Abstract
Objective
Biofilm formation and adhesion are important indicators for evaluating the beneficial effects of probiotics. However, the relationship of specific genes with the biofilm formation and adhesion of Lactiplantibacillus remains unclear. The rbk gene encodes ribokinase, which is involved in ribose metabolism and may be related to biofilm formation and adhesion. This study aims to analyze the effects of rbk overexpression on the biofilm formation and adhesion of Lactiplantibacillus paraplantrum LR-1, explore the role of this gene in the regulation of quorum sensing (QS) and expression of related genes, and reveal the influencing mechanism of rbk overexpression in bacteria from a metabolic profile perspective.
Methods
L. paraplantarum LR-1 was selected as the target strain, and the shuttle vector pMG76e was used to construct the recombinant strain rbk-pMG76e-LR-1. The overexpression of rbk was confirmed by qRT-PCR and the enzyme activity assay. Crystal violet staining, cell adhesion assay, and qRT-PCR were employed to evaluate the effects of rbk overexpression on biofilm formation, adhesion, and expression of tuf, luxS, and rpoN. Furthermore, untargeted metabolomics analysis was conducted to assess the effect of rbk overexpression on the metabolic profile. Finally, the effect on the biofilm formation and adhesion of LR-1 was verified by exogenous addition of metabolites.
Results
The overexpression of rbk increased the biofilm formation of LR-1 and the adhesion to HT-29 cells by 1.55-2.34 folds and 3.58 folds, respectively. Moreover, the overexpression of rbk up-regulated the expression levels of tuf, luxS, and rpoN by 70.30, 96.94, and 45.61 folds, respectively. The untargeted metabolomics analysis revealed that rbk overexpression led to changes in the abundance of 145 metabolites. Finally, the exogenous addition of l-proline, rhamnose, and nicotinamide adenine dinucleotide (NADH) increased the biofilm formation of LR-1 by 1.27, 1.39, and 1.25 folds and the adhesion by 1.40, 1.41, and 1.52 folds, respectively.
Conclusion
This study demonstrates that rbk can serve as a key target for enhancing the biofilm formation and adhesion of Lactiplantibacillus.
Lactic acid bacteria (LAB), especially the species of genus Lactobacillus, have recently received attention because of their generally recognized as safe (GRAS) status and their potential health-promoting effects as probiotic
Our previous research has identified a series of genes that may be associated with quorum sensing (QS) and biofilm formation in Lactobacillus, including the rbk gene. The enzyme encoded by the rbk gene is ribokinase, which has the fundamental physiological function of degrading d-ribose and ATP to produce ribose-5-phosphate and ADP. The substrate, ribose, has no toxic effects and has structural similarity with autoinducer 2 (AI-2). It is the signal molecules of the LuxS/AI-2 quorum sensing syste
This study selects Lactiplantibacillus paraplantarum LR-1 as the research subject, which is originally isolated from fermented vegetables. This strain has probiotic properties, such as alleviating coliti
1 Materials and Methods
1.1 Bacterial strains, cells, and culture conditions
The strain used in this experiment was L. paraplantarum LR-1 (CICC 24809), isolated from Sichuan pickles. The strain was cultured in de Man-Rogosa-Sharpe (MRS) broth (Beijing Land Bridge Technology Co., Ltd.) or MRS-Agar under aerobic conditions at a temperature of 37 ℃. Escherichia coli (E. coli) DH5α (TaKaRa Biotechnology (Dalian) Co., Ltd.) was cultured in Lennox broth (LB) or LB agar under aerobic conditions at a temperature of 37 ℃. E. coli DH5α containing pMG76e vector and L. paraplantarum LR-1 containing pMG76e vector were cultured in LB medium containing 200 μg/mL of erythromycin and in MRS medium containing 3 μg/mL of erythromycin, respectively.
The HT-29 cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Amresco) containing 10% (V/V) fetal bovine serum (Zhejiang TianHang Biotechnology Co., Ltd.), 1% dual antibodies (penicillin (100 IU/mL, ThermoFisher Scientific) and streptomycin (100 μg/mL, ThermoFisher Scientific)) at 37 ℃ in an atmosphere of 5% CO2/95% air at constant humidity.
1.2 Construction of plasmids and bacterial strains
The chromosomal DNA of L. paraplantarum LR-1 was extracted using a TIANamp Bacteria DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.) according to the manufacturer’s instructions. Using the genomic DNA as template, the rbk gene was amplified by PCR with primers containing Xba I and Xho I restriction sites (
Primers | Sequences (5′→3′) |
---|---|
rbk-1-F | TGCTCTAGAATGAATACGGTAACAGTG |
rbk-1-R | CCGCTCGAG TTATTTACCCTCCGCGGC |
