1,720,983 research outputs found
Sugar-inducible promoters for manipulation of core metabolic pathways in the thermophilic acetogen Thermoanaerobacter kivui
No full textABSTRACT Acetogenic bacteria form an essential part of the global carbon cycle and show promise for green chemicals production thanks to their energy-efficient carbon fixation via the Wood-Ljungdahl pathway. Energy conservation in acetogens is dependent on one of two ion-pumping membrane complexes, Rnf or Ech. While the energetics of Rnf acetogens are relatively well understood, Ech-dependent metabolism remains unclear. Like many Ech acetogens, Thermoanaerobacter kivui (T opt = 66°C) encodes two separate Ech complexes and is genetically tractable, allowing it to serve as a model of Ech-dependent metabolism. While the role of the Ech2 complex has been determined biochemically and by gene knockout, Ech1 has eluded biochemical characterization, and knockout attempts have been unsuccessful, suggesting it is essential. This work identifies two sugar-inducible promoters with very low expression under non-inducing conditions and uses them to replace the native Ech1 promoter. The resulting strains exhibit reduced growth in non-inducing “knockdown” conditions, confirming the importance of Ech1 for metabolism. Additionally, knockdown of the gene encoding formyl-THF synthetase (Fhs)—which consumes the metabolic intermediate formate—resulted in significantly more formate accumulating in the culture medium. Together, these results confirm that the characterized promoters can be used to elucidate the function of essential genes not amenable to knockout. IMPORTANCE Acetogenic bacteria are industrially relevant for conversion of synthesis gas (syngas, a mixture of hydrogen, H 2 ; carbon monoxide, CO; and carbon dioxide, CO 2 ) to products. Thermophilic bacteria have long been of interest as industrial strains due to their resistance to contamination. Thermoanaerobacter kivui is among the most thermophilic acetogens (T opt = 66°C), with very short doubling times on H 2 +CO 2 (< 2 h), and is therefore a prime candidate for synthesis gas conversion at high temperatures. Here, we expanded T. kivui ’s genetic toolkit by establishing a reporter gene operating at its temperature optimum, which was used to characterize sugar-inducible promoters. The identified promoters were used to control and engineer the metabolism of T. kivui , and may now be applied to elucidate remaining mysteries about the energetics of acetogenic metabolism.Acetogenic bacteria are industrially relevant for conversion of synthesis gas (syngas, a mixture of hydrogen, H 2 ; carbon monoxide, CO; and carbon dioxide, CO 2 ) to products. Thermophilic bacteria have long been of interest as industrial strains due to their resistance to contamination. Thermoanaerobacter kivui is among the most thermophilic acetogens (T opt = 66°C), with very short doubling times on H 2 +CO 2 (< 2 h), and is therefore a prime candidate for synthesis gas conversion at high temperatures. Here, we expanded T. kivui ’s genetic toolkit by establishing a reporter gene operating at its temperature optimum, which was used to characterize sugar-inducible promoters. The identified promoters were used to control and engineer the metabolism of T. kivui , and may now be applied to elucidate remaining mysteries about the energetics of acetogenic metabolism.ABSTRACT Acetogenic bacteria form an essential part of the global carbon cycle and show promise for green chemicals production thanks to their energy-efficient carbon fixation via the Wood-Ljungdahl pathway. Energy conservation in acetogens is dependent on one of two ion-pumping membrane complexes, Rnf or Ech. While the energetics of Rnf acetogens are relatively well understood, Ech-dependent metabolism remains unclear. Like many Ech acetogens, Thermoanaerobacter kivui (T opt = 66°C) encodes two separate Ech complexes and is genetically tractable, allowing it to serve as a model of Ech-dependent metabolism. While the role of the Ech2 complex has been determined biochemically and by gene knockout, Ech1 has eluded biochemical characterization, and knockout attempts have been unsuccessful, suggesting it is essential. This work identifies two sugar-inducible promoters with very low expression under non-inducing conditions and uses them to replace the native Ech1 promoter. The