Project description:First whole transcriptome assessment of a Bacillus megaterium strain. The B. megaterium DegU regulon was assessed for LB batch cultures with artificially induced degU expression. DegU is a pleiotropic regulator in B. subtilis governing adaptive responses such as secretory enzyme production. Overall design: 8 x 15 K customer made microarrays for gene expression analysis of B. megaterium were obtained from Agilent (Agilent Technologies, USA) with up to three probes per open reading frame of the B. megaterium DSM319 genome. Finally, the hybridization and final washing steps of the microarrays occurred as described in the Agilent manual for two color microarrays. The microarrays were scanned with the help of the Agilent C Scanner (Agilent Technologies, USA). For scanning and feature extraction the software Agilent Scan Control 8.4.1 and Feature Extraction 10.7.3.1 (Agilent Technologies, USA), respectively, was used according to the instruction. The analysis of the raw data occurred with the programming language R and Bioconductor as described in Yang and Paquet (2005). Finally, in consequence of the microarray design three different probes belonging to one gene were matched performing mean and median summarization of the logarithmic fold changes (logFC). Nevertheless, the p-values and also the logFC values were given for each probe separately, to account for different hybridization behavior of the different probes (Bunk, 2010). Only genes with a p-value < 0.01 in all replicates and an absolute |logFC| > 1 were considered as to be differentially expressed. Samples from degU expressing cells (xylose induction) of a degSU deletion mutant were compared to samples obtained from the likewise induced empty vector control strain, two time points, biological replicates: 3-5
Project description:Aspergillus flavus is one of the major fungal molds that colonize peanut in the field and during storage. The impact to human and animal health and to economy in agriculture and commerce are significant since this mold produces the most potent natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect to inhibit aflatoxin formation through down-regulating aflatoxin pathway gene expression in A. flavus as demonstrated by genechip analysis in liquid medium and peanuts. The results showed that aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98%) inhibited by B. megaterium. The expression of many of the aflatoxin biosynthetic genes in the fungus was confirmed to be turned down. Some of the target genes down-regulated by B. megaterium within the whole genome and within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX) were identified. These target genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was found to be significantly down-regulated. The effect of B. megaterium on aflatoxin biosynthesis and genes expression of pathogen was firstly tested in potato dextrose broth (PDB) and glucose minimal salts medium (MM). The cell suspension of B. megaterium (concentration in PDB and MM was finally adjusted to 108 CFU/ml) or sterile distilled water as a control was added into the 100 ml beaker flask containing 15 ml PDB or MM, respectively. Then 100 μl of spore suspension (5 × 106 spores/ml) of A. flavus were added into each beaker flask. After 48 h of incubation at 28°C at 200 rpm, mycelia were collected, fresh frozen with liquid nitrogen, ground to a fine powder in liquid nitrogen, and stored at -80°C for further analysis. The effect of B. megaterium on aflatoxin biosynthesis and genes expression in the A. flavus fungal pathogen was also tested in two types of peanut kernels, UF 715133-1 and Jinhua 1012, respectively. Peanut kernels were wounded (6 mm diameter and approximately 3 mm deep) using a sterile borer and then 20 μl of 1 × 108 CFU/ml cell suspension of B. megaterium was inoculated on wounded peanut kernels respectively. Sterile distilled water was also used for inoculation as control. Two hours after bacterial inoculation, 10 μl A. flavus spore suspension was inoculated into each wound at a concentration of 106 spores/ml. The kernels were placed in artificial weather chamber to maintain high humidity (85%) and incubated at 28°C for 7 days. Each treatment was replicated three times with 20 peanut kernels in each test. The mycelia on kernels were harvested at day 7 and fresh frozen immediately in liquid nitrogen, ground into powder, and stored at -80°C for further analysis.