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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 ΔgapB 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 ΔgapB::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 ΔgapB 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 ΔgapB 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.

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]

Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type. [ABSTRACT FROM AUTHOR]