Sulfolobus solfataricus secretes an acid-resistant ␣-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand ␣-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal ␣-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-␣-amylase antibodies raised against the purified protein immunoprecipitated secreted ␣-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the ␣-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.Sulfolobus solfataricus is a hyperthermophilic member of the archaea which inhabits acidic geothermal environments. It exhibits diverse modes of metabolism at high temperatures (70 to 90°C), including lithoautotrophy with sulfur and carbon dioxide (5, 23, 37) and chemoheterotrophy with sugars or amino acids (7,15). Despite this metabolic flexibility, mechanisms regulating carbohydrate consumption by this organism are poorly understood. On occasion, plant-derived carbohydrates such as starch and cellulose contribute to the geothermal pool carbon cycle. In the absence of plant life, however, the endogenous microbial community itself may be an important source of reduced carbon compounds. For example, since hyperthermophilic archaea accumulate glycogen as an intracellular storage polymer (24), lysis could make glycogen available for consumption by surviving cells.In hot acid environments polysaccharide degradation occurs rapidly, reflecting high rates of chemical hydrolysis and oxidation. Consequently, successful competition for these carbohydrates necessitates glycosyl hydrolase secretion. Secreted ␣-amylases promote the consumption of exogenous starch by releasing linear maltodextrins for subsequent assimilation. These enzymes have been classified into two sequence families (13 and 57) based on the presence of distinctive conserved domains (19). A growing number of glycosyl hydrolases, including ␣-amylases and pullulanases, have been characterized from hyperthermophilic archaea (8,21,32,35), including members of family 57 (10, 22). However, the lack of suitable genetic methods for hyperthermophilic archaea have precluded studies on the biological significance of these enzym...

The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic alpha-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C. The results also implicated several prototropic groups in the catalytic reaction.

Salinibacter ruber, an extremely halophilic member of the domain Bacteria, has two different cytoplasmic glutamate dehydrogenase activities, marked as GDHI and GDHII. GDHI showed a strong dependence on high salt concentrations for stability, but not for activity, displaying maximal activity in the absence of salts. GDHII depended on high salt concentrations for both activity and stability. It catalyzed amination of 2-oxoglutarate with optimal activity in 3 M KCl at pH 8. No activating effect was found when NaCl was replaced by KCl. Only GDHII displayed activity in the deamination reaction of glutamate with an optimal pH of 9.5. Both enzymes were activated by certain amino acids (L-leucine, L-histidine, L-phenylalanine) and by nucleotides such as ADP or ATP. A low-molecular-mass cytoplasmic fraction was found to be a highly effective activator of GDHII in the presence of high NaCl concentrations.

An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components. Substrate induction of synthesis of the S. solfataricus-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins.Geothermal environments are often highly acidic. Such extreme environments harbor a wide range of acidophilic hyperthermophilic organisms including members of both the bacterial and archaeal prokaryotic subdivisions. Among the archaeal representatives, members of the Sulfolobus genus have been most intensively characterized. These organisms can grow chemoheterotrophically on reduced carbon compounds (6, 9, 17) and lithoautotrophically on reduced sulfur and carbon dioxide (3,23). This metabolic versatility is thought to contribute at least in part to the low pH of their growth environment (2), and this together with the high temperature of cultivation creates unique challenges for their physiological manipulation.Studies of the biomolecules produced by acidophilic hyperthermophiles require sufficient biomass to enable biochemical studies (7,13,16,20,21). However, large-scale systems suitable for the cultivation of these organisms are not generally available. Conventional microbial fermentors or bioreactors are fabricated out of stainless steel. Stainless steel is not appropriate for cultivation of thermoacidophiles, however, because it rusts. To avoid this problem, it is necessary to minimize steel or stainless steel surface area, particularly for reactor components that cannot be replaced, such as the reactor itself. Fermentors must instead be lined with glass or ceramic materials to circumvent metal oxidation, resulting in significant added reactor cost. One solution to this problem has been the use of plastic or rubber containers placed within secondary containment vessels to allow for reactor insulation. A fabricated lid provides reactor access, and temperature is controlled externally with a hot plate or heating jacket or internally with an immersible heater and thermostat (8,14,19). Both Sulfolobus shibatae and Sulfolobus acidocaldarius were cultivated in thi...

A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.