Bacterial strains, plasmids, oligonucleotides, and culture conditions
The strains and plasmids used in the present study are summarized in Table 2. The oligonucleotide primers used are listed in Additional file 1: Table S2. Escherichia coli (E. coli) DH5α and BL21, which were used for the cloning and/or expression of genes of interest, were propagated in Luria Bertani (LB) broth at 37°C with aeration at 200 rpm/min. L. plantarum ST-III and its mutant strains were cultivated anaerobically in deMan–Rogosa–Sharpe (MRS) broth (Merck, Darmstadt, Germany) at 37°C without aeration. For the RT-qPCR analyses, wild-type and mutant bacterial strains were grown in chemically defined medium (CDM) [44, 45] containing 1% (w/v) FOS (Meiji Seika Kaisha, Tokyo, Japan) and D-ribose (Adamas, Shanghai, China). These additives were sterilized separately through a 0.2-mm filter. Where appropriate, the culture medium was supplemented with antibiotics at the following concentrations. To select antibiotic-resistant strains of E. coli, 100 μg/mL kanamycin, 50 μg/mL ampicillin, 30 μg/mL chloramphenicol, and 250 μg/mL erythromycin were added to LB. To select mutant strains of L. plantarum, 10 μg/mL chloramphenicol and 10 or 30 μg/mL (for replica plating) erythromycin were added to MRS medium.
Table 2 Relative transcript abundances of FOS-related genes in the wild-type and ΔsacR1 and ΔsacR2 strains grown in different sugarsa
Gene
|
Wild-Type Strain
|
ΔsacR1 Strain
|
ΔsacR2 Strain
|
FOS
|
GOS
|
FOS
|
GOS
|
FOS
|
GOS
|
sacPTS26b,c
|
3.10±0.32*
|
1.51±0.17
|
3.14±0.18*
|
1.22±0.05
|
3.04±0.31
|
3.27±0.16
|
sacAb,c
|
3.35±0.29*
|
1.28±0.23
|
3.18±0.14
|
3.24±0.24
|
3.42±0.12*
|
1.11±0.09
|
sacKb,c
|
3.16±0.36*
|
1.05±0.19
|
3.02±0.22
|
3.08±0.27
|
2.95±0.31*
|
0.87±0.14
|
a Data presented are mean values based on at least three replicates. Error bars indicate standard deviations.
b The relative transcription abundances of each gene in different conditions were calculated by the 2−ΔCt method and 16S rRNA was used as the internal standard.
c Asterisks indicate statistically significant differences (P < 0.05) of the corresponding values obtained from cells grown on FOS compared with those grown on GOS.
Construction of sacR1 and sacR2 mutants
The L. plantarum ST-III deletion strain was generated using the Cre-lox-based mutagenesis system [29]. The upstream and downstream DNA regions of sacR1 and sacR2 were amplified using the respective primer pairs (Additional file 1: Table S2). The resultant DNA fragments were cloned into the suicide vector pNZ5319 to yield the pNZ5319-up-down-1 and pNZ5319-up-down-2 plasmid constructs. These deletion plasmids were transfected into L. plantarum ST-III cells via electroporation, and deletion mutants were screened as described previously [12, 32]. Candidate double-crossover clones were confirmed by PCR analysis.
RNA extraction and RT-qPCR analysis
FOS and ribose were selected as carbon sources for RNA extraction. We grew overnight cultures of L. plantarum ST-III and two Lactobacillus plantarum ST-III deletion strains (ΔsacR1, ΔsacR2) via the transfer of 2% (v/v) inoculum into 100 mL of CDM supplemented with filter-sterilized solutions of 1% (w/v) FOS or ribose. Total RNA was extracted from exponentially growing wild-type and mutant cells (optical density at 600 nm [OD600] of 0.65) using TRIzol reagent (Invitrogen, Shanghai, China), as described previously [12]. Total RNA was then incubated with RNase-free DNase I and purified using a PrimeScript RT reagent kit (Takara Bio, Dalian, China). The quality and quantity of the RNA were evaluated using a Thermo Scientific Nanodrop 2000 device (Thermo, Waltham, MA, U.S.A.) and an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, U.S.A.), respectively.
For the RT-qPCR analysis, single-stranded cDNA was synthesized from total RNA using PrimeScript reverse transcriptase (Takara Bio, Dalian, China) according to the standard protocol. This synthesized cDNA was then used as a template for quantitative RT-PCR analysis, as described previously [10]. The primers used for the analysis are listed in Additional file 1: Table S2. All reactions were performed on the 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.) using previously reported PCR cycling conditions [46]. To standardize the results, the relative abundance of 16S rRNA [47] was used as the internal standard, and the relative gene expression data were calculated and analyzed using the 2−ΔΔCt method [48]. All samples were measured in triplicate.
