Transmissible spongiform encephalopathies are caused by accumulation of highly resistant misfolded amyloid prion protein PrPres and can be initiated by penetration of such pathogen molecules from infected tissue to intact organism. Decontamination of animal meal containing amyloid prion protein is proposed thanks to the use of proteolytic enzymes secreted by thermophilic bacteria Thermoanaerobacter, Thermosipho, and Thermococcus subsp. and mesophilic soil bacteria Streptomyces subsp. Keratins alpha and beta, which resemble amyloid structures, were used as the substrates for the screening for microorganisms able to grow on keratins and producing efficient proteases specific for hydrolysis of beta-sheeted proteic structures, hence amyloids. Secretion of keratin-degrading proteases was evidenced by a zymogram method. Enzymes from thermophilic strains VC13, VC15, and S290 and Streptomyces subsp. S6 were strongly active against amyloid recombinant ovine prion protein and animal meal proteins. The studied proteases displayed broad primary specificities hydrolyzing low molecular mass peptide model substrates. Strong amyloidolytic activity of detected proteases was confirmed by experiments of hydrolysis of PrPres in SAFs produced from brain homogenates of mice infected with the 6PB1 BSE strain. The proteases from Thermoanaerobacter subsp. S290 and Streptomyces subsp. S6 are the best candidates for neutralization/elimination of amyloids in meat and bone meal and other protein-containing substances and materials.
. Growth is observed from pH 5 to pH 8, the optimum being at pH 6. The salinity range for growth is 10-50 g NaCl l N1 , the optimum being at 30 g l N1 . The isolate is able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates, and growth is stimulated by L-cystine and elemental sulfur. The GMC content of the genomic DNA is 29O1 mol %. According to phylogenetic analysis of the 16S rDNA gene, the strain is placed within the order Thermotogales, in the bacterial domain. On the basis of 16S rDNA sequence comparisons and morphological, physiological and genotypic characteristics, it is proposed that the isolate be described as a novel species of the genus Marinitoga, with Marinitoga piezophila sp. nov. as the type species. The type strain is KA3 T (l DSM 14283 T l JCM 11233 T ).
Three strictly anaerobic, thermophilic bacteria (SL31T, SL30 and MLM39636) were isolated from a deep continental oil reservoir in Western Siberia (Russia). Following the mid-exponential phase of growth, the non-motile rod-shaped organisms were surrounded by a sheath-like structure. As DNA-DNA hybridizations showed that these strains were highly related genomically, only strain SL31T was studied in detail. The temperature range for growth of strain SL31T was between 45 and 75 degrees C, with optimum growth at 70 degrees C. Its optimum pH and NaCl concentration for growth were pH 7.5 and 20-30 g l(-1), respectively. The novel isolate reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide. The G+C content of the genomic DNA was 30.0 mol %. As determined by 16S rDNA sequence analysis, this organism belonged to the genus Thermosipho. DNA-DNA hybridization levels between strain SL31T and type strains of the previously described species of Thermosipho were less than 10%. On the basis of physiological and molecular properties, it is proposed that this organism should be placed in a new species, Thermosipho geolei sp. nov. The novel organism represents the first species of the genus Thermosipho that has been isolated from a petroleum reservoir. The type strain is SL31T ( = DSM 13256T = JCM 10986T).
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