Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Abstract

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...

Identification of an archaeal mercury regulon by chromatin immunoprecipitation

“…Archaeal strains, plasmids and primers are indicated (Table S1, available in the online Supplementary Material). S. solfataricus strain PBL2025 and its derivatives were grown at 80 uC with aeration in batch culture as described previously (Allen, 1959;Rolfsmeier & Blum, 1995;Worthington et al, 2003) in Allen's basal salts (Allen, 1959) as modified by Brock et al (1972) at a pH of 3.0. Liquid media were supplemented with either 0.2 % lactose (w/v), 0.2 % sucrose (w/v) or 0.2 % (w/v) tryptone as carbon and energy sources as indicated.…”

Archaeal genetics — the third way

“…solfataricus is capable of homologous recombination, and has been used for gene knock-out experiments (using selection for lactose utilisation) 86 .…”

Two novel conjugative plasmids from a single strain of Sulfolobus

“…An impressive variety of mobile genetic elements has recently been discovered in Archaea in general, and in Sulfolobus in particular: viruses, autonomous insertion sequence (IS) elements, non-autonomous miniature inverted repeat transposable elements (MITEs), small nonconjugative plasmids and large conjugative plasmids (Brugger et al, 2002;Prangishvili et al, 2001;Rice et al, 2001;Zillig et al, 1998). Although there have been impressive recent developments in Sulfolobus genetics, this remains a bottleneck (Albers et al, 2006;Bartolucci et al, 2003;Jonuscheit et al, 2003;Stedman et al, 1999;Worthington et al, 2003).…”