Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

spores germinate in the presence of a single germinant, inosine, yet with a significant lag period. spores. spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination

Characterization of selected Bacillus isolates exhibiting broad spectrum antifungal activity.

Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized

Exosporium Morphogenesis in Bacillus Cereus and Bacillus Anthracis

Bacillus cereus is a Gram-positive spore-forming bacteria that can cause food poisoning and its close relative, Bacillus anthracis is the etiological agent of anthrax. In both cases, the spore, a differentiated cell type in a dormant state, starts the infection process. Thus, the exosporium, which constitutes the surface of the spore, plays an important role during natural infection of both B. cereus and B. anthracis. Proteins from the exosporium of B. cereus ATCC 4342, a B. anthracis-like strain, were extracted with 2% β-mercaptoethanol under alkaline conditions and identified by liquid chromatography coupled with tandem mass spectrometry. A novel cell surface protein, reticulocyte binding protein (Rbp), was identified in this sample. Inactivation of rbp by insertional mutagenesis resulted in spores devoid of the hair-like nap typical of the exosporium, which suggests that Rbp could be a new component of the exosporium nap or could have a role in its assembly. GerQ, a protein that is crosslinked by transglutaminase in the spore coat of Bacillus subtilis, was also identified in the B. cereus ATCC 4342 exosporium.Absence of GerQ during sporulation resulted in a brittle exosporium in both B. cereus ATCC 4342 and B. anthracis ΔSterne spores. This suggests that transglutaminase participates in exosporium assembly and maturation by crosslinking small proteins and processed peptides, providing structural stability and resistance to degradation to the spore surface. In addition, a novel exosporium protein, ExsM, was characterized in this study. Subcellular localization of an ExsM-GFP fusion protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutants presented a tightly wrapped exosporium resulting in smaller and rounder spores than wild-type spores that have an extended exosporium. Thin-section electron microscopy revealed that exsM spores were encased by a double layer exosporium, both of which were composed of a basal layer and a hair-like nap. Compared to wild-type spores, exsM spores were more resistant to lysozyme treatment, germinated with a higher efficiency, and had a delay in outgrowth. Insertional mutagenesis of exsM in B. anthracis ΔSterne rendered spores with a partial second exosporium that were also smaller in size. These findings suggest that ExsM plays a critical role in the formation of the exosporium. B.cereus, but not B. anthracis, spores possess long appendages projecting from their surface. B. cereus ATCC 14579 appendages were isolated from spores by extraction with 2% β-mercaptoethanol under alkaline conditions, followed by CsCl gradient ultracentrifugation. Mass spectrometry analysis revealed that camelysin (CalY) and spore coat-associated protein N (CotN) were associated with the appendages sample. Both proteins are homologous to TasA, the main component of B. subtilis biofim extracellular matrix. TasA forms amyloids fibrils that hold the cells together and provide structure to the extracellular matrix. Therefore, spore appendages may have a role in biofilm formation, acting like a scaffold for the biofilm matrix and holding the spores together