The glucose effect and regulation of alpha-amylase synthesis in the hyperthermophilic archaeon Sulfolobus solfataricus

Abstract

An ␣-amylase was purified from culture supernatants of Sulfolobus solfataricus 98/2 during growth on starch as the sole carbon and energy source. The enzyme is a homodimer with a subunit mass of 120 kDa. It catalyzes the hydrolysis of starch, dextrin, and ␣-cyclodextrin with similar efficiencies. Addition of exogenous glucose represses production of ␣-amylase, demonstrating that a classical glucose effect is operative in this organism. Synthesis of [35 S]-␣-amylase protein is also subject to the glucose effect. ␣-Amylase is constitutively produced at low levels but can be induced further by starch addition. The absolute levels of ␣-amylase detected in culture supernatants varied greatly with the type of sole carbon source used to support growth. Aspartate was identified as the most repressing sole carbon source for ␣-amylase production, while glutamate was the most derepressing. The pattern of regulation of ␣-amylase production seen in this organism indicates that a catabolite repression-like system is present in a member of the archaea.Catabolite repression is a paradigm for studies concerned with global and specific gene control mechanisms (22). In prokaryotes, catabolite repression together with transient repression and inducer exclusion make up what has been termed the glucose effect or repression of catabolic enzyme synthesis by glucose (23). However, for eukaryotes, the term catabolite repression is more generally used as a pseudonym for the glucose effect. In fact, evidence for transient repression, inducer exclusion, and requisite aspects of catabolite repression, including the ability to grow most rapidly on preferred carbon sources, is not well demonstrated (for reviews, see references 29 and 31). Catabolite repression in prokaryotes and eukaryotes has received wide attention, but the existence of an analogous process in the archaea has not been addressed. One hallmark of this process in gram-negative bacteria consists of the global mode of gene regulation of catabolite-repressible genes mediated by the small molecule cyclic AMP (cAMP) (8). Although the role of cAMP in some prokaryotes is well accepted, it has been eliminated as an effector in the corresponding catabolite response in the gram-positive bacterium Bacillus subtilis (for a review, see reference 14). In eukaryotes, including the budding yeast Saccharomyces cerevisiae, cAMP plays a crucial but indirect role in mediating the glucose effect. Interestingly, cAMP has been reported in a range of archaea (21).Sulfolobus solfataricus is an extremely thermophilic organism which inhabits acidic hot springs. S. solfataricus is a member of the archaea and has been assigned to a subdivision termed the crenarchaeota by rRNA gene (rDNA) sequence analysis (32). It is capable of diverse modes of metabolism at temperatures ranging between 70 and 90ЊC. It can grow either lithoautotrophically, oxidizing sulfur (4, 15), or chemoheterotrophically on starch or other sugars as sole carbon and energy sources (7,11). Recent studies also suggest that hot springs cont...

Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon Sulfolobus solfataricus

“…To distinguish among these possibilities, Northern blot analysis was used to test whether mercury treatment depleted the abundance of specific cellular mRNAs. The rates of decay for three S. solfataricus transcripts were examined, including lacS (␤-glycosidase), a gene involved in polysaccharide hydrolysis (19), and both paralogs of the archaeal basal transcription factor, tfb-1 (35) and tfb-2 (40). Cells in early exponential growth phase were treated with 0.3 M HgCl 2 , and RNA was isolated for 30 min at the appropriate times and then analyzed for mRNA content (Fig.…”

“…S. solfataricus (38) was cultivated in batch cultures, as described previously (19,39). Cells were grown at 80°C in the medium of Allen (1) as modified by Brock et al (9) at a pH of 3.0 in 250-ml screw-cap flasks.…”

“…S. solfataricus total RNA was extracted as described previously (12) from wet cells obtained by filtration of early-exponential-phase cultures (OD 540 , 0.1) at the appropriate times. Electrophoresis of RNA, RNA transfer, and generation of lacS antisense riboprobes were performed as described previously (19,39). 7S RNA, tfb-1, and tfb-2 antisense riboprobes were prepared as described previously (5).…”

Identification and Molecular Characterization of the First α-Xylosidase from an Archaeon

“…Although the substrate specificity of these gene products remains to be identified, they could be involved in the degradation and assimilation of plant polysaccharides. The only examples of glycosyl hydrolases characterized in this genus are a secreted ␣-amylase, an intracellular ␣-glucosidase from S. solfataricus strain 98/2 (17,18), and a ␤-glycosidase that has been purified from strains MT4 and P2 (19,20). The two ␣-glycosyl hydrolases are involved in the utilization of dextrins, whereas the function in vivo of the ␤-glycosidase (LacS), which is under active study in our laboratory, is still obscure (21)(22)(23).…”

Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes

“…Another extracellular amylase has been isolated from culture supernatants of Sulfolobus solfataricus during growth on starch (21). The secreted protein has an apparent mass of 240 kDa, consisting of two identical subunits.…”