The competent cells of L. paraplantarum LR-1 were prepared as follows. Briefly, the LR-1 strain was inoculated into MRSS medium (MRS medium containing 0.3 mol/L sucrose) containing 1% glycine and cultured until the OD600 reached between 0.4 and 0.6. A 10 mL aliquot of the culture was then centrifuged at 6 000 r/min for 8 min at 4 ℃ to collect the cells. The pellet was resuspended in 2 mL of washing buffer (0.3 mol/L sucrose with 0.1 mol/L MgCl2) and centrifuged again at 6 000 r/min for 8 min at 4 ℃. Finally, the cells were resuspended in 2 mL of 30% PEG-1500 and centrifuged at 6 000 r/min for 10 min. The final pellet was resuspended in 200 μL of 30% PEG-1500 and kept on ice for further use. The recombinant plasmid rbk-pMG76e was introduced into the competent LR-1 cells by electroporation under the conditions of 1.5 kV and 400 Ω. After the electroporation, the cells were allowed to rest on ice for 5 min before adding 1 mL of MRSSM medium (MRS containing 0.3 mol/L sucrose and 0.1 mol/L MgCl2) and incubating at 37 ℃ for 2 h. The cells were then spread on MRS plates containing 3 μg/mL of erythromycin and incubated at 37 ℃ for 36-48 h. Positive clones were selected and identified by PCR, confirming the presence of the rbk-pMG76e recombinant strain.
1.3 Real-time quantitative PCR (qRT-PCR)
The strain containing the empty vector pMG76e (pMG76e-LR-1), the recombinant strain with the plasmid rbk-pMG76e (rbk-pMG76e-LR-1), and the wild-type strain LR-1 were cultured overnight at 37 ℃ in MRS medium. Total RNA was extracted using TRIzol Agent (ThermoFisher Scientific) according to the manufacturer’s instructions. The RNA quality was assessed using the A260/A280 and A260/A230 ratios. The extracted RNA was converted into cDNA using a TUREscript 1st Strand cDNA Synthesis Kit (Aidlab Biotechnologies Co., Ltd.). The expression levels of the genes rbk, tuf, luxS and rpoN were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The 16S rRNA gene was used as the reference gene. The primers for the relevant genes (
Primers | Sequences (5′→3′) |
---|---|
rbk-2-F | AGGTCCCCGCTGAACTTTTA |
rbk-2-R | CACCAGCTGACGTTGTATCG |
tuf-F | CGCAACTGATGGTCCTATGC |
tuf-R | CGCTGAACCACGGATAACAG |
luxS-F | TGATACAGCGGGCTTACACA |
luxS-R | CTTCCCACTTAGCTGGACCA |
rpoN-F | CCAAGCAATTCGGGACTACG |
rpoN-R | TTTCTTCTGCCCGGAGAACT |
16S RNA-F | CAACGAGCGCAACCCTTATT |
16S RNA-R | GCAGCCTACAATCCGAACTG |
1.4 Ribokinase activity assay
The strains rbk-pMG76e-LR-1, pMG76e-LR-1 and the wild-type LR-1 were cultured in MRS without erythromycin for 8 h. The cells were centrifuged at 5 000×g at 4 ℃ for 5 min and washed three folds with PBS. The viable cell counts of the three strains were determined using the plate counting method and standardized. The bacterial cells were then disrupted by ultrasound at 4 ℃ for 20 min (4 s work+6 s break for 120 cycles). The lysed cells were centrifuged at 5 000×g at 4 ℃ for 20 min, and the supernatant was collected.
Ribokinase activity was measured using a Ribokinase (RBKS) Enzyme-linked Immunoassay Kit (Shanghai Jingkang Bioengineering Co., Ltd.) according to the manufacturer’s instructions. Samples were added into microtiter plate coated with purified ribokinase antibodies, and then combined with horseradish peroxidase (HRP)-labeled ribokinase antibodies to form an antibody-antigen-enzyme-labeled antibody complex, which is washed and then added into Tetramethylbenzidine (TMB) to initiate color development. Under the catalytic action of HRP enzyme, TMB will be converted into blue, and finally into yellow under the action of acid. The intensity of the color is directly proportional to the ribokinase concentration in the samples. And the ribokinase activity was quantified by measuring the absorbance at 450 nm and referencing a standard curve.
1.5 Growth curves
The recombinant rbk-pMG76e-LR-1, pMG76e-LR-1 strains and the wild-type LR-1 strain were cultured in MRS without erythromycin. The cell densities were determined at 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, and 36 h by measuring the OD600 using a UV-1800 Spectrophotometer (Shimadzu).
1.6 Biofilm formation assay
The biofilm-forming ability of the strains rbk-pMG76e-LR-1, pMG76e-LR-1 and the wild-type LR-1 was determined using the crystal violet (CV) staining metho
1.7 Adhesion assay
HT-29 cells were cultured in a 24-well plates (Corning Incorporated) until a monolayer was formed, with approximately 2.5×1
Adhesion ratio=Bacterial counts after adhesion/Bacterial counts before adhesion×100% | (1) |
1.8 Untargeted metabolomics analysis
1.8.1 Sample extraction
The rbk-pMG76e-LR-1 and pMG76e-LR-1 strains were cultured in MRS without erythromycin for 8 h. Afterwards, the cells were collected by centrifugation at 5 000×g for 5 min at 4 ℃. Then, 60 mg of each sample was resuspended in 200 μL of water and mixed thoroughly for 60 s. Then, 800 μL of extraction solvent (methanol:acetonitrile=1:1) was added, and the mixture was homogenized for 60 s. This was followed by ultrasound treatment in ice-water for 30 min, which was repeated twice. The samples were then stored at -20 ℃ for 1 h to allow protein precipitation, followed by centrifugation at 12 000×g for 20 min at 4 ℃. The supernatant was dried using a vacuum concentrator and subsequently stored at -80 ℃.
