Characterization of spores of Bacillus subtilis that lack most coat layers.
Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca(2+) and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.

title: Isolation and characterization of superdormant spores of Bacillus species.
abstract: Superdormant spores of Bacillus subtilis and Bacillus megaterium were isolated in 4 to 12% yields following germination with high nutrient levels that activated one or two germinant receptors. These superdormant spores did not germinate with the initial nutrients or those that stimulated other germinant receptors, and the superdormant spores' defect was not genetic. The superdormant spores did, however, germinate with Ca(2+)-dipicolinic acid or dodecylamine. Although these superdormant spores did not germinate with high levels of nutrients that activated one or two nutrient germinant receptors, they germinated with nutrient mixtures that activated more receptors, and using high levels of nutrient mixtures activating more germinant receptors decreased superdormant spore yields. The use of moderate nutrient levels to isolate superdormant spores increased their yields; the resultant spores germinated poorly with the initial moderate nutrient concentrations, but they germinated well with high nutrient concentrations. These findings suggest that the levels of superdormant spores in populations depend on the germination conditions used, with fewer superdormant spores isolated when better germination conditions are used. These findings further suggest that superdormant spores require an increased signal for triggering spore germination compared to most spores in populations. One factor determining whether a spore is superdormant is its level of germinant receptors, since spore populations with higher levels of germinant receptors yielded lower levels of superdormant spores. A second important factor may be heat activation of spore populations, since yields of superdormant spores from non-heat-activated spore populations were higher than those from optimally activated spores.

title: Superdormant spores of Bacillus species have elevated wet-heat resistance and temperature requirements for heat activation.
abstract: Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15 degrees C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.

title: The preparation, germination properties and stability of superdormant spores of Bacillus cereus.
abstract: To determine yields, germination and stability of superdormant Bacillus cereus spores.



Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or L-alanine in 2-5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca-DPA, dodecylamine, L-alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat-activated spores were germinated. Superdormant spores stored at 4 degrees C slowly recovered some germination capacity, but recovery was slowed significantly at -20 degrees C and -80 degrees C.



Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores.



Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.

title: Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis.
abstract: Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.

title: Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide.
abstract: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance.



Bacillus megaterium was sporulated with no added MnCl(2) and up to 1 mmol l(-1) MnCl(2). The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100-fold. The Mn level had no effect on spore γ-radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H(2)O(2). However, levels of dipicolinic acid and the DNA-protective α/β-type small, acid-soluble spore proteins were the same in spores with high and low Mn levels.



Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ-radiation.



The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.

title: Mechanism of intestinal entry of infectious prion protein in the pathogenesis of variant Creutzfeldt-Jakob disease.
abstract: The pathogenesis of variant Creutzfeldt-Jakob disease (vCJD) is most likely to be dependent on intestinal entry of orally ingested infectious prion proteins, though tonsils or other oral portals of entry are possible. The exact route of entry of infectious prion proteins is uncertain but receptors for prion proteins such as laminin receptor precursor (LRP) may be expressed on intestinal brush border. Cellular prion protein (PrP(c)) is expressed on intestinal enteric nervous system and is separated by a single layer of epithelial cells from ingested infectious prion proteins. Intestinal M cells in the Peyer's patches may also transcytose prion proteins which may be transported to the lymphatic system by migrating dendritic cells underlying the M cells. The relative importance of the several potential portals of intestinal entry of infectious prion proteins is uncertain but may determine susceptibility of the population and also potential preventive strategies.

title: Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal.
abstract: The DNA binding subunit of the transcription factor NF-kappa B, p50, has been cloned. p50 appears to be synthesized as a larger protein that is then processed to its functional size. Sequence analysis reveals remarkable homology for over 300 amino acids at the amino-terminal end to the oncogene v-rel, its cellular homolog c-rel, and the Drosophila maternal effect gene dorsal. This establishes NF-kappa B as a member of the rel family of proteins, all of which display nuclear-cytosolic translocation. Protein sequence from the p65 polypeptide has established that it is not encoded in the same mRNA as p50. However, p65 appears homologous to c-rel, suggesting that c-rel may form heterodimers with p50 and rel may include a homodimerization motif.

title: Fluorescent probing of urea-induced chemical unfolding of bovine serum albumin by intramolecular charge transfer fluorescence probe E-3-(4-dimethylamino-naphthalen-1-yl)-acrylic acid.
abstract: Changes in polarity at the immediate binding site in protein bovine serum albumin (BSA) produces distinct changes in the solvent polarity-dependent emission band of fluorescence probe E-3-(4-dimethylamino-naphthalen-1-yl)-acrylic acid. Steady-state spectroscopy and time-resolved spectroscopy have been used to investigate this binding process. Attaching the probe to BSA and then monitoring its spectral changes with increasing urea concentration and raising temperature has also tracked the denaturation of BSA chemically and thermally. The polarity of the microenvironment was investigated employing the Reichardt E(T)(30) scale. Fluorescence anisotropy, red edge excitation shifts and acrylamide-induced quenching of fluorescence have been exploited to gain better insight into this binding process.

title: Evaluation of Registered Visually Disabled Individuals in a District of West Bengal, India
abstract: To identify the sociodemographic characteristics, degree and cause of visual disability among certified visually disabled individuals in a rural district of West Bengal, India and to identify possible lacunae, if any, in the existing certification system.  A cross-sectional study by secondary data analysis of medical records of 155 visually disabled individuals and their 310 eyes. Demographical features, diagnosis, percentage of visual disability and work activity status of each individual were analyzed.  One hundred and thirty one (84.52%) individuals had 100% disability. The number of males was significantly higher than that of females. Fifty eight (37.42%) individuals were below 21 years of age. Phthisis bulbi was the most common cause followed by microphthalmos. Further, 81.29% patients had the same lesion bilaterally.  Patients with higher grades of disability have attended certification boards. A large number of disabled individuals comprised children and young adults. Male gender bias demands concern.

title: Fluorescent Probing of Protein Bovine Serum Albumin Stability and Denaturation Using Polarity Sensitive Spectral Response of a Charge Transfer Probe
abstract: The polarity sensitive photo-induced intra-molecular charge transfer (ICT) fluorescence probe (E)-3-(4-methylamino-phenyl)-acrylic acid ethyl ester (MAPAEE) has been used to study the model protein Bovine Serum Albumin (BSA) in its native and thermal and urea induced denatured states. The interaction between BSA and the regular surfactant Sodium Dodecyl Sulphate (SDS) as well as the biologically relevant steroid-based amphiphile Sodium Deoxycholate (NaDC) has also been very keenly followed using this ICT probe. The variation of micellar properties of both SDS and NaDC with increasing ionic strengths and in presence of the chaotrope urea has also been well documemted by the same probe. Steady-state spectroscopy, FRET, and fluorescence anisotropy measurements have been used to gain better insight into these processes and the molecule MAPAEE to be a full-bodied fluorescent probe for studying such intricate biological systems, their properties and interactions.