Production of extracellular amylase contributes to the colonization of Bacillus cereus 0-9 in wheat roots

Abstract Background: Bacteria usually secrete a variety of extracellular enzymes to degrade extracellular macromolecules to meet their nutritional needs and enhance their environmental adaptability. Bacillus cereus 0-9, a biocontrol bacterial strain isolated from the wheat root, has three genes annotated as amylase in the genome, but its function is unknown, and whether it is involved in its colonization process remains to be further studied. Methods: The gene mutant strains and fluorescently tagged strains were constructed by homologous recombination, and the amylase protein obtained by the prokaryotic expression system Escherichia coli BL21(DE3). Iodine staining method was used to measure the activities of amylase proteins. We further observed the colonization abilities of test strains in wheat roots through frozen section technology. Results: The results showed that there were three amylase-encoding genes of amyC , amyP and amyS in the B. cereus 0-9 genome. Among the three amylases, only the Δ amyS mutant did not produce extracellular amylase, and the secretion ability was related to the signal peptide at positions 1-27. AmyS protein encoded by amyS gene is an α-amylase. The growth of R. cerealis was inhibited 84.7% by B. cereus 0-9, but the inhibited rate of Δ amyS strain decreased to 43.8%, and that of Δ amyS / amyS was restored when the amyS gene complementation. Further, the inhibited rate of Δ amySec strain was decreased to 46.8%, almost the same as Δ amyS mutant. Due to the deletion of amyS gene, the colonization capacity of Δ amyS (RFP) and Δ amySec (RFP) in wheat roots declined, while that of Δ amyS / amyS (RFP) was restored after the amyS gene was complemented, indicating that amyS gene influences the colonization of B. cereus 0-9 in wheat roots.For another, the colonization and biocontrol ability of the mutant was also restored after the addition of sugars such as glucose and maltose. Conclusions: B. cereus 0-9 has three genes of amyC , amyP and amyS annotated as amylase. Among them, only the deletion of amyS gene with signal peptide did not produce extracellular amylase. AmyS protein encoded by amyS gene is an α-amylase. Our results indicated the amyS gene is closely related to the colonization abilities of B. cereus 0-9 in wheat root, and the biocontrol abilities of B. cereus 0-9 to fight against R. cerealis . The extracellular amylase produced by B. cereus 0-9 can hydrolyze starch, and use the glucose, maltose and other nutrients to meet the needs of bacterial growth and multiply. Therefore, it is very possible that the secretion and hydrolytic activity of extracellular amylase can promote the colonization of B. cereus 0-9 in wheat roots, and play an important role in the prevention and control of plant diseases. Our results contribute to explore the mechanisms of microbial colonization in plant roots.