1.8.2 Mass spectrometry
The analyses were performed using UHPLC (Agilent Technologies) coupled with a quadrupole time-of-flight (Sciex) at Shanghai Applied Protein Technology Co., Ltd. For HILIC separation, the samples were analyzed using a 2.1 mm×100 mm ACQUIY UPLC BEH 1.7 µm column (Waters Corporation). In both ESI positive and negative modes, the mobile phase consisted of A: 25 mmol/L ammonium acetate and 25 mmol/L ammonium hydroxide in water, and B: acetonitrile. For RPLC separation, a 2.1 mm×100 mm ACQUIY UPLC HSS T3 1.8 µm column (Waters Corporation) was utilized. In ESI positive mode, the mobile phase comprised A: water with 0.1% formic acid and B: acetonitrile with 0.1% formic acid. In ESI negative mode, the mobile phase consisted of A: 0.5 mmol/L ammonium fluoride in water and B: acetonitrile. For MS/MS analysis, the ESI source conditions were set as follows: Ion Source Gas1 (Gas1) as 60, Ion Source Gas2 (Gas2) as 60, curtain gas (CUR) as 30, source temperature: 600 ℃, and IonSpray Voltage Floating (ISVF)±5 500 V.
1.8.3 Data analysis
The raw MS data (wiff.scan files) were converted to MzXML files using ProteoWizard MS Convert v3.0.6458 before importing into freely available XCMS software. After normalizing total peak intensity, the processed data were uploaded into SIMCA-P (v14.1), where they underwent multivariate data analysis, including Pareto-scaled principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA), and orthogonal PLS-DA (OPLS-DA). Metabolites with variable importance for the projection (VIP) value>1 were further analyzed for significance, with a threshold of P<0.05 was considered statistically significant.
1.8.4 Bioinformatic analysis
The metabolites were matched against the online Kyoto encyclopedia of genes and genomes (KEGG) database (http://geneontology.org/). KEGG pathway enrichment analyses were conducted using Fisher’s exact test, with pathways yielding a P-value less than 0.05 considered significant. The relative expression data of the analyzed metabolites were utilized to perform hierarchical clustering analysis using Cluster 3.0 (http://bonsai.hgc.jp/~ mdehoon/software/cluster/software.htm).
1.9 Evaluation of the effects of exogenous metabolites on biofilm formation and adhesion of LR-1
Based on the metabolomic analysis results, this experiment selected 8 metabolites related to bacterial biofilm formation and adhesion. These include l-histine, l-proline, l-arginine, l-serine (Solarbio), Rhamnose, N-acetyl-d-lactosamine, NADH (Shandong Sparkjade Biotechnology Co., Ltd.), and d-mannose-6-phosphate (Rhawn). Solutions of these metabolites were prepared at a concentration of 50 mg/mL, and the pH was adjusted to neutral.
The CV staining method was used to assess the impact of the aforementioned 8 metabolites on the biofilm formation of LR-1. Briefly, the bacterial suspension was adjusted to an OD600 of 0.1 and inoculated into 96-well cell culture plates with 100 μL per well. Subsequently, 100 μL of the metabolites solution diluted with MRS broth was added, resulting in a final concentration of 100 mg/mL. After incubation at 37 ℃ for 48 h, the biofilms were detected as aforementioned in section 1.6.
The adhesion experiment set up two treatment methods: one involved culturing LR-1 with exogenous metabolites before extracting the bacterial cells and co-culturing them with HT-29 cells, referred to as the met-LR-1-post-LR-1 group. The other method involved simultaneous co-culturing of exogenous metabolites, LR-1, and HT-29 cells, referred to as the met-LR-1-HT-29 group. The specific procedure for the met-LR-1-post-LR-1 group was as follows: 8 exogenous metabolites were added to fresh LR-1 bacterial suspension to achieve a final concentration of 10 mg/mL, with a bacterial concentration of 1
1.10 Statistical analysis
Significant differences were determined using analysis of variance (ANOVA) in R (v4.1.1). Data are presented as mean±standard deviation (SD). P-values less than 0.05 were considered significant, with *** representing P<0.001, ** representing P<0.01, * representing P<0.05, and ns indicating no significance. Each experiment was conducted in triplicate, with each test involving at least three separate measurements. In the case of untargeted metabolomics analysis, six samples were analyzed. The relationships between significantly different metabolites and the phenotype of strains were calculated using the Pearson correlation coefficient with the OmicShare tools, a free online platform for data analysis (http://www.omicshare.com/tools). Cytoscape (v3.8.2) was employed to visualize the interaction networks among the significantly different metabolites and the phenotype of the strains.
2 Results and Discussion
2.1 The successful construction of the recombinant strain rbk-pMG76e-LR-1 with overexpression of the rbk gene
The PCR identification results of strains containing the recombinant plasmids rbk-pMG76e and pMG76e are shown in Figure

