Detection of Aspergillus Flavus in Wheat Grains Using Anti-Mannoprotein (MP1) and Spore Proteins Polyclonal Antibodies

Cell wall mannoprotein (MP1) gene of an aflatoxigenic strain of Aspergillus flavus, isolated from stored wheat grains, was cloned and sequenced. MP1 protein was expressed in E. coli in soluble form and purified. Polyclonal antibodies were raised against recombinant MP1 protein and inactivated spores of this fungus in rabbit, and purified by ammonium sulphate precipitation, Protein A sepharose and antigen affinity chromatography. The minimum concentration of purified mycelial or spore proteins that could be detected by ELISA was determined as 100 ng using 2 µg of these antibodies. The anti-MP1 antibody was found more sensitive than anti-spore protein antibody. Western blot and immunofluorescence analysis showed reactivity of these antibodies to various proteins (30 kDa to 200 kDa) distributed throughout the surface of mycelia and spore of A. flavus. Cross reactivity of these antibodies was detected with fungi belonging to different Aspergillus, Rhizopus and Alternaria species out of fourteen different fungal species tested. In fungal contaminated wheat grains these antibodies could detect presence of as low as 1 µg mycelia or 103 spores per gram of wheat grains using ELISA. The results suggest that the developed antibodies could be successfully applied for the detection of predominant fungal infestation in stored wheat grains.

Hot Resistance of Spores from the Thermophilic Bacillus horneckiae SBP3 of Shallow Hydrothermal Vent Origin Elucidated by Spectroscopic Analyses

Spores from Bacillus horneckiae SBP3 (SBP3) of shallow hydrothermal vent origin have recently been reported to survive extreme conditions more often than their close phylogenetic relatives B. horneckiae DSM 23495T (BHO) and B. subtilis 168 (BSU) used in biodosimetry and the space microbiology model. To investigate the structures of unheated spores, Fourier-transform infrared spectroscopy (FTIR) analysis was used. The FTIR spectra of the spores from the strains SBP3, BHO and BSU mainly differed in the region that referred to lipids and amino acids or polypeptides, indicating that the SBP3 spores were richer in saturated fatty acids, and the protein structures of SBP3 and BHO spores were more aggregated and complex than those of BSU. SBP3 spores were more resistant (LD90 = 4.2 ± 0.3 min) to wet heat treatment (98 °C) than BHO (LD90 = 1.8 ± 0.2 min) and BSU (LD90 = 2.9 ± 0.5 min) spores were. In comparison to the untreated spores, the Raman spectra of the wet-heat-treated SBP3 spores showed minor variations in the bands that referred to proteins, whereas major changes were observed in the bands that referred to lipids and amide I in the heated BSU spores and to both lipids and proteins bands in the treated BHO spores. These results suggest that the major stability of SBP3 spore proteins could explain their greater resistance to wet heat compared to BHO and BSU. Our findings provide basic information for further comparative studies into spore responses to natural and laboratory stresses, which are useful in several different fields, such as astrobiology.

Analysis of 5′-NAD capping of mRNAs in dormant spores of Bacillus subtilis

ABSTRACT Spores of Gram-positive bacteria contain 10s–1000s of different mRNAs. However, Bacillus subtilis spores contain only ∼ 50 mRNAs at > 1 molecule/spore, almost all transcribed only in the developing spore and encoding spore proteins. However, some spore mRNAs could be stabilized to ensure they are intact in dormant spores, perhaps to direct synthesis of proteins essential for spores’ conversion to a growing cell in germinated spore outgrowth. Recent work shows that some growing B. subtilis cell mRNAs contain a 5′-NAD cap. Since this cap may stabilize mRNA in vivo, its presence on spore mRNAs would suggest that maintaining some intact spore mRNAs is important, perhaps because they have a translational role in outgrowth. However, significant levels of only a few abundant spore mRNAs had a 5′-NAD cap, and these were not the most stable spore mRNAs and had likely been fragmented. Even higher levels of 5′-NAD-capping were found on a few low abundance spore mRNAs, but these mRNAs were present in only small percentages of spores, and had again been fragmented. The new data are thus consistent with spore mRNAs serving only as a reservoir of ribonucleotides in outgrowth.