Prediction of the binding sites of SacR1 and SacR2
RSAT was used to analyze the consensus motif of the TFBS for SacR1 and SacR2. The motifs were identified by scanning all upstream regions in the genome of L. plantarum ST-III based on the profiles of gene binding sites (Lp_0188 and Lp_3221) in L. plantarum WCSF1 via the RegPrecise database [2]. A PFM was constructed to collect TFBSs, and putative TFBSs in the upstream regions of sacPTS1 and sacPTS26 clusters were searched. The scores of candidate sites were calculated as the sums of the positional nucleotide weights, as previously described [49], and values >5 were considered indicative of potential TFBSs of SacR1 and SacR2.
Purification of SacR1 and SacR2 proteins expressed in E. coli
Expression of the sacR1 gene to produce recombinant protein was performed using the pTolo-EX5 vector (TOLO Biotech, Shanghai, China). Briefly, a 981bp sequence of the sacR1 gene was PCR amplified using the primer pair sacR1-F and sacR1-R, which includes the same XhoI site at the 5' end of the primers (Additional file 1: Table S2). Subsequently, the amplified DNA was digested by XhoI and inserted into the corresponding site of the pTolo-EX5 vector. A 1,002 bp sequence of the sacR2 gene was PCR amplified using the primer pair sacR2-F and sacR2-R, which include the NheI and HindIII sites at the 5' end of the primers, respectively (Additional file 1: Table S2). Expression of the sacR2 gene was achieved by digesting amplified DNA using the two restriction endonucleases, followed by insertion into the corresponding sites of the pET-28a (+) expression vector. The resulting plasmids, pTolo-EX5-sacR1 and pET-28a-sacR2, contained the target gene fused to an N-terminal His-tag sequence. The recombinant plasmids were transformed as described previously [50], and the strain harboring these plasmids were named E. coli BL21- sacR1 and E. coli BL21- sacR2.
E. coli BL21(DE3) cells transformed with the two recombinant plasmids were grown at 37°C in 100 mL of LB medium supplemented with kanamycin (150 μg /mL). When the OD600 reached 0.4–0.6, expression of the recombinant gene was induced by the addition of 1 mM isopropyl-b-D-thioisopropyl-b-D-thiogalactoside (IPTG). After an 8–h incubation at 25°C, the cells were harvested by centrifugation. The His6-tagged proteins were extracted and purified by nickel ion affinity chromatography on a Chelating Sepharose Fast Flow column (GE Healthcare, Waukesha, WI, U.S.A.) according to the manufacturer’s instructions. The purified proteins were desalted and concentrated using Amicon Ultra-0.5 centrifugal filter devices (Millipore, Billerica, MA, U.S.A.). The resultant proteins were used in EMSAs.
Electrophoretic mobility-shift assay (EMSA)
EMSAs were performed using 1 nM double-stranded DNA fragments (Ppts1−sacA, Pagl4, and PsacR2, ~200 bp) that were generated by PCR using specific primer pairs (Additional file 1: Table S2). The DNA fragments were located in the four promoter regions of the sacPTS1 and sacPTS26 clusters. The DNA probes were incubated with increasing quantities of the selected proteins in binding buffer (50 mM Tris-HCl, pH 8.0; 100 mM KCl; 2.5 mM MgCl2, 0.2 mM dithiothreitol [DTT]; 2 μg polydIdC; 10% [v/v] glycerol) in a total reaction volume of 20 μL for 30 min at 30°C. The samples were loaded onto 2% agarose gels containing 0.5× Tris-borate-EDTA buffer (TBE). To verify the specific binding of SacR1 and SacR2 to the TFBSs, each putative TFBS generated from the RSAT analysis according to the consensus motif was mutated and named TFBS-MUT (Additional file 1: Table S1). The mutations were introduced as previously reported [12].
Chromatin immunoprecipitation assay (ChIP)
The respective sacR1and sacR2 overexpression plasmids pSIP409-Flag-sacR1 and pSIP409-Flag-sacR2 were constructed by inserting the purified sacR1 or sacR2 coding sequence into a restriction enzyme-digested pSIP409 vector as described previously (Additional file 1: Table S2) [50]. Next, the recombinant plasmids were electroporated into L. plantarum ST-III, which were used to produce 409-Flag-sacR1 and 409-Flag-sacR2 for ChIP.