Figure 1 The rbk gene was successfully overexpressed in LR-1. PCR validation of the rbk-pMG76e-LR-1 (A) and pMG76e-LR-1 (B) recombinant strains. Detection of the rbk gene transcriptional levels (C), ribokinase activity (D) and growth curves (E) in LR-1 (wild-type), pMG76e-LR-1, and rbk-pMG76e-LR-1 strains. The data obtained from three separate experiments are presented as mean±SD. ***: P<0.001; **: P<0.01; ns: No significant.
The rbk gene encodes ribokinase and is involved in the phosphorylation metabolic process of ribos
2.2 The overexpression of the rbk gene enhanced the biofilm formation and adhesion capabilities of the LR-1 strain
The biofilm formation results are illustrated in Figure

Figure 2 The overexpression of the rbk gene enhanced the biofilm formation and adhesion abilities of the LR-1 strain. The biofilm formation of LR-1, pMG76e-LR-1, and rbk-pMG76e-LR-1 in MRS (A), MRS with pH 4.0 (B), MRS with 0.2% (W/V) bile salt (C), and MRS with 8.0% (W/V) NaCl (D). The adhesion of the three strains to HT-29 cells (E). The data obtained from three separate experiments are presented as mean±SD. **: P<0.01; *: P<0.05; ns: No significant.
Bacteria in a biofilm state possess a stronger ability to resist adverse external environment
2.3 The overexpression of the rbk gene upregulated the expression of tuf, luxS and rpoN genes in the LR-1 strain
Our previous research has identified the tuf, luxS and rpoN genes as three genes closely related to biofilm formation in Lactobacillu

