Biofilm Formation Mechanisms Of Bacillus Spp.

Bacillus spp. have long been used as a kind of plant growth-promoting rhizobacteria (PGPR) in agriculture, functioning as the biological control agents by colonizing the plant roots. However, the exact mechanisms for biocontrol of plant diseases have not been clearly elucidated. Previous researches have confirmed that Bacillus spp. could stably colonize the surface and interior of the plant roots by forming biofilms in response to environmental signals. So studying on the mechanisms and regulatory pathways of biofilm formation are not only beneficial for revealing the colonization and biocontrol mechanisms, but also for providing theoretical support for the research on Bacillus biopesticide, and enhancing the commercial development of Bacillus spp..In this study, we carried out molecular studies of biofilm formation mechanisms of B. cereus 905 and B. subtilis NCIB 3610 with the supports from molecular technologies and complete genome sequence data. We first reported that B. cereus 905 could form two kinds of biofilms by using two independent mechanisms when cultured in different media; We also described how environmental conditions influence the "cross-talk" between two pairs of bacterial tyrosine kinases (BY-kinases) and. their cognate activators (EpsB/EpsA and PtkA/TkmA) in B. subtilis NCIB 3610, regulating biofilm formation by regulating the EPS biosynthesis at post-translational level. The main experimental results are as follows:(1) In this part of experiment, we first determined that B. cereus 905 could make visible floating pellicles at the air-liquid interface in MSgg medium, and could also form submerged, surface-associated biofilms in TSBG medium. By interrogating the B. cereus 905 genome we identified homologs for many of the genes employed by B. subtilis in pellicle formation. By constructing the dedicated mutants and testing biofilm phenotype, our experimental results confirmed that spoOA, sinl, sipW, calYl and calY2 are involved in pellicle formation, indicating that B. cereus 905 formed pellicles in a similar manner to that observed for B. subtilis NCIB 3610 in MSgg medium; strikingly, we found that mild acid (pH≤6.5) was required for submerged biofilms formation without participation of spoOA, sinl, sipW, calYl and calY2, suggesting that B. cereus 905 could also form submerged biofilms by a process that has several of the hallmarks of Staphylococcus aureus biofilm formation. In addition, we further showed that biofilm matrix composed of extracellular protein and extracellular DNA. B. cereus group of bacteria might do not induce expression of eps under biofilm forming conditions and do not require exopolysaccharide for biofilm formation.(2) Tyrosine kinase has been proved involved in B. subtilis biofilm formation. In this part of study, we described the "cross-talk" between two pairs of bacterial tyrosine kinases (BY-kinases) and their cognate activators, EpsB/EpsA and PtkA/TkmA, in B. subtilis. Combined our experimental results and previous studies, we presented evidences that the kinase activator TkmA likely activated non-cognate EpsB as well as its cognate BY-kinase PtkA, and that this activation contributed to biofilm formation. In LBGM medium, we observed that deletion of tkmA (△tkmA) resulted in severe biofilm defect. But when testing in MSgg medium, △tkmA didn’t prevent biofilm formation although it affected the biofilm morphology. These results suggested that the importance of TkmA in biofilm formation was seemed like medium-dependent. The effect of TkmA on biofilm formation was likely through activation of EpsB, as over-expression of epsAB in △tkmA background restored biofilm formation. By mapping the spontaneous suppressor of AtkmA-ugd and measuring the activities of lacZ reporter fusion, we confirmed that SpoOA and DegU regulate tkmA-ugd and epsA-O operons. The molecular basis for the medium dependence is likely due to differential expression of tkmA and epsA in the two different media (LBGM and MSgg) regulated by SpoOA and DegU, with TkmA’s contribution more apparent in rich LBGM medium as the high activities of SpoOA and DegU increasing TkmA expression, and EpsA’s contribution more obvious in minimal MSgg medium since that the expression of EpsA is in high level.