resulting strains exhibit reduced growth in non-inducing “knockdown” conditions, confirming the importance of Ech1 for metabolism. Additionally, knockdown of the gene encoding formyl-THF synthetase (Fhs)—which consumes the metabolic intermediate formate—resulted in significantly more formate accumulating in the culture medium. Together, these results confirm that the characterized promoters can be used to elucidate the function of essential genes not amenable to knockout. IMPORTANCE Acetogenic bacteria are industrially relevant for conversion of synthesis gas (syngas, a mixture of hydrogen, H 2 ; carbon monoxide, CO; and carbon dioxide, CO 2 ) to products. Thermophilic bacteria have long been of interest as industrial strains due to their resistance to contamination. Thermoanaerobacter kivui is among the most thermophilic acetogens (T opt = 66°C), with very short doubling times on H 2 +CO 2 (< 2 h), and is therefore a prime candidate for synthesis gas conversion at high temperatures. Here, we expanded T. kivui ’s genetic toolkit by establishing a reporter gene operating at its temperature optimum, which was used to characterize sugar-inducible promoters. The identified promoters were used to control and engineer the metabolism of T. kivui , and may now be applied to elucidate remaining mysteries about the energetics of acetogenic metabolism.Acetogenic bacteria are industrially relevant for conversion of synthesis gas (syngas, a mixture of hydrogen, H 2 ; carbon monoxide, CO; and carbon dioxide, CO 2 ) to products. Thermophilic bacteria have long been of interest as industrial strains due to their resistance to contamination. Thermoanaerobacter kivui is among the most thermophilic acetogens (T opt = 66°C), with very short doubling times on H 2 +CO 2 (< 2 h), and is therefore a prime candidate for synthesis gas conversion at high temperatures. Here, we expanded T. kivui ’s genetic toolkit by establishing a reporter gene operating at its temperature optimum, which was used to characterize sugar-inducible promoters. The identified promoters were used to control and engineer the metabolism of T. kivui , and may now be applied to elucidate remaining mysteries about the energetics of acetogenic metabolism.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Bundesministerium für Bildung und Forschung http://dx.doi.org/10.13039/501100002347Bundesministerium für Bildung und Forschung http://dx.doi.org/10.13039/50110000234
Adaptive laboratory evolution of a thermophile toward a reduced growth temperature optimum
No full textThermophily is an ancient trait among microorganisms. The molecular principles to sustain high temperatures, however, are often described as adaptations , somewhat implying that they evolved from a non-thermophilic background and that thermophiles, i.e., organisms with growth temperature optima (T OPT ) above 45°C, evolved from mesophilic organisms (T OPT 25–45°C). On the contrary, it has also been argued that LUCA, the last universal common ancestor of Bacteria and Archaea , may have been a thermophile, and mesophily is the derived trait. In this study, we took an experimental approach toward the evolution of a mesophile from a thermophile. We selected the acetogenic bacterium T. kivui (T OPT 66°C) since acetogenesis is considered ancient physiology and cultivated it at suboptimal low temperatures. We found that the lowest possible growth temperature (T MIN ) under the chosen conditions was 39°C. The bacterium was subsequently subjected to adaptive laboratory evolution (ALE) by serial transfer at 45°C. Interestingly, after 67 transfers (approximately 180 generations), the adapted strain Adpt45_67 did not grow better at 45°C, but a shift in the T OPT to 60°C was observed. Growth at 45°C was accompanied by a change in the morphology as shorter, thicker cells were observed that partially occurred in chains. While the proportion of short-chain fatty acids increased at 50°C vs. 66°C in both strains, Adpt45_67 also showed a significantly increased proportion of plasmalogens. The genome analysis revealed 67 SNPs compared to the type strain, among these mutations in transcriptional regulators and in the cAMP binding protein. Ultimately, the molecular basis of the adaptation of T. kivui to a lower T OPT remains to be elucidated. The observed change in phenotype is the first experimental step toward the evolution of thermophiles growing at colder temperatures and toward a better understanding of the cold adaptation of thermophiles on early Earth.Volkswagen Foundation http://dx.doi.org/10.13039/501100001663Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/50110000165
