| 1. |
- ter Steege, Hans, et al.
(författare)
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Mapping density, diversity and species-richness of the Amazon tree flora
- 2023
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Ingår i: COMMUNICATIONS BIOLOGY. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Using 2.046 botanically-inventoried tree plots across the largest tropical forest on Earth, we mapped tree species-diversity and tree species-richness at 0.1-degree resolution, and investigated drivers for diversity and richness. Using only location, stratified by forest type, as predictor, our spatial model, to the best of our knowledge, provides the most accurate map of tree diversity in Amazonia to date, explaining approximately 70% of the tree diversity and species-richness. Large soil-forest combinations determine a significant percentage of the variation in tree species-richness and tree alpha-diversity in Amazonian forest-plots. We suggest that the size and fragmentation of these systems drive their large-scale diversity patterns and hence local diversity. A model not using location but cumulative water deficit, tree density, and temperature seasonality explains 47% of the tree species-richness in the terra-firme forest in Amazonia. Over large areas across Amazonia, residuals of this relationship are small and poorly spatially structured, suggesting that much of the residual variation may be local. The Guyana Shield area has consistently negative residuals, showing that this area has lower tree species-richness than expected by our models. We provide extensive plot meta-data, including tree density, tree alpha-diversity and tree species-richness results and gridded maps at 0.1-degree resolution. A study mapping the tree species richness in Amazonian forests shows that soil type exerts a strong effect on species richness, probably caused by the areas of these forest types. Cumulative water deficit, tree density and temperature seasonality affect species richness at a regional scale.
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| 2. |
- Householder, John Ethan, et al.
(författare)
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One sixth of Amazonian tree diversity is dependent on river floodplains
- 2024
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Ingår i: NATURE ECOLOGY & EVOLUTION. - 2397-334X.
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Tidskriftsartikel (refereegranskat)abstract
- Amazonia's floodplain system is the largest and most biodiverse on Earth. Although forests are crucial to the ecological integrity of floodplains, our understanding of their species composition and how this may differ from surrounding forest types is still far too limited, particularly as changing inundation regimes begin to reshape floodplain tree communities and the critical ecosystem functions they underpin. Here we address this gap by taking a spatially explicit look at Amazonia-wide patterns of tree-species turnover and ecological specialization of the region's floodplain forests. We show that the majority of Amazonian tree species can inhabit floodplains, and about a sixth of Amazonian tree diversity is ecologically specialized on floodplains. The degree of specialization in floodplain communities is driven by regional flood patterns, with the most compositionally differentiated floodplain forests located centrally within the fluvial network and contingent on the most extraordinary flood magnitudes regionally. Our results provide a spatially explicit view of ecological specialization of floodplain forest communities and expose the need for whole-basin hydrological integrity to protect the Amazon's tree diversity and its function.
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| 3. |
- Luize, Bruno Garcia, et al.
(författare)
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Geography and ecology shape the phylogenetic composition of Amazonian tree communities
- 2024
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Ingår i: JOURNAL OF BIOGEOGRAPHY. - 0305-0270 .- 1365-2699.
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Tidskriftsartikel (refereegranskat)abstract
- Aim: Amazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types. Location: Amazonia. Taxon: Angiosperms (Magnoliids; Monocots; Eudicots). Methods: Data for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran's eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny. Results: In the terra firme and v & aacute;rzea forest types, the phylogenetic composition varies by geographic region, but the igap & oacute; and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R-2 = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R-2 = 28%). A greater number of lineages were significant indicators of geographic regions than forest types. Main Conclusion: Numerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions.
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| 4. |
- Peripato, Vinicius, et al.
