The Molecular Mechanisms Of Biofilm Formation And Cucumber Root Colonization Of Bacillus Amyloliquefaciens SQR9

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum J. H. Owen (FOC), results in considerable yield losses for cucumber plants. Chemical controls, such as the use of chemical fertilizer and the fumigant methylbromide, often result in environmental and food quality problems. As an alternative approach, the application of biocontrol agents to suppress soil-borne pathogens has been widely used. Bacillus amyloliquefaciens SQR9, isolated and identified by our laboratory, was proved to be an outstanding PGPR strain by its abilities to inhibit the growth of FOC and decrease the incidence of Fusarium wilt of cucumber. This study is focus on the molecular mechanism of antifungal activity and biofilm formation. Firstly, we isolated and purified and the antifungal componds produced by SQR9. Then, the effect and mechanism of bamD deletion on biofilm formation and root colonization of SQR9 was studied by Solexa high-throughout transcriptome sequence. At last, mechanism of DegU phosphorylation controls biocontrol activity of Bacillus amyloliquefaciens SQR9 was studied. The main results obtained are listed as follows.1. To elucidate the antagonistic mechanism of the lipopeptides (LPs) on the fungi, the LPs products of B. amyloliquefaciens SQR9 were investigated by RP-HPLC. Three-day-old supernatant were separated by RP-HPLC. Six lipopeptide fractions were detected in the cultures of B. amyloliquefaciens SQR9. The two fractions were identified as bacillomycin D and fengycin based on the comparisons of these MS data with fractions previously identified by MS analysis in other B. amyloliquefaciens strains. Biochemical and genetic approaches demonstrated bacillomycin D is the major compound responsible for the inhibition of F. oxysporum growth. In the in vitro bioassay evaluating the supernatant of B. amyloliquefaciens SQR9 mutants for their capacities of inhibiting F. oxysporum conidial germination. Significant differences of inhibition rates were obtained after 12 h incubation, which were 98%,40% and 96% for SQR9, SQR9M1 and SQR9M2 respectively.2. Biofilm assay with microtitre plates showed that SQR9M1 formed thin and fragile biofilm compared with wild type strain SQR9 which had no difference with the biofilm of strain SQRM2. Quantitative analysis of the biofilm biomass indicated the bacillomycin D mutant exhibited significantly lower levels of biofilm biomass. Additionally, the three strains showed indistinctive growth curves. When bacillomycin D (purified by RP-HPLC and identified by LC-MS) was added to strain SQR9M1 medium, the biofilm was restored to the level of wild type strain SQR9, these results indicate that bacillomycin D is necessary for SQR9 to form biofilm. Transcription levels of three genes, epsD, yqxM and kinC, in SQR9M1 and SQR9M2 relative to B. amyloliquefaciens SQR9 were evaluated. The transcripts of epsD, yqxM and kinC in SQR9M1 decreased significantly with average expression levels decreased 7,3 and 4 times, respectively. However, the transcripts of epsD, yqxM and kinC in SQR9M2 showed no significant difference from B. amyloliquefaciens SQR9. Addition of bacillomycin D significantly increased the transcriptional levels of these three genes in SQR9M1. Increasing the extracellular potassium concentration did not result in diminished biofilm formation, which indicates that KinC activation is not triggered by losing of potassium. The quantitative analysis revealed that 4 days after inoculation, the population of SQR9 and SQR9M1 maintained at about 107 CFU·g-1 soil and 104 CFU·g-1 soil. These results confirmed that bacillomycin D was necessary for rhizosphere colonization.3. The transcriptional profiling of the biofilm formation of SQR9 and SQR9M1 at 24 h time point were analysed by Solexa high-throughout transcriptome sequence. The results show that a total of 1261 genes expression changes, including 574 genes were up-regulated (P<0.01),687 genes were down-regulated (P<0.01). Results indicated the main regulated genes belonged to metabolism (Inorganic ion, amino acids, lipid and nucleotide), protein translation and folding, membrane transport (ABC transporters), signal transduction (two-component system) and cell chemotaxis and motility. Low concentrations of iron ions can promote the biofilm formation of SQR9, but high iron concentrations perturb biofilm formation and promote dissociation of a preformed biofilm. When ferric chloride (10μM) was added to strain SQR9M1 medium, the biofilm was restored to the level of wild type strain SQR9.4. There are five major pathways for