The energy-converting hydrogenase Ech2 is important for the growth of the thermophilic acetogen Thermoanaerobacter kivui on ferredoxin-dependent substrates
No full textABSTRACTThermoanaerobacter kivui is the thermophilic acetogenic bacterium with the highest temperature optimum (66°C) and with high growth rates on hydrogen (H2) plus carbon dioxide (CO2). The bioenergetic model suggests that its redox and energy metabolism depends on energy-converting hydrogenases (Ech). Its genome encodes two Echs, Ech1 and Ech2, as sole coupling sites for energy conservation during growth on H2 + CO2. During growth on other substrates, its redox activity, the (proton-gradient-coupled) oxidation of H2 may be essential to provide reduced ferredoxin (Fd) to the cell. While Ech activity has been demonstrated biochemically, the physiological function of both Ech’s is unclear. Toward that, we deleted the complete gene cluster encoding Ech2. Surprisingly, the ech2 mutant grew as fast as the wild type on sugar substrates and H2 + CO2. Hence, Ech1 may be the essential enzyme for energy conservation, and either Ech1 or another enzyme may substitute for H2-dependent Fd reduction during growth on sugar substrates, putatively the H2-dependent CO2 reductase (HDCR). Growth on pyruvate and CO, substrates that are oxidized by Fd-dependent enzymes, was significantly impaired, but to a different extent. While ∆ech2 grew well on pyruvate after four transfers, ∆ech2 did not adapt to CO. Cell suspensions of ∆ech2 converted pyruvate to acetate, but no acetate was produced from CO. We analyzed the genome of five T. kivui strains adapted to CO. Strikingly, all strains carried mutations in the hycB3 subunit of HDCR. These mutations are obviously essential for the growth on CO but may inhibit its ability to utilize Fd as substrate.IMPORTANCEAcetogens thrive by converting H2+CO2 to acetate. Under environmental conditions, this allows for only very little energy to be conserved (∆G′<–20 kJ mol−1). CO2 serves as a terminal electron acceptor in the ancient Wood-Ljungdahl pathway (WLP). Since the WLP is ATP neutral, energy conservation during growth on H2 + CO2 is dependent on the redox metabolism. Two types of acetogens can be distinguished, Rnf- and Ech-type. The function of both membrane-bound enzyme complexes is twofold—energy conversion and redox balancing. Ech couples the Fd-dependent reduction of protons to H2 to the formation of a proton gradient in the thermophilic bacterium Thermoanaerobacter kivui. This bacterium may be utilized in gas fermentation at high temperatures, due to very high conversion rates and the availability of genetic tools. The physiological function of an Ech hydrogenase in T. kivui was studied to contribute an understanding of its energy and redox metabolism, a prerequisite for future industrial applications
DNA uptake from a laboratory environment drives unexpected adaptation of a thermophile to a minor medium component
No full textDNA uptake is widespread among microorganisms and considered a strategy for rapid adaptation to new conditions. While both DNA uptake and adaptation are referred to in the context of natural environments, they are often studied in laboratories under defined conditions. For example, a strain of the thermophile Thermoanaerobacter kivui had been adapted to growth on high concentrations of carbon monoxide (CO). Unusual phenotypes of the CO-adapted strain prompted us to examine it more closely, revealing a horizontal gene transfer (HGT) event from another thermophile, Thermoanaerobacter sp. strain X514, being cultured in the same laboratory. The transferred genes conferred on T. kivui the ability to utilize trehalose, a trace component of the yeast-extract added to the media during CO-adaptation. This same HGT event simultaneously deleted a native operon for thiamine biosynthesis, which likely explains why the CO-adapted strain grows poorly without added vitamins. Attempts to replicate this HGT by providing T. kivui with genomic DNA from Thermoanaerobacter sp. strain X514 revealed that it is easily reproducible in the lab. This subtle form of “genome contamination” is difficult to detect, since the genome remains predominantly T. kivui, and no living cells from the original contamination remain. Unexpected HGT between two microorganisms as well as simultaneous adaptation to several conditions may occur often and unrecognized in laboratory environments, requiring caution and careful monitoring of phenotype and genotype of microorganisms that are naturally-competent for DNA uptake