(författare)
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More than 10,000 pre-Columbian earthworks are still hidden throughout Amazonia
- 2023
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Ingår i: Science (New York, N.Y.). - 1095-9203. ; 382:6666, s. 103-109
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Tidskriftsartikel (refereegranskat)abstract
- Indigenous societies are known to have occupied the Amazon basin for more than 12,000 years, but the scale of their influence on Amazonian forests remains uncertain. We report the discovery, using LIDAR (light detection and ranging) information from across the basin, of 24 previously undetected pre-Columbian earthworks beneath the forest canopy. Modeled distribution and abundance of large-scale archaeological sites across Amazonia suggest that between 10,272 and 23,648 sites remain to be discovered and that most will be found in the southwest. We also identified 53 domesticated tree species significantly associated with earthwork occurrence probability, likely suggesting past management practices. Closed-canopy forests across Amazonia are likely to contain thousands of undiscovered archaeological sites around which pre-Columbian societies actively modified forests, a discovery that opens opportunities for better understanding the magnitude of ancient human influence on Amazonia and its current state.
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| 5. |
- Kattge, Jens, et al.
(författare)
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TRY plant trait database - enhanced coverage and open access
- 2020
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Ingår i: Global Change Biology. - : Wiley-Blackwell. - 1354-1013 .- 1365-2486. ; 26:1, s. 119-188
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Tidskriftsartikel (refereegranskat)abstract
- Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives.
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| 6. |
- Kehoe, Laura, et al.
(författare)
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Make EU trade with Brazil sustainable
- 2019
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 364:6438, s. 341-
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 7. |
- Bolivar, Juan M, et al.
(författare)
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Coating of soluble and immobilized enzymes with ionic polymers : full stabilization of the quaternary structure of multimeric enzymes
- 2009
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 10:4, s. 742-747
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Tidskriftsartikel (refereegranskat)abstract
- This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).
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| 8. |
- Bolivar, Juan M, et al.
(författare)
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Immobilization-stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus
- 2008
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 80:1, s. 49-58
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Tidskriftsartikel (refereegranskat)abstract
- The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.
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| 9. |
- Cooper, Declan L.M., et al.
(författare)
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Consistent patterns of common species across tropical tree communities
- 2024
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Ingår i: Nature. - 0028-0836 .- 1476-4687. ; 625:7996, s. 728-734
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Tidskriftsartikel (refereegranskat)abstract
- Trees structure the Earth’s most biodiverse ecosystem, tropical forests. The vast number of tree species presents a formidable challenge to understanding these forests, including their response to environmental change, as very little is known about most tropical tree species. A focus on the common species may circumvent this challenge. Here we investigate abundance patterns of common tree species using inventory data on 1,003,805 trees with trunk diameters of at least 10 cm across 1,568 locations 1–6 in closed-canopy, structurally intact old-growth tropical forests in Africa, Amazonia and Southeast Asia. We estimate that 2.2%, 2.2% and 2.3% of species comprise 50% of the tropical trees in these regions, respectively. Extrapolating across all closed-canopy tropical forests, we estimate that just 1,053 species comprise half of Earth’s 800 billion tropical trees with trunk diameters of at least 10 cm. Despite differing biogeographic, climatic and anthropogenic histories 7, we find notably consistent patterns of common species and species abundance distributions across the continents. This suggests that fundamental mechanisms of tree community assembly may apply to all tropical forests. Resampling analyses show that the most common species are likely to belong to a manageable list of known species, enabling targeted efforts to understand their ecology. Although they do not detract from the importance of rare species, our results open new opportunities to understand the world’s most diverse forests, including modelling their response to environmental change, by focusing on the common species that constitute the majority of their trees.
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| 10. |
- Hidalgo, Aurelio, et al.
(författare)
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Thermus thermophilus as a cell factory for the production of a thermophilic Mn-dependent catalase which fails to be synthesized in an active form in Escherichia coli
- 2004
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 70:7, s. 3839-3844
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Tidskriftsartikel (refereegranskat)abstract
- Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H(2)O(2)-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.
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| 11. |
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| 12. |
- Muscarella, Robert, et al.