Figure 3 The overexpression of rbk in LR-1 upregulates the expression of tuf, luxS, and rpoN genes. The data obtained from three separate experiments are presented as mean±SD. ***: P<0.001; **: P<0.01; ns: No significant.
The tuf, luxS and rpoN genes are three genes that we previously identified through high-throughput screening and preliminary validation to have a significant positive correlation with biofilm formation in Lactobacillu
2.4 The effect of rbk overexpression on the metabolic profile of the LR-1 strain
This study employed PCA, PLS-DA, and OPLS-DA to conduct multivariate statistical analysis on the metabolites of rbk-pMG76e-LR-1 and pMG76e-LR-1, with the results shown in Figure

Figure 4 The PCA (A), PLS-DA (B) and OPLS-DA (C) analysis of the metabolites from rbk-pMG76e-LR-1 and pMG76e-LR-1. POS: Positive ion model; NEG: Negative ion model.
The differential metabolites of the rbk-pMG76e-LR-1 and pMG76e-LR-1 samples are shown in Figure

Figure 5 The differential metabolites of the rbk-pMG76e-LR-1 and pMG76e-LR-1 samples. The heatmap analysis of significantly differential metabolites in rbk-pMG76e-LR-1 and pMG76e-LR-1 in the positive ion model (A) and in the negative ion model (B). The fold changes of the differential metabolites in rbk-pMG76e-LR-1 compared to pMG76e-LR-1 (C). POS: Positive ion model; NEG: Negative ion model.
This study further predicted the correlation between differential metabolites and phenotypes, with results shown in

Figure 6 The Pearson correlation network diagram between differential metabolites and biofilm formation as well as adhesion is presented. The absolute value of correlation coefficient>0.9, P<0.01. Red circles represent differential metabolites that have a positive correlation with biofilm formation and adhesion, while green circles represent differential metabolites that are negatively correlated with these phenotypes. Blue circles indicate the phenotypes of biofilm formation and adhesion capacity. The larger the circle, the more associated substances it contains. Red lines indicate positive correlations, while green lines indicate negative correlations.
2.4.1 Central carbon metabolism changes
Among the 145 differential metabolites, 18 substances (12.41%) belong to the central carbon metabolism pathway, as shown in

Figure 7 Changes in metabolites in the central carbon metabolism pathway following rbk gene introduction are highlighted. Metabolites with increased abundance are marked in red, while those with decreased abundance are marked in green. P: Phosphate; PRPP: 5-phospho-alpha-d-ribose 1-diphosphate.
In the EMP pathway, the introduction of the rbk gene results in the upregulation of α-d-glucose (2.05 folds), α-d-glucose-6P (3.80 folds), β-d-fructose-1,6P2 (2.43 folds), and glyceraldehyde-3P (3.28 folds). Additionally, these four substances are located upstream of the EMP pathwa
In the HMP pathway, rbk overexpression led to a significant upregulation of d-ribulose-5P (49.99 folds), the most prominently upregulated metabolite among all the substance
2.4.2 Amino acid metabolism changes
In the context of the 145 differential metabolites induced by rbk overexpression, 15 of these metabolites (10.34%) were identified to be located in the amino acid metabolism pathway, as shown in

Figure 8 Changes in metabolites in the amino acid metabolism pathway following rbk gene introduction are highlighted. Metabolites with increased abundance are marked in red, while those with decreased abundance are marked in green. P: Phosphate.
The results indicate that the majority of amino acid levels increased in the rbk-pMG76e-LR-1 recombinant strain, suggesting that the overexpression of the rbk gene enhances amino acid metabolism. Given that amino acids are important biochemical precursors, the increased activity of amino acid metabolism may lead to significant changes in physiological phenotypes. For instance, previous studies have shown that L. plantarum can resist acid stress by upregulating the abundance of aspartate and arginin
2.4.3 Energy metabolism changes

