| 1. |
- Brouns, Stan J. J., et al.
(författare)
-
Identification of the missing links in prokaryotic pentose oxidation pathways : evidence for enzyme recruitment
- 2006
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:37, s. 27378-27388
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 2. |
- van de Werken, Harmen, et al.
(författare)
-
Hydrogenomics of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus
- 2008
-
Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 74:21, s. 6720-6729
-
Tidskriftsartikel (refereegranskat)abstract
- Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO2, and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocelluloseutilizing, biofuel-producing bacterium.
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
- Brouns, Stan J, et al.
(författare)
-
Small CRISPR RNAs guide antiviral defense in prokaryotes
- 2008
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 321:5891, s. 960-964
-
Tidskriftsartikel (refereegranskat)abstract
- Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.
|
|
| 6. |
- Ettema, Thijs J G, et al.
(författare)
-
Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2.
- 2006
-
Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 152:Pt 7, s. 1969-79
-
Tidskriftsartikel (refereegranskat)abstract
- Using a comparative genomics approach, a copper resistance gene cluster has been identified in multiple archaeal genomes. The cop cluster is predicted to encode a metallochaperone (CopM), a P-type copper-exporting ATPase (CopA) and a novel, archaea-specific transcriptional regulator (CopT) which might control the expression of the cop genes. Sequence analysis revealed that CopT has an N-terminal DNA-binding helix-turn-helix domain and a C-terminal TRASH domain; TRASH is a novel domain which has recently been proposed to be uniquely involved in metal-binding in sensors, transporters and trafficking proteins in prokaryotes. The present study describes the molecular characterization of the cop gene cluster in the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The polycistronic copMA transcript was found to accumulate in response to growth-inhibiting copper concentrations, whereas copT transcript abundance appeared to be constitutive. DNA-binding assays revealed that CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the copMA gene cluster.
|
|
| 7. |
- Ettema, Thijs J. G., et al.
(författare)
-
The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus : a key-enzyme of the semi-phosphorylative branch of the Entner-Doudoroff pathway
- 2008
-
Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1431-0651 .- 1433-4909. ; 12:1, s. 75-88
-
Tidskriftsartikel (refereegranskat)abstract
- Archaea utilize a branched modification of the classical Entner-Doudoroff (ED) pathway for sugar degradation. The semi-phosphorylative branch merges at the level of glyceraldehyde 3-phosphate (GAP) with the lower common shunt of the Emden-Meyerhof-Parnas pathway. In Sulfolobus solfataricus two different GAP converting enzymes-classical phosphorylating GAP dehydrogenase (GAPDH) and the non-phosphorylating GAPDH (GAPN)-were identified. In Sulfolobales the GAPN encoding gene is found adjacent to the ED gene cluster suggesting a function in the regulation of the semi-phosphorylative ED branch. The biochemical characterization of the recombinant GAPN of S. solfataricus revealed that-like the well-characterized GAPN from Thermoproteus tenax-the enzyme of S. solfataricus exhibits allosteric properties. However, both enzymes show some unexpected differences in co-substrate specificity as well as regulatory fine-tuning, which seem to reflect an adaptation to the different lifestyles of both organisms. Phylogenetic analyses and database searches in Archaea indicated a preferred distribution of GAPN (and/or GAP oxidoreductase) in hyperthermophilic Archaea supporting the previously suggested role of GAPN in metabolic thermoadaptation. This work suggests an important role of GAPN in the regulation of carbon degradation via modifications of the EMP and the branched ED pathway in hyperthermophilic Archaea.
|
|
| 8. |
- van der Oost, John, et al.
(författare)
-
CRISPR-based adaptive and heritable immunity in prokaryotes
- 2009
-
Ingår i: TIBS -Trends in Biochemical Sciences. Regular ed.. - : Elsevier BV. - 0968-0004 .- 1362-4326. ; 34:8, s. 401-407
-
Forskningsöversikt (refereegranskat)abstract
- The recently discovered CRISPR (clustered regularly interspaced short palindromic repeat) defense system protects bacteria and archaea against mobile genetic elements. This immunity system has the potential to continuously adjust its reach at the genomic level, implying that both gain and loss of information is inheritable. The CRISPR system consists of typical stretches of interspaced repetitive DNA (CRISPRs) and associated cas genes. Three distinct stages are recognized in the CRISPR defense mechanism: (i) adaptation of the CRISPR via the integration of short sequences of the invaders as spacers; (ii) expression of CRISPRs and subsequent processing to small guide RNAs; and (iii) interference of target DNA by the crRNA guides. Recent analyses of key Cas proteins indicate that, despite some functional analogies, this fascinating prokaryotic system shares no phylogenetic relation with the eukaryotic RNA interference system.
|
|
