Jasper Walther

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for d-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on d-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that d-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a d-arabinose dehydrogenase, a d-arabinonate dehydratase, a novel 2-keto-3-deoxy-d-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.

Transcriptome analysis of infection of the archaeon Sulfolobus solfataricus with Sulfolobus turreted icosahedral virus

ABSTRACT Microarray analysis of infection by Sulfolobus turreted icosahedral virus (STIV) revealed insights into the timing and extent of virus transcription, as well as differential regulation of host genes. Using a microarray containing genes from both the host and the virus, the infection cycle of STIV was studied. Following infection of Sulfolobus solfataricus strain 2-2-12 with STIV, transcription of virus genes was first detected at 8 h postinfection (p.i.), with a peak at 24 h p.i. Lysis of cells was first detected at 32 h p.i. There was little temporal control of the transcription of virus genes, although the three open reading frames on the noncoding strand were transcribed later in the infection process. During the infection, 177 host genes were determined to be differentially expressed, with 124 genes up-regulated and 53 genes down-regulated. The up-regulated genes were dominated by genes associated with DNA replication and repair and those of unknown function, while the down-regulated genes, mostly detected at 32 h p.i., were associated with energy production and metabolism. Examination of infected cells by transmission electron microscopy revealed alterations in cell ultrastructure consistent with the microarray analysis. The observed patterns of transcription suggest that up-regulated genes are likely used by the virus to reprogram the cell for virus replication, while the down-regulated genes reflect the imminent lysis of the cells.

Characterization and mode of action of an exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritima

An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin‐hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo‐active and thermostable, with a melting temperature of 105 °C and a temperature optimum of 80 °C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 U·mg−1). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl‐esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo‐active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed.

Effect of O2 concentrations on Sulfolobus solfataricus P2

Sulfolobus solfataricus P2 was grown aerobically at various O(2) concentrations. Based on growth parameters in microcosms, four types of behavior could be distinguished. At 35% O(2) (v/v; gas phase), the cultures did not grow, indicating a lethal dose of oxygen. For 26-32% O(2), the growth was significantly affected compared with the reference (21%), suggesting a moderate toxicity by O(2). For 16-24% O(2), standard growth was observed. For 1.5-15% O(2), growth was comparable with the reference, but the yield on O(2) indicated a more efficient use of oxygen. These results indicate that S. solfataricus P2 grows optimally in the range of 1.5-24% O(2), most likely by adjusting its energy-transducing machinery. To gain some insight into control of the respiratory system, transcriptomes of the strain cultivated at different O(2) concentrations, corresponding to each behavior (1.5%, 21% and 26%), were compared using a DNA microarray approach. It showed differential expression of several genes encoding terminal oxidases, indicating an adaptation of the strains respiratory system in response to fluctuating oxygen concentrations.

Functional genomics of the thermo-acidophilic archaeon Sulfolobus solfataricus

Publisher Summary The chapter describes ongoing developments at the level of functional genomics of archaeal and bacterial thermophiles, with particular emphasis on Archaea of the genus Sulfolobus. At present, important developments in biochemical and molecular genetics analyses are under way, allowing novel approaches for analysis in vitro (heterologous expression, directed and random mutagenesis, study of protein/protein and protein/DNA interactions), and in vivo (chromosomal knockouts, in trans overexpression, phenotype characterization). Recent developments of transcriptomics and proteomics tools for thermophiles in general, and Sulfolobus in particular are reviewed. Developments in thermophile genomics proceed at an impressive pace: additional hyperthermophile genomes are released on a regular basis, comparative tools become more sophisticated and functional predictions become more reliable, deoxyribo nucleic acid (DNA) microarrays are available, and recent breakthroughs illustrate the role of mass spectrometry-based proteomics and metabolomics as an indispensable tool for molecular and cellular biology, and for the emerging field of systems biology. Now the focus is on the integration of classical and modern technologies to acquire knowledge in the functioning of metabolic networks and of the regulatory circuits of these systems.