Putative −35/−10 hexamers and RBS were indicated

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Construction of BmpB Expression Vectors Using Lactococcal Promoters

We constructed BmpB expression vectors to examine promoter activity in E. coli and LAB. Four promoters were fused to modified bmpB, respectively. Each fused fragment was subcloned into pGEM-T easy and pIL.CatT to generate two expression vectors (Fig. 1). For no apparent reason, we failed to construct an E. coli expression vector containing PdnaJ. All LAB expression vectors were transformed into L. lactis IL1403, L. plantarum Lb25, and L. reuteri ATCC23272.

BmpB Expression by Lactococcal Promoters and Host-Range of Promoters

In order to examine the activity of the four lactococcal promoters, cells of all wild-type and recombinant strains were cultivated and harvested. Protein was extracted, and BmpB expression induced by these promoters was detected by western blot assay (Fig. 3). As expected, BmpB expression was induced by all promoters when L. lactis IL1403 was used as the expression host. The promoterless vector did not induce BmpB expression (data not shown). Ptuf activity was strongest among the promoters examined in IL1403. In IL1403, activity of PpfkA was stronger than that of PdnaJ or Pusp45. This result was restricted to IL1403, and we could not expect the same result in other bacteria. Therefore, we examined these promoters using other LAB species and unrelated bacteria (E. coli). As shown in Fig. 3, these promoters have different host-range. Only Pusp45 could weakly induce BmpB expression in L. plantarum Lb25, the others were weakly active in L. reuteri ATCC23272. In E. coli Top10F’, Ptuf and PpfkA were strongly active, but Pusp45 was not active.

Fig. 3
figure 3

Detection of BmpB induced by lactococcal promoters. BmpB expression in different bacteria harboring different plasmids was detected. Arrow indicates BmpB. This figure is a representative result from three independent experiments. dnaJ, pILPdnaJ.Mb; tuf, pILPtuf.Mb or pT.Ptuf.MbF; pfkA, pILPpfk.Mb or pT.Ppfk.MbF; usp45, pILPusp.Mb or pT.Pusp.MbF; WT, no plasmid

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Promoter Activity was Dependent on Growth Phase

We showed that Ptuf and PpfkA were strong in IL1403. Since expression of foreign genes by strong promoter can sometimes cause problems, such as growth inhibition of host cells [4], we examined the growth pattern of wild-type and two recombinant IL1403 strains (Fig. 4a), but there was no difference in the growth pattern between them. Some genes are expressed in a growth phase-dependent manner in LAB [5]. Therefore, we investigated the growth phase-dependent activity of the two promoters (Fig. 4b). We sampled LAB cells at indicated times during cultivation (Fig. 4a), and promoter activity was estimated by BmpB expression at each time. Maximal activity was observed at late log phase for Ptuf and at early stationary phase for PpfkA, and the activity was maintained at early stationary phase.

Fig. 4
figure 4

Growth curve and growth phase-dependent BmpB expression. a Growth curve of three strains; wild-type IL1403 and its recombinants harboring pILPtuf.Mb or pILPpfk.Mb. Arrows indicate sample-collecting times. b Growth phase-dependent BmpB expression induced by two promoters. These figures are representative results from three (a) or two (b) independent experiments

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Correlation Between Promoter Activity and Microarray Data

Since the promoters used in this study were derived and cloned from IL1403, we wondered whether the level of BmpB expression induced by each promoter correlates with the expression level in the microarray data. The microarray data (Series GSE5761) were sorted by the normalized signal, and then regression of the normalized signal (Y) on the rank (X) was analyzed (Ŷ = 7.9752 −  0.9398 ln(X), R 2 = 0.9809, P < 0.0001). In order to linearize the curvilinear relation, we transformed the value of the normalized signal, and then regression of the transformed signal (Y) on the rank (X) was analyzed (Ŷ = 168.33 + 0.8894 X, R 2 = 0.9963, P < 0.0001). Finally, we analyzed the correlation between the level of BmpB expression (Y) by each promoter and the transformed signal (X) of each cognate gene (Ŷ = 2.2964 − 0.0021 X, R 2 = 0.7186, P = 0.06968, Fig. 5). Since there was no value of pfkA in the microarray data, we substituted values of ldh and pyk genes for a value of pfkA. This was because the two genes lie downstream of pfkA and are under the control of PpfkA. Although the sample number is low, we observed a correlation tendency. This result suggests that a gene with high microarray signal may have a strong promoter and that a gene with low signal may have a weak one.

Fig. 5
figure 5

Correlation between the level of BmpB expression and the value of transformed signal. Relative BmpB expression indicates intensity of each BmpB band divided by the intensity of the BmpB band induced by PdnaJ

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Discussion

Previously, bacterial Ptuf had been investigated only in Chlamydia trachomatis [18] and Lactobacillus johnsonii [25]. There are no reports of Ptuf in L. lactis. We have now identified and cloned a novel Ptuf of L. lactis IL1403.

Based on our present results regarding IL1403 Ptuf, we do not know whether they need trans-elements. However, they worked without any additional trans-elements in IL1403. On the contrary, the nisin-inducible promoter needs additional trans-elements such as NisK and NisR proteins [6]. Therefore, the short IL1403 Ptuf can be easily and simply used in IL1403.

Generally, promoters have their own host-range. As shown in Fig. 3, Ptuf and PpfkA did not work in L. plantarum, but they worked in L. lactis, L. reuteri, and E. coli. Therefore, these promoters also have their own host-range. One previous study showed that LAB promoters working in L. lactis have similar activity in E. coli [8]. We also showed a similar result using Ptuf and PpfkA.

Several proteomic studies of L. lactis reported that the product of IL1403 or MG1363 tuf gene was detected at high level by two-dimensional gel electrophoresis (2-DE) [7, 14]. These previous reports are coincident with the strong activity of Ptuf observed in this study. The las operon of L. lactis consists of pfkA, ldh and pyk and is involved in glycolysis. This operon has a strong promoter in L. lactis MG1363 [20], and products of these IL1403 genes were also detected at high levels by 2-DE [7]. These previous reports are coincident with the strong PpfkA activity observed in this study.

Since genetically engineered microorganisms may have potential risks and bio-hazards, many studies have focused on food-grade gene expression systems which consist of genetic elements derived from food-grade microorganisms [4, 8, 11]. LAB are representative food-grade microorganisms. Therefore, the promoters examined in this study are all food-grade genetic elements, and we can use them as food-grade promoters to develop LAB-based applications and as genetic tools to investigate physiology of LAB.