Post-genomic characterization of metabolic pathways in Sulfolobus solfataricus

Abstract

The physiological functions and mode of actions of different biomolecules are of continuous interest and a prerequisite to fully understand and appreciate the potential of Archaea and their molecules. We chose to study Sulfolobus solfataricus for its stable (heat-resistant) enzymes and specific metabolic potential, the ease of cultivation of this organism, and the relative large amount of knowledge about this heat-loving acidophilic organism. We selected a systems approach to study the behaviour of this organism trying to make steps forward into the unknown, whenever possible trying to link exploration to exploitation. The cultivation of S.solfataricus is an essential element in all systems approaches that link genotype to phenotype. Hence, specific attention is given to the advanced culturing systems for this extremophile that have been used in all experimental studies described here (Chapters 3-6). Systems analysis includes the integration of all available omics data and is increasingly used in the analysis of Archaea (Chapters 3 and 4). However, most attention has been given to archaeal transcriptome analysis and hence the most important literature on heat-loving Archaea is summarized (Chapter 2). In the experimental chapters (Chapters 3-6) various systems approaches are applied to gain understanding of metabolic pathways in Sulfolobus. Chapter 3 describes the study of the central carbon pathways, consisting of the (non-) phosphorilated Entner-Douderoff (ED) pathway and the citric acid cycle. Different functional genomic approaches were applied on the model organism Sulfolobus solfataricus to study the response of growth on different carbon sources, D-Glucose vs. Tryptone and Yeast Extract. The complete transcriptome was studied using PCR-based microarrays. In addition the proteome was studied using 2D-electrophoresis map in combination with 13N- labelling technique to determine protein fluctuations. Despite the large difference in medium, very few significant differences on protein or RNA level were observed for the two conditions. Therefore regulation of these pathways does in all probability not occur through changes in protein abundance but presumably rather by direct changes in enzyme activity. This is unlike two thermophilic Euryarchaea: Thermococcus kodaaraensis (Kanai, Akerboom et al. 2007)and Pyrococcus furiosus (Schut, Brehm et al. 2003)where extensive regulation of glycolytic genes was observed in a similar situation. Chapter 4describes the study of the degradation of D-arabinose through a similar approach as was described in chapter 3. S. solfataricus was grown on either D-arabinose or D-glucose and a comprehensive transcriptome and proteome study was carried out. The result of these studies was not only elucidation of the D-arabinose degradation route, but also a general prokaryotic pentose, hexaric acids and hydroxyproline degradation route, which supports the theory of metabolic pathway genesis by enzyme recruitment. Also this study predicted a cis-regulatory element to induce the arabinose degrading pathway when needed. The enzymes involved in the proposed pathway were cloned, expressed and their function was biochemically measured. This showed that using these enzymes, D-arabinose can be degraded to 2-oxogluterate, one of the metabolites that are part of the citric acid cycle. Chapter 5reports on the effects of different oxygen concentrations on the behaviour of Sulfolobus solfataricus. The oxygen amount can be controlled relatively easily in a bioreactor, which is crucial for rapid and reproducible growth. Based on growth experiments in microcosms, different types of behaviour could be seen. At 35% (v/v gas phase) the cultures did not grow, indicating that S. solfa-taricus experiences a lethal dose of oxygen. At 26-32% growth was impaired, suggesting a moderate toxicity compared to the reference (21%). In the ranges 16-24% of oxygen, standard growth was observed, suggesting that S. solfataricus is comfortable in these oxygen ranges. For the lower amounts of oxygen (1.5-15%), the growth was comparable to the reference, but the respiratoryefficiency was increased. To get some more insight into this behaviour, we looked at the transcriptome. It showed differential expression of several genes, including genes encoding terminal oxidases, indicating that the organism adapts to lower oxygen concentrations by adapting its respiratory machinery. Chapter 6 describes the zeaxanthin pathway in the Sulfolobus species. Zeaxanthin is a colorant and of vital importance for the function of the human eye. In this chapter the genes responsible for zeaxanthin production are presented. For this, DNA microarrays, bioinformatics as well as molecular genetics techniques were used. A crtx-like gene is operational in most of the known Sulfolobus species that is able to attach sugar-like molecules to zeaxanthin, which improves its solubility in water, which is very important in many food uses. We have cloned this crtx-like gene of S. solfataricus, S. shibatae, and S. acidocaldarius in a zeaxanthin overproducing E. coli strain. It has been demonstrated that the gene products of S. shibatae and S. acidocaldarius were responsible for attaching sugar-like molecules to zeaxanthin. The ctrx-like gene of S. solfataricus was not operating in E. coli. This is probably due to the fact that the gene is truncated. This chapter has further improved the understanding of archaeal carotenoid pathways and it has shown that the Sulfolobus species are able to modify zeaxanthin, although each species produces different zeaxanthin modifications. </p