Unraveling the significance of calcium as a biofilm promotion signal for Bacillus licheniformis strains isolated from dairy products.

Bacillus licheniformis, a quick and strong biofilm former, is served as a persistent microbial contamination in the dairy industry. Its biofilm formation process is usually regulated by environmental factors including the divalent cation Ca. This work aims to investigate how different concentrations of Ca change biofilm-related phenotypes (bacterial motility, biofilm-forming capacity, biofilm structures, and EPS production) of dairy B. licheniformis strains. The Ca ions dependent regulation mechanism for B. licheniformis biofilm formation was further investigated by RNA-sequencing analysis. Results revealed that supplementation of Ca increased B. licheniformis biofilm formation in a dose-dependent way, and enhanced average coverage and thickness of biofilms with complex structures were observed by confocal laser scanning microscopy. Bacterial mobility of B. licheniformis was increased by the supplementation of Ca except the swarming ability at 20 mM of Ca. The addition of Ca decreased the contents of polysaccharides but promoted proteins production in EPS, and the ratio of proteins/polysaccharides content was significantly enhanced with increasing Ca concentrations. RNA-sequencing results clearly indicated the variation in regulating biofilm formation under different Ca concentrations, as 939 (671 upregulated and 268 downregulated) and 951 genes (581 upregulated and 370 downregulated) in B. licheniformis BL2-11 were induced by 10 and 20 mM of Ca, respectively. Differential genes were annotated in various KEGG pathways, including flagellar assembly, two-component system, quorum sensing, ABC transporters, and related carbohydrate and amino acid metabolism pathways. Collectively, the results unravel the significance of Ca as a biofilm-promoting signal for B. licheniformis in the dairy industry.