(författare)
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The global abundance of tree palms
- 2020
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Ingår i: Global Ecology and Biogeography. - : Wiley. - 1466-822X .- 1466-8238. ; 29:9, s. 1495-1514
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Tidskriftsartikel (refereegranskat)abstract
- AimPalms are an iconic, diverse and often abundant component of tropical ecosystems that provide many ecosystem services. Being monocots, tree palms are evolutionarily, morphologically and physiologically distinct from other trees, and these differences have important consequences for ecosystem services (e.g., carbon sequestration and storage) and in terms of responses to climate change. We quantified global patterns of tree palm relative abundance to help improve understanding of tropical forests and reduce uncertainty about these ecosystems under climate change.LocationTropical and subtropical moist forests.Time periodCurrent.Major taxa studiedPalms (Arecaceae).MethodsWe assembled a pantropical dataset of 2,548 forest plots (covering 1,191 ha) and quantified tree palm (i.e., ≥10 cm diameter at breast height) abundance relative to co‐occurring non‐palm trees. We compared the relative abundance of tree palms across biogeographical realms and tested for associations with palaeoclimate stability, current climate, edaphic conditions and metrics of forest structure.ResultsOn average, the relative abundance of tree palms was more than five times larger between Neotropical locations and other biogeographical realms. Tree palms were absent in most locations outside the Neotropics but present in >80% of Neotropical locations. The relative abundance of tree palms was more strongly associated with local conditions (e.g., higher mean annual precipitation, lower soil fertility, shallower water table and lower plot mean wood density) than metrics of long‐term climate stability. Life‐form diversity also influenced the patterns; palm assemblages outside the Neotropics comprise many non‐tree (e.g., climbing) palms. Finally, we show that tree palms can influence estimates of above‐ground biomass, but the magnitude and direction of the effect require additional work.ConclusionsTree palms are not only quintessentially tropical, but they are also overwhelmingly Neotropical. Future work to understand the contributions of tree palms to biomass estimates and carbon cycling will be particularly crucial in Neotropical forests.
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| 13. |
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| 14. |
- Agudo, Antonio, et al.
(författare)
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Polymorphisms in metabolic genes related to tobacco smoke and the risk of gastric cancer in the European prospective investigation into cancer and nutrition
- 2006
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Ingår i: Cancer Epidemiology Biomarkers & Prevention. - 1538-7755. ; 15:12, s. 2427-2434
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Tidskriftsartikel (refereegranskat)abstract
- Metabolizing enzymes, which often display genetic polymorphisms, are involved in the activation of compounds present in tobacco smoke that may be relevant to gastric carcinogenesis. We report the results of a study looking at the association between risk of gastric adenocarcinoma and polymorphisms in genes CYP1A1, CYP1A2, EPHX1, and GSTT1. A nested case-control study was carried out within the European Prospective Investigation into Cancer and Nutrition, developed in 10 European countries. The study includes 243 newly diagnosed cases of histologically confirmed gastric adenocarcinoma and 946 controls matched by center, age, sex, and date of blood collection. Genotypes were determined in nuclear DNA from WBCs. We found an increased risk of gastric cancer for homozygotes for C (histidine) variant in Y113H of EPHX1 (odds ratio, 1.91; 95% confidence interval, 1.19-3.07) compared with subjects with TC/TT. There was also a significant increased risk for smokers carrying at least one variant allele A in Ex7+129C > A (m4) of CYP1A1 and never smokers with null GSTT1 and allele A in the locus -3859G > A of CYP1A2. Most of these genes are involved in the activation and detoxification of polycyclic aromatic hydrocarbons, suggesting a potential role of these compounds in gastric carcinogenesis.
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| 15. |
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| 16. |
- Alvarez, Laura, et al.
