Overexpression of rbk affects the biofilm formation, adhesion, and metabolic profile of Lactiplantibacillus paraplantarum LR-1

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.

Key words:Lactiplantibacillus;rbk overexpression;biofilm;adhesion;untargeted metabolomics