Cultivation systems for anaerobic single and mixed cultures
No full textThe promising, yet barely investigated anaerobic strain Phocaeicola vulgatus (formerly Bacteroides vulgatus) plays a vital role for human gut health and effectively produces the industrially relevant acid succinate. Cultivating anaerobic bacteria is challenging, and a detailed understanding of P. vulgatus growth and metabolism is required to improve succinate production. Currently, P. vulgatus is mainly studied in mixed cultures, as it is its natural occurrence. In this work, axenic cultures were studied in microtiter plate, shake flask, and 2 L fermenter scales. First, the influence of initial pH, buffer concentration, osmolality, product inhibition was characterized. Second, the influence of different CO2 and O2 concentrations was tested. Third, the effect of initial glucose and NH4Cl concentration and different carbon and nitrogen sources on growth and organic acid production by P. vulgatus were examined. Cultivations were performed in an in-house built device for anaerobic online monitoring of fluorescence and scattered light (BioLector) in microtiter plate scale, a fermenter equipped with online gas monitoring and the in-house developed Respiratory Activity Monitoring System (RAMOS) in shake flask scale, which provides online monitoring of CO2, O2, and pressure under gassed conditions. HPLC analysis generated closed carbon balances in shake flask and fermenter scale, accounting for all produced acids. Total gas and CO2 transfer rates revealed that 65 % of produced gas was attributed to H2, while just 35 % was connected to CO2 production. A minimum buffer concentration of 50 mM MOPS and an initial pH of 7.3 were determined to reduce pH inhibition when cultivating P. vulgatus in a defined minimal medium and glucose as substrate. The initial addition of lactate showed an inhibitory effect, starting at a concentration of 1 g L-1. In contrast, the initial addition of acetate benefited organic acid production. A comparison between a pH-buffered and a pH-controlled 2-liter fermentation showed a switch in acid production to succinate under pH control. Thus, pH control increased succinate production from 0.05 g L-1 h-1 in the pH-uncontrolled shake flask to 0.12 g L-1 h-1 succinate in the 2-L fermenter. Changing the CO2 concentration in the gas supply revealed a CO2 optimum of 3.0 vol% for total organic acid production and 15.0 vol% for succinate production. It was demonstrated that the organic acid composition changed depending on the CO2 concentration. Furthermore, unrestricted growth of P. vulgatus up to an O2 concentration of 0.7 vol% in the gas supply was proven. The viability decreased rapidly at concentrations larger than or equal to 1.3 vol% O2. Results revealed that the highest succinate yields were reached with a standard media composition of 8 g L-1 initial glucose, low osmolalities, and 0.25 g L-1 NH4Cl. However, succinate yields were even higher when using maltose and lactose as carbon sources. The organic acid composition changed with changing carbon and nitrogen sources. Furthermore, a microtiter plate called Link-Plate, was developed in this work to co-cultivate two strains spatially separated by a membrane, making strain-resolved offline and online analysis possible. Online analysis was pursued by a BioLector and the in-house built micro(μ)-scale respiration activity monitoring system (µRAMOS). In the Link-Plate experiments, it was possible to determine the OTR and scattered light strain resolved in a co-cultivation experiment. However, there are still challenges to overcome in the manufacturing process
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Physiologische und molekularbiologische Charakterisierung der anaeroben Oxidation von Methan an einem mesophilen Flachwasserstandort.
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