(författare)
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A new family of nitrate/nitrite transporters involved in denitrification
- 2019
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Ingår i: International Microbiology. - : Springer-Verlag New York. - 1139-6709 .- 1618-1905. ; 22:1, s. 19-28
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Tidskriftsartikel (refereegranskat)abstract
- Denitrifying bacteria carry out nitrate and nitrite respiration inside and outside the cell, respectively. In Thermus thermophilus, nitrate and nitrite transport processes are carried out by major facilitator superfamily (MFS) transporters. The sequence of the nar operon of nitrate-only respiring strains of T. thermophilus includes two tandemly organized MFS transporter genes (narK and narT) of the NarK1 and NarK2 families. Both can function as nitrate/nitrite antiporters, but NarK has been proposed as more specific for nitrate whereas NarT more specific for nitrite. In some nitrate- and nitrite-respiring strains of the same species, a single MFS transporter (NarO) belonging to a different MFS subfamily appears. To analyze the role of this single MFS in the same genetic context, we transferred the two types of nar operon to the aerobic strain HB27, and further included in both of them the ability to respire nitrite. The new denitrifying strains HB27dn, with two MFS, and HB27dp, with a single one, were used to isolate mutants devoid of transporters. Through in trans complementation experiments, we demonstrate that the NarO single MFS works efficiently in the transport of both nitrate and nitrite.
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| 17. |
- Alvarez, Laura, et al.
(författare)
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Hierarchical control of nitrite respiration by transcription factors encoded within mobile gene clusters of Thermus thermophilus
- 2017
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Ingår i: Genes. - : MDPI. - 2073-4425. ; 8:12
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Tidskriftsartikel (refereegranskat)abstract
- Denitrification in Thermus thermophilus is encoded by the nitrate respiration conjugative element (NCE) and nitrite and nitric oxide respiration (nic) gene clusters. A tight coordination of each cluster's expression is required to maximize anaerobic growth, and to avoid toxicity by intermediates, especially nitric oxides (NO). Here, we study the control of the nitrite reductases (Nir) and NO reductases (Nor) upon horizontal acquisition of the NCE and nic clusters by a formerly aerobic host. Expression of the nic promoters PnirS, PnirJ, and PnorC, depends on the oxygen sensor DnrS and on the DnrT protein, both NCE-encoded. NsrR, a nic-encoded transcription factor with an iron-sulfur cluster, is also involved in Nir and Nor control. Deletion of nsrR decreased PnorC and PnirJ transcription, and activated PnirS under denitrification conditions, exhibiting a dual regulatory role never described before for members of the NsrR family. On the basis of these results, a regulatory hierarchy is proposed, in which under anoxia, there is a pre-activation of the nic promoters by DnrS and DnrT, and then NsrR leads to Nor induction and Nir repression, likely as a second stage of regulation that would require NO detection, thus avoiding accumulation of toxic levels of NO. The whole system appears to work in remarkable coordination to function only when the relevant nitrogen species are present inside the cell.
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| 18. |
- Aragão, Luiz E. O. C., et al.
(författare)
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21st Century drought-related fires counteract the decline of Amazon deforestation carbon emissions
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Tropical carbon emissions are largely derived from direct forest clearing processes. Yet, emissions from drought-induced forest fires are, usually, not included in national-level carbon emission inventories. Here we examine Brazilian Amazon drought impacts on fire incidence and associated forest fire carbon emissions over the period 2003-2015. We show that despite a 76% decline in deforestation rates over the past 13 years, fire incidence increased by 36% during the 2015 drought compared to the preceding 12 years. The 2015 drought had the largest ever ratio of active fire counts to deforestation, with active fires occurring over an area of 799,293 km(2). Gross emissions from forest fires (989 +/- 504 Tg CO2 year(-1)) alone are more than half as great as those from old-growth forest deforestation during drought years. We conclude that carbon emission inventories intended for accounting and developing policies need to take account of substantial forest fire emissions not associated to the deforestation process.
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| 19. |
- Blas-Galindo, Emilio, et al.
(författare)
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Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus
- 2007
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 73:16, s. 5138-5145
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Tidskriftsartikel (refereegranskat)abstract
- A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.
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| 20. |
- Castán, Pablo, et al.
(författare)
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The periplasmic space in Thermus thermophilus : evidence from a regulation-defective S-layer mutant overexpressing an alkaline phosphatase
- 2002
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Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1431-0651 .- 1433-4909. ; 6:3, s. 225-232
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Tidskriftsartikel (refereegranskat)abstract
- The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8(Delta)UTR1) defective in the regulation of its S-layer (surface crystalline layer). This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase. Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH(2)-containing material between the external envelopes and the peptidoglycan. A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components. To demonstrate this, we cloned a gene ( phoA) from T. thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector. Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction. All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria. The potential application of the HB8(Delta)UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed.
