Biological Function Of Bacillus Subtilis Biofilm During Biocontrol Of Ralstonia Wilt Disease And Cyclic-DI-Gmp Signaling Pathway In Bacillus Subtilis

Bacillus subtilis and other Bacilli have long been used as biological control agents (BCAs) against plant bacterial diseases. The exact mechanisms for plant biocontrol have not been clearly addressed. There are too many questions needed to address in this field. For example, what are major genes involved in biocontrol efficacy against plant diseases in B. subtilis? Which kind of cell morphology exists in the rhizosphere after application? How do B. subtilis cells move towards plant roots and colonizate on the root surface? Is there a signaling transduction pathway between B. subtilis and plants during the biocontrol? If there is a signal pathway, what are the signaling molecules, and how does B. subtilis or plant recognize the signal and regulate genes expression? It will be beneficial for deeply investigating the B. subtilis biocontrol mechanism and accelerating the commercial procedure of B. subtilis biopesticide.In this study, we established the B. subtilis-tomato interaction system to gain a better insight into B. subtilis biocontrol mechanism. We showed that in those wild strains, both biofilm formation and production of surfactin, an antimicrobial agent, were necessary for plant biocontrol but neither one was sufficient. B. subtilis kinase KinD can recognize the signaling molecules from tomato root exudates, then active the biofilm formation. We also present the function of C-di-GMP signaling pathway in gram-positive bacterium B. subtilis. C-di-GMP controlled the motility and biofilm formation in B. subtilis.1. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation and surfactinOur goal in this study is to first isolate spore-forming B. subtilis wild strains from natural environments that exhibit high biocontrol efficacy. And second, we hope to investigate the molecular mechanisms of plant biocontrol in those wild strains. We screened a total of sixty distinct isolates collected from various locations countrywide and obtained six wild strains of B. subtilis that demonstrated above50%biocontrol efficacy on tomato plants against the plant pathogen Ralstonia solanacearum under greenhouse conditions. These wild strains were able to form robust biofilms both in defined media and on tomato plant roots, and exhibited strong antagonistic activities against various plant pathogens in plate assays. We further showed that in those wild strains, both biofilm formation and production of surfactin, an antimicrobial agent, were necessary for plant biocontrol but neither one was sufficient. Loss of either feature due to genetic mutations resulted in a substantial decrease in biocontrol efficacy. Biofilm formation and surfactin production may act synergistically to enhance biocontrol efficacy. In addition, we showed that biofilm formation strongly promoted colonization of B. subtilis cells to tomato root surfaces, during which the biofilm matrix may function as an adhesion. Finally, we have established a model system for studies of B. subtilis-tomato plant interactions at molecular levels.2. A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plantsThe soil bacterium Bacillus subtilis is widely used in agriculture as a biocontrol agent able to protect plants from a variety of pathogens. Protection is thought to involve the formation of bacterial communities-biofilms-on the roots of the plants. Here we used confocal microscopy to visualize biofilms on the surface of the roots of tomato seedlings and demonstrated that biofilm formation requires genes governing the production of the extracellular matrix that holds cells together. We further show that biofilm formation was dependent on the sensor histidine kinase KinD and in particular on an extracellular CACHE domain implicated in small molecule sensing. Finally, we report that exudates of tomato roots strongly stimulated biofilm formation ex planta and that an abundant small molecule in the exudates, L-malic acid, was able to stimulate biofilm formation at high concentrations in a manner that depended on the KinD CACHE domain. We propose that small signaling molecules released by the roots of tomato plants are directly or indirectly recognized by KinD, triggering biofilm formation.3. Evidence for cyclic di-GMP-mediated signaling in Bacillus subtilis Cyclic-di-GMP (C-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. Studies of C-di-GMP signaling have chiefly focused on gram-negative bacteria. Here we investigated C-di-GMP signaling in the gram-positive bacterium Bacillus subtilis by constructing deletion mutations for genes predicted to be involved in the synthesis, breakdown, or response to the second messenger. We found that a putative C-di-GMP degrading phosphodiesterase YuxH and a putative C-di-GMP receptor YpfA had a strong influence on motility and that these effects depended on sequences similar to canonical EAL and RxxxR---D/NxSxxG motifs, respectively. Evidence indicates that YpfA inhibits motility by interacting with the flagellar motor protein MotA and that yuxH is under the negative control of the master regulator SpoOA-P. Based on these findings, we propose that YpfA inhibits motility in response to rising levels of c-di-GMP during entry into stationary phase due to the down regulation of yuxH by SpoOA-P. We also present evidence that YpfA has a mild influence on biofilm formation. In toto, our results demonstrate the existence of a functional C-di-GMP signaling system in B. subtilis that directly inhibits motility and directly or indirectly influences biofilm formation.