Sweets for my sweet - carbohydrate transport and its regulation in Sulfolobus acidocaldarius
Carbohydrates are energy-providing nutrients which are utilized by organisms in all three domains of life. Sugar transport into the cell is crucial for the metabolism of these nutrient as they cannot cross the membrane by simple diffusion. The hyperthermophilic crenarchaeon Sulfolobus acidocaldarius...
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|Zusammenfassung:||Carbohydrates are energy-providing nutrients which are utilized by organisms in all three domains of life. Sugar transport into the cell is crucial for the metabolism of these nutrient as they cannot cross the membrane by simple diffusion. The hyperthermophilic crenarchaeon Sulfolobus acidocaldarius can utilize different carbohydrates including L-arabinose, dextrin, D-glucose, maltose, sucrose, starch or D-xylose (Grogan, 1989; Brouns et al., 2006). However, only the ABC transporter system for maltose and maltodextrin was elucidated in S. acidocaldarius by sequence homology to the maltose transporter system of S. solfataricus. In the closely related S. solfataricus several ABC transporter systems for different carbohydrates. However, no homologues of these transporter systems are present in S. acidocaldarius and the mode of sugar transport in this organism was a mystery. In order to study the principle of sugar transport and its regulation in S. acidocaldarius, I developed a genetic tool box for this organism. The genetic system, described in this thesis, is based on the uracil auxotrophic strain MW001 constructed from the S. acidocaldarius DSM639 wild type strain. Multiple markerless gene deletions, in genome tagging, ectopic integrations, complementations in cis and in trans as well as inducible homologues expression can be done by this system in MW001. This enables fundamental research as well as metabolic engineering of S. acidocaldarius. Using that system, the regulation of the maltose transporter in S. acidocaldarius was analysed first. Transcription of the maltose gene cluster was upregulated in the presence of maltose or maltodextrin indicating a regulatory mechanism. The transcriptional regulator MalR was identified among the maltose gene cluster. Characterization of the deletion mutant ΔmalR confirmed the regulatory function of this protein which acts as an activator for transcription of the mal regulon. MalR was the first identified positive regulator involved in sugar transport in the domain of Archaea. Point mutations in MalR indicated that sugar binding to MalR as well as the phosphorylation state of MalR influences its activity, but however, the elucidation of the complete regulatory mechanism in that system is still in progress. Because of the absence of other sugar related archaeal ABC transporters in S. acidocaldarius, transport mechanism for other carbohydrates were not known for long time. However, we could identify an ABC transporter system responsible for D-xylose and L-arabinose uptake. Transcription of the transporter operon was induced in the presence xylose or arabinose. Deletion mutants of the arabinose/xylose sugar transporter units confirmed the function of the transporter which is relevant if only low carbohydrate concentrations are present. However, evidence for a second D-xylose/L-arabinose transporter is provided by growth of the mutants in the presence of higher sugar concentrations. D-xylose and L-arabinose activated not only transcription of the ABC transporter operon, but also induced transcription of two genes involved in the aldolase independent pentose degradation. Deletion of these metabolizing enzymes α-ketoglutarate semialdehyde dehydrogenase (KGSADH) and 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD/KDAD) indicated the probable absence of the aldolase dependent pentose degradation pathway from S. acidocaldarius. Taken together, the work presented in this thesis give first and new insights into carbohydrate transport and it regulation in S. acidocaldarius. The knowledge about the sugar transport and metabolism together with the developed genetic tool box enables the utilization S. acidocaldarius as chassis for metabolic engineering and industrial usage.|