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| 21. |
- Cava, Felipe, et al.
(författare)
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A cytochrome c containing nitrate reductase plays a role in electron transport for denitrification in Thermus thermophilus without involvement of the bc respiratory complex
- 2008
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 70:2, s. 507-518
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Tidskriftsartikel (refereegranskat)abstract
- The bc(1) respiratory complex III constitutes a key energy-conserving respiratory electron transporter between complex I (type I NADH dehydrogenase) and II (succinate dehydrogenase) and the final nitrogen oxide reductases (Nir, Nor and Nos) in most denitrifying bacteria. However, we show that the expression of complex III from Thermus thermophilus is repressed under denitrification, and that its role as electron transporter is replaced by an unusual nitrate reductase (Nar) that contains a periplasmic cytochrome c (NarC). Several lines of evidence support this conclusion: (i) nitrite and NO are as effective signals as nitrate for the induction of Nar; (ii) narC mutants are defective in anaerobic growth with nitrite, NO and N2O; (iii) such mutants present decreased NADH oxidation coupled to these electron acceptors; and (iv) complementation assays of the mutants reveal that the membrane-distal heme c of NarC was necessary for anaerobic growth with nitrite, whereas the membrane-proximal heme c was not. Finally, we show evidence to support that Nrc, the main NADH oxidative activity in denitrification, interacts with Nar through their respective membrane subunits. Thus, we propose the existence of a Nrc-Nar respiratory super-complex that is required for the development of the whole denitrification pathway in T. thermophilus.
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| 22. |
- Cava, Felipe, et al.
(författare)
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A new type of NADH dehydrogenase specific for nitrate respiration in the extreme thermophile Thermus thermophilus
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:44, s. 45369-45378
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Tidskriftsartikel (refereegranskat)abstract
- A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.
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| 23. |
- Cava, Felipe, et al.
(författare)
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Binding to pyruvylated compounds as an ancestral mechanism to anchor the outer envelope in primitive bacteria
- 2004
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 52:3, s. 677-690
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Tidskriftsartikel (refereegranskat)abstract
- Electron microscopy of isolated cell walls of the ancient bacterium Thermus thermophilus revealed that most of the peptidoglycan (PG) surface, apart from the septal region, was shielded against specific alphaPG antibodies. On the other hand, an antiserum raised against S-layer-attached cell wall fragments (alphaSAC) bound to most of the surface except for the septal regions. Treatments with alpha-amylase and pronase E made the entire cell wall surface uniformly accessible to alphaPG and severely decreased the binding of alphaSAC. We concluded that a layer of strongly bound secondary cell wall polymers (SCWPs) covers most of the cell wall surface in this ancient bacterium. A preliminary analysis revealed that such SCWPs constitute 14% of the cell wall and are essentially composed of sugars. Enzyme treatments of the cell walls revealed that SCWP was required in vitro for the binding of the S-layer protein through the S-layer homology (SLH) motif. The csaB gene was necessary for the attachment of the S-layer-outer membrane (OM) complex to the cell wall in growing cells of T. thermophilus. In vitro experiments confirmed that cell walls from a csaB mutant bound to the S-layer with a much lower affinity ( approximately 1/10) than that of the wild type. CsaB was found to be required for pyruvylation of components of the SCWP and for immunodetection with alpha-SAC antiserum. Therefore, the S-layer-OM complex of T. thermophilus binds to the cell wall through the SLH motif of the S-layer protein via a strong interaction with a highly immunogenic pyruvylated component of the SCWP. Immuno-cross-reactive compounds were detected with alphaSAC on cell walls of other Thermus spp. and in the phylogenetically related microorganism Deinococcus radiodurans. These results imply that the interaction between the SLH motif and pyruvylated components of the cell wall arose early during bacterial evolution as an ancestral mechanism for anchoring proteins and outer membranes to the cell walls of primitive bacteria.