The ChIP procedure was modified from existing protocols [12]. Briefly, for the strains 409-Flag-sacR1 and 409-Flag-sacR2, the cells were cultured at an OD600 of 0.3 and then induced with peptide pheromone IP-673 (synthesized by Invitrogen, Shanghai, China) in a final concentration of 50 ng/mL and allowed to grow for 2 hours at 37°C. Subsequently, in vivo cross-linking in the cultures was performed using 1% (v/v) formaldehyde for 20 min, and subsequently quenched by the addition of glycine to a final concentration of 0.125 M at room temperature for 5 min. The bacterial cells were collected by centrifugation at 5,000 × g and 4°C for 5 min and washed twice with ice-cold 5 mM Tris-HCl (pH 8.0). The pellet was resuspended in 5 mM Tris-HCl (pH 8.0) containing 5 μL of protease inhibitors. Bacterial chromatin was sheared by ultrasonic disintegration (Bioraptor plus, Diagenode, Belgium) for 5 min at 4°C with input setting 6. After centrifugation, 5 mL of supernatant were transferred to a fresh tube as the input sample, and the remaining supernatant was added to the FLAG-binding beads overnight at 4°C on a rotating wheel. On the next day, the beads were removed from the supernatant via magnetic separation (DynaMagTM-2, Invitrogen, UK). The beads were washed four times in wash buffer (500 mM EDTA, 5M NaCl, 1M Tri-HCl, pH 8.0) and resuspended in 200 μL of elution buffer. The resulting supernatant was collected after magnetic bead separation, mixed with 5 M NaCl, and heated to 65°C for 12 h to reverse cross-links. DNA was purified via phenol:chloroform extraction and ethanol precipitation [51]. The purified DNA samples were analyzed by qPCR using specific primers (Additional file 1: Table S2). Normal rabbit IgG was used as a negative control. All qPCRs were performed on the 7300 Fast Real-Time PCR System using a three-step PCR procedure (initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 54°C for 25 s, and synthesis at 60°C for 25 s). Product specificity was confirmed by a melting curve analysis. The qPCR results of each ChIP sample were normalized to a region of the 16S rRNA gene. Relative target levels were calculated using the fold enrichment method [52]. The results are reported as the average enrichment for three biological replicates.
Statistical analysis
The data shown herein are representative of at least three independent experiments. Student’s t-test was used to determine statistical differences. Differences between samples with a P-value of ≤0.05 were considered statistically significant.
Table 3 Strains and plasmids used in this study.
Strain and plasmid
|
Relevant featurea
|
Source or reference
|
Strains
|
|
|
L. plantarum
|
|
CGMCC 0847
|
ST-III
|
Wild type
|
|
ΔsacR1 ::cat
|
Derivative of ST-III containing a lox66-P32-cat-lox71 replacement of sacR1
|
This study
|
ΔsacR2 ::cat
|
Derivative of ST-III containing a lox66-P32-cat-lox71 replacement of sacR2
|
This study
|
ΔsacR1
|
Derivative of ST-III containing a lox72 replacement of sacR1
|
This study
|
ΔsacR2
|
Derivative of ST-III containing a lox72 replacement of sacR2
|
This study
|
409-Flag-sacR1
|
Derivative of ST-III harboring pSIP409-Flag-sacR1
|
This study
|
409-Flag-sacR2
|
Derivative of ST-III harboring pSIP409-Flag-sacR2
|
This study
|
E.coli
|
|
|
DH5α
|
For general gene cloning and plasmid construction
|
Promega
|
BL21
|
For protein expression
|
Novagen
|
BL21-sacR1
|
E. coli BL21 (DE3) harboring pTolo-EX5-sacR1
|
This study
|
BL21-sacR2
|
E. coli BL21 (DE3) harboring Pet28a-sacR2
|
This study
|
Plasmid
|
|
|
pTolo-EX5
|
ApR, for cloning and protein expression, included His-tag
|
Tolobio
|
pET-28a (+)
|
KanaR, for cloning and protein expression, included His-tag
|
Novagen
|
pTolo-EX5-sacR1
|
ApR, pTolo-EX5 with sacR1 gene cloned into XhoI sites
|
This study
|
pET-28-sacR2
|
KanaR, pET-28a (+) with sacR2 gene cloned into NheI/HindШ sites
|
This study
|
pNZ5319
|
CmR, EmR; for multiple gene replacements in Gram-positive bacteria
|
[29]
|
pNZ5319-up-down-1
|
CmR, EmR; pNZ5319 derivative containing homologous regions up and downstream of sacR1
|
This study
|
pNZ5319-up-down-2
|
CmR, EmR; pNZ5319 derivative containing homologous regions up and downstream of sacR2
|
This study
|
pNZ5348
|
EmR; contains cre under the control of the lp_1144 promoter
|
[29]
|
pSIP409
|
EmR; for shuttle vector in E.coil, gusA controlled by PsppQ
|
[53]
|
pSIP409-Flag-sacR1
|
EmR; pSIP409 derivative; gusA replaced by Flag-tagged sacR1
|
This study
|
pSIP409-Flag-sacR2
|
EmR; pSIP409 derivative; gusA replaced by Flag-tagged sacR2
|
This study
|
a KanaR, kanamycin resistant; ApR, ampicillin resistant; CmR chloramphenicol resistant; EmR, erythromycin resistant.