Figure 9 Changes in metabolites in the energy metabolism pathway following rbk gene introduction are highlighted. Metabolites with increased abundance are marked in red, while those with decreased abundance are marked in green. P: Phosphate; PAP: Adenosine 3′,5′-diphosphate.
The total NAD(H) plays a crucial role in whole-cell biochemical redox transformation
These results suggest that energy metabolism is likely more active in the rbk-pMG76e-LR-1 strain. The biofilm formation is process is known to be energy-consuming, as the synthesis of the biofilm matrix requires significant metabolic resource
2.4.4 Nucleotide metabolism changes

Figure 10 Changes in metabolites in the nucleotide metabolism pathway following rbk gene introduction are highlighted. Metabolites with increased abundance are marked in red, while those with decreased abundance are marked in green.
In terms of pyrimidine metabolism, metabolites such as uracil, UMP, cytosine, UDP, cytidine, and CMP increased to 1.41, 2.17, 1.08, 2.39, 6.83 and 1.27 folds, respectively, in the rbk-pMG76e-LR-1 recombinant strain. However, dihydrouracil, thymine, dTMP, and dTDP showed decreases of 0.36, 0.20, 0.41 and 0.68 folds, respectively. Notably, uracil has been reported to influence all three known QS pathways in Pseudomonas aeruginosa, and mutations related to uracil can disrupt biofilm formation by this strai
2.4.5 Changes in other metabolites
Some metabolites were not clearly categorized within the previously mentioned pathways but are closely linked to biofilm formation and stress resistance. For instance, l-rhamnose increased to 28.66 folds in the rbk-pMG76e-LR-1 recombinant strain. This metabolite is considered crucial for the growth of Streptococcus mutans, and disruption in its biosynthesis can heighten the strain’s susceptibility to acid and oxidative stres
Moreover, several substances with potential health benefits were found to be elevated in the overexpressed rbk strain. Levels of γ-amino butyric acid (GABA), glutathione (GSH), and S-adenosyl methionine (SAM) increased to 1.78, 16.42 and 2.74 folds, respectively, in rbk-pMG76e-LR-1. GSH and SAM are recognized for their liver-protective properties, helping to prevent and treat various liver injuries and disease
2.5 Metabolites enhance the biofilm formation and adhesion ability of LR-1 strain
The influence of 8 metabolites on the biofilm formation ability of LR-1 strain is shown in

Figure 11 Effect of exogenously added metabolites on biofilm formation (A) and adhesion ability (B) of LR-1 strain. The data obtained from three separate experiments are presented as mean±SD. ****: P<0.000 1; ***: P<0.001; **: P<0.01; *: P<0.05; ns: No significant.
The LR-1 strains treated by two treatment methods were used for adhesion to HT-29 cells, and the results are shown in
The results showed that proline and arginine could significantly enhance the adhesion ability of LR-1, which was consistent with the finding of Wan et al. that arginine and proline would significantly affect the adhesion ability of Lactobacillus plantarum ATCC 1491
3 Conclusion
This study demonstrates that the overexpression of the rbk gene enhances the biofilm formation and adhesion capabilities of L. paraplantarum LR-1 while also upregulating the transcriptional levels of the tuf, luxS, and rpoN genes. Additionally, untargeted metabolomic analysis reveals that this overexpression leads to significant changes in 145 metabolites of L. paraplantarum LR-1, with 112 of these metabolites showing a strong correlation with biofilm formation and adhesion. Furthermore, the abundance of most metabolites related to central carbon, amino acid, energy, and nucleotide metabolism is higher in the rbk-pMG76e-LR-1 strain. These findings suggest that the overexpression of the rbk gene enhances the activity of these metabolic pathways. In addition, it was verified by exogenous addition of metabolites that some of the metabolites could significantly increase the biofilm formation and adhesion ability of LR-1 strain, which also indicates on the side that the overexpression of the rbk gene would have an effect on its biofilm formation and adhesion ability. Overall, this study indicates that the rbk gene plays a crucial role in metabolic regulation, significantly promoting the biofilm formation and adhesion abilities of Lactobacillus strains.
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