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| 24. |
- Cava, Felipe, et al.
(författare)
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Control of the respiratory metabolism of Thermus thermophilus by the nitrate respiration conjugative element NCE
- 2007
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 64:3, s. 630-646
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Tidskriftsartikel (refereegranskat)abstract
- The strains of Thermus thermophilus that contain the nitrate respiration conjugative element (NCE) replace their aerobic respiratory chain by an anaerobic counterpart made of the Nrc-NADH dehydrogenase and the Nar-nitrate reductase in response to nitrate and oxygen depletion. This replacement depends on DnrS and DnrT, two homologues to sensory transcription factors encoded in a bicistronic operon by the NCE. DnrS is an oxygen-sensitive protein required in vivo to activate transcription on its own dnr promoter and on that of the nar operon, but not required for the expression of the nrc operon. In contrast, DnrT is required for the transcription of these three operons and also for the repression of nqo, the operon that encodes the major respiratory NADH dehydrogenase expressed during aerobic growth. Thermophilic in vitro assays revealed that low DnrT concentrations allows the recruitment of the T. thermophilus RNA polymerase sigma(A) holoenzyme to the nrc promoter and its transcription, whereas higher DnrT concentrations are required to repress transcription on the nqo promoter. In conclusion, our data show a complex autoinducible mechanism by which DnrT functions as the transcriptional switch that allows the NCE to take the control of the respiratory metabolism of its host during adaptation to anaerobic growth.
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| 25. |
- Cava, Felipe, et al.
(författare)
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Expression and use of superfolder green fluorescent protein at high temperatures in vivo : a tool to study extreme thermophile biology
- 2008
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Ingår i: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 10:3, s. 605-613
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Tidskriftsartikel (refereegranskat)abstract
- Superfolder GFP (sGFP) is a variant of the Green Fluorescent Protein that folds efficiently when fused to poorly folded proteins. In this study, we show that sGFP, but not enhanced GFP, is functional in vivo at 70 degrees C in the extreme thermophile Thermus thermophilus (Tth); thus, permitting the use of sGFP as a localization tag in vivo. We created a suite of plasmids that allow the expression of carboxy-terminal sGFP fusion proteins in both Escherichia coli and Tth. In order to demonstrate the facility of sGFP as an in vivo localization tag in Tth, we tagged GroES (the small subunit of the bacterial GroES/GroEL chaperone), NarC (a membrane component of the nitrate respiration apparatus) and PhoA (a TAT-secreted periplasmic protein), and visualized the distribution of the sGFP fusion proteins using confocal microscopy. Fusions to NarC and PhoA produced enzymatically active proteins that complemented both the narC and the phoA strains respectively. Observation of the distribution of the GroES-sGFP protein by confocal microscopy revealed a homogeneous fluorescence in the cells, which is in full agreement with the cytoplasmic nature of GroES, whereas the NarC-sGFP protein was localized to the membrane. Finally, a combination of confocal microscopy and biochemistry revealed that PhoA is localized in the periplasm. We suggest that sGFP will be broadly applicable in characterizing various extreme thermophile systems.
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| 26. |
- Cava, Felipe, et al.
(författare)
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The role of the nitrate respiration element of Thermus thermophilus in the control and activity of the denitrification apparatus
- 2008
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Ingår i: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 10:2, s. 522-533
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Tidskriftsartikel (refereegranskat)abstract
- The nitrate conjugative element (NCE) encodes the ability to respire nitrate in the facultative Thermus thermophilus NAR1 strain. This process is carried out by two heterotetrameric enzymes that catalyse the oxidation of NADH (Nrc) and the reduction of nitrate (Nar), whose expression is activated by the NCE-encoded transcription factors DnrS and DnrT. We report the presence of NCE in other facultative strains of T. thermophilus and analyse its role in subsequent steps of the denitrification pathway. We encountered that nrc mutants of denitrifying strains show a decrease in anaerobic growth rates not only with nitrate, but also with nitrite, NO and N(2)O, which is concomitant to their lower NADH oxidation activities in vitro. We show that nitrate, nitrite and NO are activating signals for transcription of nrc in these strains. Finally, we demonstrate that DnrS and DnrT are required for anaerobic growth not only with nitrate, but also with nitrite, NO and N(2)O. These data allow us to conclude that: (i) Nrc constitutes the main electron donor for the four reductases of the denitrification pathway, and (ii) the NCE controls the expression of the whole denitrification pathway and the repression of the aerobic respiration through the transcription factors DnrS and DnrT.
