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In this study, eight rap -related genes were found in the Bacillus cereus 0–9 genome; five rap genes were located on chromosomes and three on large plasmids. A total of five Rap proteins in B. cereus 0–9 were annotated as 'tetratricopeptide repeat proteins'. SMART Server analysis showed that the eight Rap proteins had typical tetrapeptide repeat sequence (TPR) domains. Biofilm assays and crystal violet staining showed that overexpression of the rapp1 and rap5 genes affected the biofilm formation of B. cereus 0–9, and the activities of Rapp1 and Rap5 proteins were inhibited by their corresponding cognate Phr, suggesting that the Rap–Phr quorum sensing (QS) system might also exist in the B. cereus 0–9 strain. In addition, overexpression of rap1 genes inhibited in the extracellular amylase decomposition capacity of B. cereus 0–9. The results of the sporulation assay indicated that overexpression of the eight rap genes inhibited the spore formation of B. cereus 0–9 to varying degrees. These results provide a reference for research on the regulation of the Rap–Phr QS system in B. cereus. [ABSTRACT FROM AUTHOR]

Bacillus cereus 0-9, a Gram-positive endospore-forming bacterium isolated from healthy wheat roots, has biological control capacity against several soil-borne plant diseases of wheat such as sharp eyespot and take-all. The bacterium can produce various biofilms that differ in their architecture and formation mechanisms, possibly for adapting to different environments. The gapB gene, encoding a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), plays a key role in B. cereus 0-9 biofilm formation. We studied the function of GapB and the mechanism of its involvement in regulating B. cereus 0-9 biofilm formation. GapB has GAPDH activities for both NAD+- and NADP+-dependent dehydrogenases and is a key enzyme in gluconeogenesis. Biofilm yield of the Δ gapB strain decreased by 78.5% compared with that of wild-type B. cereus 0-9 in lysogeny broth supplemented with some mineral salts (LBS), and the Δ gapB :: gapB mutants were recovered with gapB gene supplementation. Interestingly, supplementing the LBS medium with 0.1–0.5% glycerol restored the biofilm formation capacity of the Δ gapB mutants. Therefore, GapB regulates biofilm formation relative to its function in gluconeogenesis. To illustrate how GapB is involved in regulating biofilm formation through gluconeogenesis, we carried out further research. The results indicate that the GapB regulated the B. cereus 0-9 biofilm formation independently of the exopolysaccharides and regulatory proteins in the typical SinI/R system, likely owing to the release of extracellular DNA in the matrix. Transcriptome analysis showed that the gapB deletion caused changes in the expression levels of only 18 genes, among which, lrgAB was the most significantly increased by 6.17-fold. We confirmed this hypothesis by counting the dead and living cells in the biofilms and found the number of living cells in the biofilm formed by the Δ gapB strain was nearly 7.5 times than that of wild-type B. cereus 0-9. Therefore, we concluded that the GapB is involved in the extracellular DNA release and biofilm formation by regulating the expression or activities of LrgAB. These results provide a new insight into the regulatory mechanism of bacterial biofilm formation and a new foundation for further studying the stress resistance of B. cereus. [ABSTRACT FROM AUTHOR]