iron uptake in SQR9:YwbMNL, YfmCDEF, FhuBGC, YfiYZ and FeuABC. The genes feuBC, yusV, ywbM, yfmC,fhuB, and yfiY were disrupted. Iron ABC transporter FeuABC is required to regulate complex colony architecture. On solid medium, the wild-type strain formed rough colonies in which it was difficult to discern individual fruiting bodies. The feuBC and yusV mutant, on the other hand, produced flat, featureless colonies devoid of fruiting bodies. In liquid medium, the wild strain grew as bundled chains and formed robust rugose pellicles. These structures were hardy and difficult to disrupt mechanically when probed with a toothpick. In contrast, the yusV mutant lacked bundled chains and was completely defective in biofilm formation. Mutant AfeuBC can not form biofilms. Transcription levels of genes, epsD, yqxM and SinI, in AfeuBC and AyusV relative to B. amyloliquefaciens SQR9 were evaluated. The transcripts of epsD, yqxM and SinI in AfeuBC and AyusV are significantly decreased. Addition of bacillomycin D and ferric chloride significantly increased the transcriptional levels of these three genes in AfeuBC. The Iron ABC transporter FeuABC is involved in responding to bacillomycin D and iron. Histidine-tagged FeuA was purified by Ni-chelating affinity chromatography from sonicated cells of the positive transformant. Bacillomycin D was found to bind to the peripasmic binging protein FeuA.5. To investigate the effect of the level of DegU-P on the biocontrol efficiency, B. amyloliquefaciens SQR9 derivatives with different levels of DegU-P were constructed (SQR9M4, SQR9Q and SQR9SUQ). DegU-P is required to regulate complex colony architecture. SQR9M4 showed impaired complex colony architecture:upon closer examination of the degQ deletion mutant, it was apparent that the central aerial structures observed on colonies formed by the wild-type strain were not present and the colony remained flattened against the agar surface. The four strains showed indistinctive growth curves, but in liquid medium, the degQ mutant formed thin and fragile biofilm compared with wild type strain SQR9. In contrast, the strain SQR9SUQ in high level of DegU-P formed robust rugosebiofilms. After six days post-cocultivation of cucumber roots and Green Fluorescent Protein (GFP)-labeled B. amyloliquefaciens strains (SQR9-gfp, SQR9M4-gfp, SQR9Q-gfp and SQR9SUQ-gfp) in a hydroponic system, roots were viewed by confocal scanning laser microscopy (CSLM). A segment of the primary root, located around 2-4 cm distant from the root tip, was found heavily colonized by SQRSUQ, which formed micro-colonies on the surface of the outer epidermis cells of the primary root. However, in the same position, only few and small regions of the roots were colonized by the degQ mutant cells, and significantly reduced biofilm formation was observed compared with other strains. Potential lipopeptides and polyketides antibiotics (bacillomycin D、 fengycin、surfactin、 macrolactin、difficidin、and bacillaene) predicted by genomic analysis were verified through HPLC-ESI-MS. The transcription of the bamD, sftA, dfnA, baeB, mlnA and fenA genes was investigated by real-time qPCR in B. amyloliquefaciens strains SQR9, SQR9M4, SQR9Q and SQR9SUQ. At 12 h time points, compared with SQR9, the bamD and dfnA transcriptions in strain SQR9SUQ were increased by 5- and 7-fold, respectively. However, compared with SQR9, a high level of DegU-P (in strain SQR9SUQ) inhibits the mlnA and fenA transcriptions by 4 and 5 times, respectively. The low level of DegU-P in strain SQR9M4 stimulates the transcription of sftA, baeB, mlnA and fenA 4-,6-, 4-and 17-fold, respectively, when compared with SQR9. At 36 h time points, the transcription of bamD and dfnA in strain SQRSUQ increased significantly with average transcription levels and was 10 and 6 times higher than that in SQR9, respectively. Greenhouse experiments were carried out to compare the abilities of the different B. amyloliquefaciens SQR9 strains in disease control. Increasing the levels of DegU-P in B. amyloliquefaciens SQR9 can improve the biocontrol activity.6. In MSNc medium, the biofilm-forming abilities of SQR9 is significantly higher than B. subtilis 168, B. subtilis 3610 and B. amyloliquefaciens FZB42. Cucumber plant extracts and root exudates induce biofilm formation. The master regulator SpoOA and the SinI are involved in responding to plant polysaccharides (arabinogalactan, pectin and xylan). The master regulator DegU are involved in responding to cucumber root exudates. Histidine-tagged DegU was induced by 1 mM IPTG and purified by Ni-chelating affinity chromatography from sonicated cells of the positive transformant.