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| 27. |
- Cava, Felipe, et al.
(författare)
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Thermus thermophilus as biological model
- 2009
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Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1431-0651 .- 1433-4909. ; 13:2, s. 213-31
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Tidskriftsartikel (refereegranskat)abstract
- Thermus spp is one of the most wide spread genuses of thermophilic bacteria, with isolates found in natural as well as in man-made thermal environments. The high growth rates, cell yields of the cultures, and the constitutive expression of an impressively efficient natural competence apparatus, amongst other properties, make some strains of the genus excellent laboratory models to study the molecular basis of thermophilia. These properties, together with the fact that enzymes and protein complexes from extremophiles are easier to crystallize have led to the development of an ongoing structural biology program dedicated to T. thermophilus HB8, making this organism probably the best so far known from a protein structure point view. Furthermore, the availability of plasmids and up to four thermostable antibiotic selection markers allows its use in physiological studies as a model for ancient bacteria. Regarding biotechnological applications this genus continues to be a source of thermophilic enzymes of great biotechnological interest and, more recently, a tool for the over-expression of thermophilic enzymes or for the selection of thermostable mutants from mesophilic proteins by directed evolution. In this article, we review the properties of this organism as biological model and its biotechnological applications.
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| 28. |
- Chahlafi, Zahra, et al.
(författare)
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The role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus
- 2018
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Ingår i: Environmental Microbiology. - : John Wiley & Sons. - 1462-2912 .- 1462-2920. ; 20:10, s. 3851-3861
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Tidskriftsartikel (refereegranskat)abstract
- In many Thermus thermophilus strains, nitrate respiration is encoded in mobile genetic regions, along with regulatory circuits that modulate its expression based on anoxia and nitrate presence. The oxygen‐responsive system has been identified as the product of the dnrST (dnr) operon located immediately upstream of the nar operon (narCGHJIKT), which encodes the nitrate reductase (NR) and nitrate/nitrite transporters. In contrast, the nature of the nitrate sensory system is not known. Here, we analyse the putative nitrate‐sensing role of the bicistronic drp operon (drpAB) present downstream of the nar operon in most denitrifying Thermus spp. Expression of drp was found to depend on the master regulator DnrT, whereas the absence of DrpA or DrpB increased the expression of both DnrS and DnrT and, concomitantly, of the NR. Absence of both proteins made expression from the dnr and nar operons independent of nitrate. Polyclonal antisera allowed us to identify DrpA as a periplasmic protein and DrpB as a membrane protein, with capacity to bind to the cytoplasmic membrane. Here, we propose a role for DrpA/DrpB as nitrate sensors during denitrification.
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| 29. |
- Chautard, Hélène, et al.
(författare)
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An activity-independent selection system of thermostable protein variants
- 2007
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 4:11, s. 919-921
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Tidskriftsartikel (refereegranskat)abstract
- We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.
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| 30. |
- Gómez-Campo, Cristina L., et al.
(författare)
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Differential requirement for RecFOR pathway components in Thermus thermophilus
- 2024
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Ingår i: Environmental Microbiology Reports. - : John Wiley & Sons. - 1758-2229. ; 16:3
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Tidskriftsartikel (refereegranskat)abstract
- Recombinational repair is an important mechanism that allows DNA replication to overcome damaged templates, so the DNA is duplicated timely and correctly. The RecFOR pathway is one of the common ways to load RecA, while the RuvABC complex operates in the resolution of DNA intermediates. We have generated deletions of recO, recR and ruvB genes in Thermus thermophilus, while a recF null mutant could not be obtained. The recO deletion was in all cases accompanied by spontaneous loss of function mutations in addA or addB genes, which encode a helicase-exonuclease also key for recombination. The mutants were moderately affected in viability and chromosome segregation. When we generated these mutations in a Δppol/addAB strain, we observed that the transformation efficiency was maintained at the typical level of Δppol/addAB, which is 100-fold higher than that of the wild type. Most mutants showed increased filamentation phenotypes, especially ruvB, which also had DNA repair defects. These results suggest that in T. thermophilus (i) the components of the RecFOR pathway have differential roles, (ii) there is an epistatic relationship of the AddAB complex over the RecFOR pathway and (iii) that neither of the two pathways or their combination is strictly required for viability although they are necessary for normal DNA repair and chromosome segregation.
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| 31. |
- Laptenko, Oleg, et al.
(författare)
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pH-dependent conformational switch activates the inhibitor of transcription elongation
- 2006
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 25:10, s. 2131-2141
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Tidskriftsartikel (refereegranskat)abstract
- Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N-terminal coiled-coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C-terminal domain (CTD). We show that depending on pH, Gfh1-CTD exists in two alternative orientations. At pH above 7, it assumes an inactive 'flipped' orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1-CTD switches to an active 'Gre-like' orientation, which enables Gfh1 to bind to and inhibit RNAP.
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| 32. |
- Moreno, Renata, et al.
(författare)
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Development of a gene expression vector for Thermus thermophilus based on the promoter of the respiratory nitrate reductase
- 2003
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Ingår i: Plasmid. - 0147-619X .- 1095-9890. ; 49:1, s. 2-8
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Tidskriftsartikel (refereegranskat)abstract
- A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA region containing the promoter (Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of Thermus thermophilus HB8. Two genes, encoding a thermophilic beta-galactosidase and an alkaline phosphatase were cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively. The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T. thermophilus HB27 to which the gene cluster for nitrate respiration was transferred by conjugation. Overexpressions in the range of approximately 200-fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to approximately 20-fold, with respect to their intrinsic respective activities.
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| 33. |
- Perales, Celia, et al.
(författare)
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Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein
- 2003
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 31:22, s. 6473-6480
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial single-stranded DNA-binding proteins (SSBs) are required for DNA replication and repair. We have over-expressed and purified the native form and two His-tagged fusions of the SSB from Thermus thermophilus (TthSSB). The three proteins were found as dimers in solution. They bound in vitro to single-stranded DNA specifically over a temperature range of 4-80 degrees C, and the wild-type protein could withstand incubation at 94 degrees C for 2 min. Addition of TthSSB to PCR halved the elongation time required for the DNA polymerases of T.thermophilus (Tth) and Pyrococcus furiosus (Pfu) to synthesise DNA fragments in PCRs. The presence of TthSSB increased the fidelity of the proof- reading-free DNA polymerase of T.thermophilus. TthSSB was also able to bind single-stranded RNA, allowing a dramatic enhancement of the reverse transcription activity of its cognate Tth DNA polymerase during cDNA synthesis.
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| 34. |
- Zafra, Olga, et al.
(författare)
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A cytochrome c encoded by the nar operon is required for the synthesis of active respiratory nitrate reductase in Thermus thermophilus
- 2002
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 523:1-3, s. 99-102
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Tidskriftsartikel (refereegranskat)abstract
- A cytochrome c (NarC) is encoded as the first gene of the operon for nitrate respiration in Thermus thermophilus. NarC is required for anaerobic growth and for the synthesis of active nitrate reductase (NR). The alpha and delta subunits (NarG, NarJ) of the NR were constitutively expressed in narC::kat mutants, but NarG appeared in the soluble fraction instead of associated with the membranes. Our data demonstrate for NarC an essential role in the synthesis of active enzyme and for the attachment to the membrane of the respiratory NR from T. thermophilus.
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| 35. |
- Zafra, Olga, et al.
(författare)
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Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
- 2005
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 187:12, s. 3990-3996
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Tidskriftsartikel (refereegranskat)abstract
- The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.
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