The euryarchaea Picrophilus torridus and Picrophilus oshimae are able to grow around pH 0 at up to 65°C, thus they represent the most thermoacidophilic organisms known. Several features that may contribute to the thermoacidophilic survival strategy of P. torridus were deduced from analysis of its 1.55-megabase genome. P. torridus has the smallest genome among nonparasitic aerobic microorganisms growing on organic substrates and simultaneously the highest coding density among thermoacidophiles. An exceptionally high ratio of secondary over ATP-consuming primary transport systems demonstrates that the high proton concentration in the surrounding medium is extensively used for transport processes. Certain genes that may be particularly supportive for the extreme lifestyle of P. torridus appear to have been internalized into the genome of the Picrophilus lineage by horizontal gene transfer from crenarchaea and bacteria. Finally, it is noteworthy that the thermoacidophiles from phylogenetically distant branches of the Archaea apparently share an unexpectedly large pool of genes.

This paper describes a molecular-based method which is able to discriminate between viable and inactivated Bacillus subtilis spores by utilizing the DNA-intercalating dye propidium monoazide. The approach should be valuable in our attempt to employ molecular methods to streamline the evaluation of process validation using bacterial endospores.There is a continued need for the development and application of rapid methods for the detection and enumeration of bacterial endospores, especially as investigators seek to evaluate the efficacy of emerging food-processing technologies. For such thermal-process validation studies, surrogates of Clostridium botulinum, including Bacillus subtilis and Clostridium sporogenes, are commonly used. Currently, standard plating methods remain the "gold standard" for enumeration of survivors of these processes. Molecular amplification approaches, such as quantitative real-time PCR (qPCR), have shown promise but have not been applied to the enumeration of surviving spores because (i) release of nucleic acid from spores is difficult and (ii) the detection of DNA does not necessarily equate with the presence of viable spores.Recently, the DNA-intercalating agents ethidium monoazide and propidium monoazide (PMA) have been used in conjunction with qPCR for the selective detection of live cells of food-borne pathogens (2, 3, 5, 6, 9). These compounds selectively penetrate the membranes of dead cells and form stable DNA monoadducts upon photolysis, resulting in DNA which cannot be amplified by PCR (3). To our knowledge, this technique has not yet been applied to bacterial spores. The purpose of this study was to demonstrate that DNA-intercalating agents could be used in conjunction with qPCR for the selective enumeration of viable, but not inactivated, spores of B. subtilis.All media were supplied by Difco (Franklin Lakes, NJ), and chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Bacillus subtilis ATCC 35021 (American Type Culture Collection, Manassas, VA) was grown overnight in 10 ml of brain heart infusion broth at 37°C. Five-hundred-microliter aliquots of the vegetative cells were spread onto 150-by 15-mm petri dishes containing sporulation agar comprised of 13 g/liter nutrient broth, 15 g/liter agar, 0.51 g/liter MgSO 4 ⅐ 7H 2 O, 0.97 g/liter KCl, 0.2 g/liter CaCl 2 ⅐ 2H 2 O, 3 mg/liter MnSO 4 ⅐ H 2 0, and 0.5 mg/liter FeSO 4 ⅐ 7H 2 O. The plates were incubated aerobically at 37°C for 3 to 5 days until more than 95% of cells had sporulated, as determined by phase-contrast microscopy. Spores were harvested in cold, sterile distilled water (dH 2 O) and washed repeatedly (5 to 10 times). Before final resuspension, the spores were treated with 80 U/ml DNase (SigmaAldrich, St. Louis, MO) at 37°C for 90 min (to degrade residual DNA), with subsequent DNase inactivation by heating at 65°C for 10 min. The final populations were approximately 10 7 spores/ml, and crops were stored at 4°C until use. Using the capillary tube method (8), the D 121°C value (decimal reduction time, i.e., tim...

The taproot from sugar beet (Beta vulgaris L.) undergoes a specific developmental process to function as a food storage organ. Suppression Subtractive Hybridization (SSH) was utilized for the isolation of cDNA fragments for taproot expressed genes. Isolation and molecular analysis of six cDNAs encoding the complete gene product revealed that these genes comprise homologues of a drought-inducible linker histone, a homologue of a major latex-like protein, a phosphoenolpyruvate carboxylase kinase, a putative vacuolar processing enzyme, a thaumatin-like protein and an alanine- and glutamic acid-rich protein. All genes are transcribed in taproots while transcription in leaves is low or undetectable.

Thermophilic acidophiles are microorganisms that are able to grow in pH and temperature ranges of 0.0–4.0 and 55–95 °C, respectively. Most of these microorganisms, which usually live in solfataric fields, belong to the archaea and include the genera Acidianus, Desulfurolobus, Metallosphaera, Stygiolobus, Sulfolobus, Sulfurisphaera, Sulfurococcus, Thermoplasma and Picrophilus. The sequencing of five complete genomes of thermoacidophilic organisms has allowed more detailed investigation regarding the evolution of organisms sharing extreme growth conditions of a unique niche, especially with respect to horizontal gene transfer. From such novel microorganisms, robust enzymes with potential biotechnological applications can be isolated. Enzymes that are optimally active at high temperature and extremely low pH are very rare and most of them are extracellular and involved in polymer degradation. Heat and acid stable α‐amylase, cyclomaltodextrinase (neopullulanase), maltose binding protein and endoglucanase have been purified and characterized from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. Archaeal glycosyl hydrolases have been characterized from Picrophilus torridus, Picrophilus oshima, Thermoplasma acidophilum and Sulfolobus solfataricus. Biotransformation reactions can be performed even at pH close to zero and temperatures of 100 °C.

A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in its active state. Amylolytic activity was measured over a wide range of temperature (40-90 degrees C) and pH (pH 3.5-7) and was maximal at 75 degrees C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate, including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the presence of 5 mM CaCl(2), the half-life increased from 15 min to 2 h at 80 degrees C.