Spore

Aims:  To compare the properties of the spores of Bacillus subtilis prepared in liquid and on plates.Methods and Results:  The spores of B. subtilis were prepared at 37°C using a nutrient exhaustion medium either in liquid or on agar plates. The levels of core water, dipicolinic acid (DPA) and small, acid-soluble spore proteins (SASP) were essentially identical in spores made in liquid or on plates. Spores prepared in liquid were killed ∼threefold more rapidly at 90°C in water than the spores prepared on plates, and the spores prepared in liquid were more sensitive to nitrous acid and a diluted stable superoxidized water. Spores prepared in liquid also germinated more rapidly with several agents than those prepared on plates. Pellets of spores prepared on plates were darker than spores prepared in liquid, and spores prepared in liquid had more readily extracted coat protein. However, there were no major differences in the relative levels of individual coat proteins or the cross-linking of the coat protein GerQ in the two types of spores, although the inner membrane of spores prepared on plates had a higher ratio of anteiso- to iso-fatty acids.Conclusions:  The preparation in liquid yielded spores with some different properties than those made on agar plates. Spores made in liquid had lower resistance to heat and several chemicals, and germinated more readily with several agents. There were also differences in the composition of the inner membrane of spores prepared under these two conditions. However, there were no major differences in the levels of DPA, core water, SASP and individual coat proteins or the cross-linking of a coat protein in spores made in liquid and on plates.Significance and Impact of the Study:  This work demonstrates that the preparation method can affect the resistance and germination properties of bacterial spores, even if an identical medium and temperature are used. Evidence was also obtained consistent with the role of the inner membrane in spore resistance and germination, and that some factor in addition to core water, DPA and SASP content plays a role in spore resistance to wet heat.

Knowledge and conservation of diversity in symbiotic arbuscular mycorrhizal fungi is of crucial importance for their efficient use in environmental conservation and sustainable plant production systems. The recalcitrance of these microorganisms to pure culture, and the absence of information about plasmogamy, karyogamy or meiosis, means that information about the genetical bases of any variations in their population biology ecological specificity or symbiotic activity is generally lacking. A multidisciplinary approach is necessary to identify significant differences between populations, species or isolates. An overview is presented of some of the morphological, biological and molecular aspects of arbuscular mycorrhizal fungi that may contribute to defining and understanding biodiversity in this group of symbiotic microorganisms.

A monoclonal antibody that recognises components of the wall of sporangia of Peronospora destructor was raised. Tests using spores of higher fungi and other species of mildew demonstrated the specificity of the monoclonal. The antibody was used to develop lateral flow devices for sporangia of P. destructor. A competitive lateral flow format was developed which could detect onion downy mildew sporangia. Five-microliter gold anti-mouse IgM solution pre-mixed with 10 μl of P. destructor monoclonal antibody (EMA 242) proved the optimal concentration for detection of sporangia of P. destructor when applied to sample pads of lateral flow devices. Limits of approximately 500 sporangia of P. destructor could be detected by the absence of a test line on the lateral flow device within test samples. Using a scanning densitometer improved the sensitivity of detection. Further development and validation of the test is required if it is to be used for risk assessments of onion downy mildew in the field.

Bacterial lipid profiles are dynamic features of the cell that can change rapidly in response to an organism’s growth environment. Within a forensic setting, these can provide unique clues as to the origin of microbial threat agents that are recovered as evidence, particularly Bacillus spores. Phospholipids contain a hydrophobic tail comprised of long-chain fatty acids. The types of fatty acid structures and their relative abundance within the cell are heavily influenced by the composition of the growth medium and, specifically, the source of complex carbon/nitrogen. Additives such as tryptone (enzymatic digest of casein) and soy extract cause an increase in the abundance of branched-odd fatty acids whereas other additives such as peptone (digest of animal muscle) and brain-heart infusion lead to an increase in the proportion of straight-chained fatty acids. Other aspects of the growth medium including blood supplements, trace metal content, and the concentration of complex nutrients have a predictable effect on the abundance of individual fatty acid markers (18:1ω9c, 15:0anteiso, and 17:0iso, respectively). Because medium recipes are usually specific to the host facility or a single researcher, this information can help establish leads or exclude suspects during a forensic investigation.
Another advantage to lipid profiling of Bacillus spores is that it can be non-destructive, allowing it to be combined with other forensic analyses without any sample loss. While most of the cell’s fatty acids are localized within the membrane bilayer, preliminary experiments in our laboratory indicate that free fatty acids in the growth medium accumulate on the external surface of spores during batch culturing. Their composition is consistent with profiles generated from membrane lipids. These external fatty acids can be analyzed without affecting spore viability through the use of soft-ionization mass spectrometry (i.e. DART-MS) or chemical extraction of surface constituents followed by GC-MS profiling.

This study includes some details of the biostratigraphy of the Northern Thrace Basin, Turkey based on the fossil fungal and algal palynomorphs. These fossils are encountered in the (?) Middle-Upper Eocene turbiditic-deltaic to Oligocene deltaic-Iagoonal-Iacustrine-paludal and Miocene-Pliocene lacustrine-fluvial sediments. Seven new morphological species belonging to six form-genera are erected. Three already established species are also emended.

In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution. Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination. Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment. We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components. Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV. The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment.