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Objectives: The four-dimensional ultrasound (4D-US) enables imaging of the aortic segment and simultaneous determination of the wall expansion. The method shows a high spatial and temporal resolution, but its in vivo reliability is so far unknown for low-measure values. The present study determines the intraobserver repeatability and interobserver reproducibility of 4D-US in the atherosclerotic and non-atherosclerotic infrarenal aorta. Methods: In all, 22 patients with non-aneurysmal aorta were examined by an experienced examiner and a medical student. After registration of 4D images, both the examiners marked the aortic wall manually before the commercially implemented speckle tracking algorithm was applied. The cyclic changes of the aortic diameter and circumferential strain were determined with the help of custom-made software. The reliability of 4D-US was tested by the intraclass correlation coefficient (ICC). Results: The 4D-US measurements showed very good reliability for the maximum aortic diameter and the circumferential strain for all patients and for the non-atherosclerotic aortae (ICC >0.7), but low reliability for circumferential strain in calcified aortae (ICC = 0.29). The observer- and masking-related variances for both maximum diameter and circumferential strain were close to zero. Conclusions: Despite the low-measured values, the high spatial and temporal resolution of the 4D-US enables a reliable evaluation of cyclic diameter changes and circumferential strain in non-aneurysmal aortae independent from the observer experience but with some limitations for calcified aortae. The 4D-US opens up a new perspective with regard to noninvasive, in vivo assessment of kinematic properties of the vessel wall in the abdominal aorta.
Genetic engineering of Saccharomyces cerevisiae for improved cytosolic isobutanol biosynthesis
(2021)
The finite nature of fossil resources and the environmental problems caused by their excessive usage requires alternative approaches. The transformation from a fossil based economy to one based on renewable biomass is called a “bioeconomy”. To substitute fossil resources, various microorganisms have already been modified for the biosynthesis of valuable chemicals from biomass. However, the development of such efficient microorganisms at an industrial scale, remains a major challenge. The most prominent and robust microorganism for industrial production is the yeast Saccharomyces cerevisiae, which is known to produce ethanol that is used as renewable biofuel. However, S. cerevisiae is also naturally able to produce isobutanol in small amounts. Isobutanol is favoured as a biofuel compared to ethanol due to its higher octane number and lower hygroscopicity, which makes it more suitable for application in conventional combustion engines. In S. cerevisiae, the biosynthesis of isobutanol is permitted by the combination of mitochondrial valine synthesis (catalysed by Ilv2, Ilv5 and Ilv3) and its cytosolic degradation (catalysed by Aro10 and Adh2). The different compartmentalisation of the two pathways limit isobutanol biosynthesis. Thus, Brat et al. (2012) were able to increase the isobutanol yield up to 15 mg/gGlc by cytosolic re localisation of the enzymes Ilv2Δ54, Ilv5Δ48 and Ilv3Δ19 (cyt-ILV), with simultaneous deletion of ilv2. This corresponds to approximately 3.7% of the theoretical yield of 410 mg/gGlc, implying existing limitations in isobutanol biosynthesis, which have been investigated in this work.
For yet unknown reasons, isobutanol was only produced by S. cerevisiae in a valine free medium, according to Brat et al. (2012). This work shows that this can be attributed to the catalytic activity of Ilv2Δ54, which acted as growth inhibitor to S. cerevisiae. By this logic, a negative selection on the ILV2∆54 gene was exerted, which made the ilv2 deletion and simultaneous valine exclusion necessary to maintain the functional expression of toxic ILV2∆54. Furthermore, it was shown that valine exclusion is not mandatory due to the feedback regulation of Ilv2, permitted by Ilv6. Rather, increased isobutanol yield was observed when cytosolic Ilv6∆61 was expressed in the valine free medium, which is explained by the enhanced regulation of Ilv2Δ54 by Ilv6∆61 when BCAA are absent. Isobutanol biosynthesis is neither redox nor NAD(P)H co factor balanced. It was seen that co factor imbalance could be mitigated by the expression of an NADH oxidase (NOX), but not by expression of the NADH dependent ilvC6E6, since the latter showed low in vivo activity. Furthermore, it was seen that NAD(H) imbalance did already limit isobutanol biosynthesis, but the NADP(H) imbalance did not. Another limitation of cytosolic isobutanol biosynthesis is the secretion of the intermediate 2‑dihydroxyisovalerate, which then no longer is taken up by S. cerevisiae, causing a reduced isobutanol yield. This is attributed to insufficient Ilv3∆19 activity, due to poor iron sulphur cluster apo protein maturation. Therefore, it was aimed to replace Ilv3∆19 by heterologous dihydroxyacid dehydratases. Even though some of the enzymes were functionally expressed, none showed better in vivo activity than Ilv3∆19. Therefore, the Ilv3∆19 apo protein maturation was improved. This was achieved by the genomic deletion of fra2 or pim1 as well as by the cytosolic expression of Grx5∆29.
In addition to the isobutanol pathway, S. cerevisiae was optimised for isobutanol biosynthesis by rational and evolutionary engineering. For this purpose, the genes which are necessary for isobutanol production were integrated into the ilv2 locus, and the resulting strain was evolved in a medium containing the toxic amino acid analogue norvaline. Evolved single colonies were isolated, which presented improved growth and increased isobutanol yields (0.59 mg/gGlc) in a valine free medium, as compared to the initial strain. This is explained by a gene dosage effect which occurred during the evolutionary engineering experiment. In collaboration with Dr. Wess, the genes ilv2, bdh1/2, leu4/9, ecm31, ilv1, adh1, gpd1/2 and ald6 were cumulatively deleted in CEN.PK113 7D to block competing metabolic pathways. The resulting strain JWY23 achieved isobutanol yields up to 67.3 mg/gGlc, when expressing the cyt ILV enzymes from a multi copy vector. The most promising approaches of this work, namely the deletion of fra2 and the expression of Grx5∆29, Ilv6∆61, and NOX, were confirmed in this JWY23 strain. The highest isobutanol yield from this work was observed at 72 mg/gGlc for Ilv6∆61 and cyt ILV enzymes expressing JWY23, which corresponds to 17.6% of the theoretical isobutanol yield.
Isobutyric acid (IBA) is a by product of isobutanol biosynthesis, but it is also considered a valuable platform chemical. Therefore, the approaches that improved isobutanol biosynthesis were applied to the biosynthesis of IBA in S. cerevisiae. The highest IBA yield of 9.8 mg/gGlc was observed in a valine free medium by expression of cyt ILV enzymes, NOX and Ald6 in JWY04 (CEN.PK113 7D Δilv2; Δbdh1; Δbdh2; Δleu4; Δleu9; Δecm31; Δilv1). This corresponded to an 8.9 fold increase compared with the control and is, to our best knowledge, the highest IBA yield reported to date for S. cerevisiae.
Die Studien im Rahmen dieser Arbeit wurden am Modellorganismus Anabaena sp. PCC 7120 (Anabaena) durchgeführt, einem filamentösen Süßwasser-Cyanobakterium. Cyanobakterien sind photosynthetische, Gram-negative Organismen. Sie besitzen eine das Zytosol begrenzende Plasmamembran und eine Äußere Membran. TonB-abhängige Transporter (TBDTs) und Porine der Äußeren Membran bewerkstelligen und regulieren die Aufnahme von Nährstoffen. Typischerweise wenig abundante Substrate für den TBDT-vermittelten, aktiven Transport sind beispielsweise eisenhaltige Siderophore oder VitaminB12. Kleinere gelöste und abundante Stoffe wie Salze oder andere Ionen gelangen hingegen passiv durch Porine in das Periplasma.
In Anabaena wurden neun putative Porine identifiziert. Sieben hiervon wiesen eine porinspezifische Domänenstruktur auf (Alr0834, Alr2231, All4499, Alr4550, Alr4741, All5191 und All7614), und wurden im Rahmen dieser Arbeit näher betrachtet. Die Expression dieser sieben Gene wurde vergleichend untersucht, nachdem der Wildtyp in Standardmedium oder in Medium indem jeweils Mangan, Eisen, Kupfer oder Zink fehlte angezogen wurde. Außerdem wurde das Wachstum der einzelnen Porinmutanten im Vergleich zum Wildtyp auf Festmedium mit hohen Konzentrationen von Salzen, Antibiotika oder anderen Stoffen analysiert. Hierbei konnten den einzelnen Mutanten teilweise spezifische phänotypische Eigenschaften zugeschrieben werden. Zusammengefasst kann anhand der Analysenergebnisse vermutet werden, dass Alr4550 eine besondere Rolle in der Wahrung der Zellhüllenstabilität oder -integrität spielt, wohingegen das Fehlen von Alr5191 auf unbekannte Weise die Fixierung von Stickstoff zu erschweren scheint. Die alr2231-Mutante zeigte eine Resistenz gegenüber hohen Zinkkonzentrationen, was die Vermutung zulässt, dass Zink ein Substrat von Alr2231 darstellt. Für weitere Porine kann ebenfalls ein Zusammenhang zum Transport von Kupfer oder Mangan vermutet werden.
Neben Porinen wurden ebenfalls TonB-ähnliche Proteine in Anabaena untersucht. TonB ist ein plasmamembranständiges Protein, das in Komplex mit ExbB und ExbD die Energie für Transportprozesse über die Äußere Membran bereitstellt. Hierfür bindet TonB C-terminal an TBDTs und induziert dort Strukturänderungen, welche den Substratimport ins Periplasma ermöglichen. Als Energiequelle wird der Protonengradient genutzt, der über die Plasmamembran besteht. In Anabaena wurden vier putative TonB Proteine identifiziert, die sich jeweils in Länge und Domänenstruktur unterscheiden. Im Rahmen dieser Arbeit konnte durch Substrattransport-Experimente und Wachstumsanalysen gezeigt werden, dass TonB3 an der Aufnahme zweier Siderophore (Schizokinen und dem Xenosiderophor Ferrichrom) beteiligt ist, da die entsprechende Mutante sich als unfähig erwies diese zu als Eisenquelle nutzbar zu machen. Daneben wies TonB3 weitere Merkmale auf, die auch TonB-Proteinen anderer Organismen zugeschrieben wurden (Wachstumsdefizit der Mutante unter Eisenmangel, eisenabhängiges Expressionsprofil). Interessanterweise zeigte sich, dass das Siderophor Ferrichrom ebenfalls nicht als Eisenquelle für die tonB4-Mutante zur Verfügung stand, was zum Beispiel auf eine Beteiligung von TonB4 an dessen Transport hinweisen könnte.
TonB1, welches sich durch ein inkomplettes TBDT-Interaktionsmotiv auszeichnet, und TonB2 konnte keine Beteiligung am Siderophoretransport zugeschrieben werden, jedoch zeigten Mutanten der einzelnen Gene spezifische phänotypische Eigenschaften. Die tonB1-Mutante stach hervor durch ein vergleichsweise stark verzögertes Wachstum unter diazotrophen Bedingungen. Es konnte gezeigt werden, dass sowohl die Nitrogenaseaktivität als auch die expression vermindert war im tonB1-Mutantenstamm. Außerdem zeigten die Heterozysten dieser Mutante, die auf die Stickstoffixierung spezialisierten Zellen, eine abnormale Morphologie. Da die Expression von tonB1 jedoch nach dem Überführen von Wildypzellen in stickstoffreies Medium nicht erhöht war, kann eine direkte Beteiligung von TonB1 an der Heterozystendifferenzierung als unwahrscheinlich betrachtet werden. Die Zelleinschnürungen zwischen Heterozysten und vegetativen Zellen waren in I-tonB1 weniger ausgeprägt als im Wildtyp, was durch eine Anfärbung der Zellwand mit einem Fluoreszenzmarker gezeigt werden konnte. Ebenfalls konnte anhand des fluoreszierenden Markers Calcein gezeigt werden, dass die molekulare Diffusionsgeschwindigkeit zwischen Heterozysten und vegetativen Zellen, und auch zwischen zwei benachbarten vegetativen Zellen, in der tonB1-Mutante erhöht ist. Deswegen kann hier vermutlich vermehrt die Nitrogenase schädigender Sauerstoff in Heterozysten eindringen. Die aufgezählten Ergebnisse deuten auf eine Funktion von SjdR im Aufbau der Septumsstrukturen hin, beispielsweise durch Regulation der Peptidoglykansynthese oder -verteilung, weswegen TonB1 umbenannt wurde in SjdR (Septal junction disc regulator).
Die Untersuchung der tonB2-Mutante zeigte bei dieser eine veränderte Pigmentierung, eine vermehrte Lipopolysaccharidproduktion und Filamentaggregation sowie eine erhöhte Resistenz gegenüber bestimmten Antibiotika oder Detergenzien. Letzteres könnte auf die ebenfalls in der tonB2-Mutante beobachtete verringerte Porinexpression zurückgeführt werden. Es wurde außerdem eine vermehrte Anreicherung von Kupfer und Molybdän in der Mutante gemessen, was ein Grund für die Veränderte Pigmentierung sein könnte und ebenfalls die Porinexpression beeinflussen könnte. Insgesamt scheint sich das Fehlen von TonB2 auf die Integrität der Äußeren Membran auszuwirken. Daher kann für TonB2, eine Funktion in Anlehnung an das Tol-system vermutet werden.
Climate change causes increased tree mortality leading to canopy loss and thus sun-exposed forest floors. Sun exposure creates extreme temperatures and radiation, with potentially more drastic effects on forest organisms than the current increase in mean temperature. Such conditions might potentially negatively affect the maturation of mushrooms of forest fungi. A failure of reaching maturation would mean no sexual spore release and, thus, entail a loss of genetic diversity. However, we currently have a limited understanding of the quality and quantity of mushroom-specific molecular responses caused by sun exposure. Thus, to understand the short-term responses toward enhanced sun exposure, we exposed mushrooms of the wood-inhabiting forest species Lentinula edodes, while still attached to their mycelium and substrate, to artificial solar light (ca. 30°C and 100,000 lux) for 5, 30, and 60 min. We found significant differentially expressed genes at 30 and 60 min. Eukaryotic Orthologous Groups (KOG) class enrichment pointed to defense mechanisms. The 20 most significant differentially expressed genes showed the expression of heat-shock proteins, an important family of proteins under heat stress. Although preliminary, our results suggest mushroom-specific molecular responses to tolerate enhanced sun exposure as expected under climate change. Whether mushroom-specific molecular responses are able to maintain fungal fitness under opening forest canopies remains to be tested.
1. Plant-fungal interactions are important for plant community assembly, but quantifying these relationships remains challenging. High throughput sequencing of fungal communities allows us to identify plant-fungal associations at a high level of resolution, but often fails to provide information on taxonomic and functional assignment of fungi. 2. We transplanted seeds of Pinus cembra across an elevational gradient (1850–2250 m a.s.l.) and identified environmental factors and known fungal associates important for seedling establishment and survival. We then applied null model tests to identify taxonomically unassigned fungi associated with pine recruitment. 3. Early seedling establishment was determined by abiotic environmental factors, while seedling survival was predominantly affected by biotic environmental factors (i.e., the abundance of a fungal pathogen known from literature and the distance to adult trees). Null model tests identified known mycorrhizal partners and a large number of unknown operational taxonomic units (OTUs) associated with seedling survival, including saprotrophic and pathogenic species. These results highlight that unknown fungal OTUs, which are usually discarded from analyses, could play a crucial role for plant survival. 4. Synthesis. We conclude that high throughput metabarcoding paired with null model tests, is a valuable approach for identifying hidden plant-fungal associations within large and complex DNA metabarcoding datasets. Such an approach can be an important tool in illuminating the black box of plant-microbe interactions, and thus understanding ecosystem dynamics.
The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2 + CO2, formate, pyruvate, lactate or alanine. In the absence of Na+, the Wood–Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities which significantly contributes to its persistence in hospital environments. The vast majority of bacteria are able to prevent loss of cellular water by amassing osmoactive compatible solutes or their precursors into the cytoplasm. One such precursor of an osmoprotectant is choline that is taken up from the environment and oxidized to the compatible solute glycine betaine. Here, we report the identification of the osmotic stress operon betIBA in A. baumannii. This operon encodes the choline oxidation pathway important for the production of the solute glycine betaine. The salt-sensitive phenotype of a betA deletion strain could not be rescued by addition of choline, which is consistent with the role of BetA in choline oxidation. We found that BetA is a choline dehydrogenase but also mediates in vitro the oxidation of glycine betaine aldehyde to glycine betaine. BetA was found to be associated with the membrane and to contain a flavin, indicative for BetA donating electrons into the respiratory chain. The choline dehydrogenase activity was not salt dependent but was stimulated by the compatible solute glutamate.
An exploration of the relationship between recruitment communication and foraging in stingless bees
(2021)
Social information is widely used in the animal kingdom and can be highly adaptive. In social insects, foragers can use social information to find food, avoid danger, or choose a new nest site. Copying others allows individuals to obtain information without having to sample the environment. When foragers communicate information they will often only advertise high-quality food sources, thereby filtering out less adaptive information. Stingless bees, a large pantropical group of highly eusocial bees, face intense inter- and intra-specific competition for limited resources, yet display disparate foraging strategies. Within the same environment there are species that communicate the location of food resources to nest-mates and species that do not. Our current understanding of why some species communicate foraging sites while others do not is limited. Studying freely foraging colonies of several co-existing stingless bee species in Brazil, we investigated if recruitment to specific food locations is linked to 1) the sugar content of forage, 2) the duration of foraging trips, and 3) the variation in activity of a colony from 1 day to another and the variation in activity in a species over a day. We found that, contrary to our expectations, species with recruitment communication did not return with higher quality forage than species that do not recruit nestmates. Furthermore, foragers from recruiting species did not have shorter foraging trip durations than those from weakly recruiting species. Given the intense inter- and intraspecific competition for resources in these environments, it may be that recruiting species favor food resources that can be monopolized by the colony rather than food sources that offer high-quality rewards.
Obligate endoparasitic oomycetes are known to ubiquitously occur in marine and freshwater diatoms, but their diversity is still largely unexplored. Many of these parasitoids are members of the early-diverging oomycete lineages (Miracula, Diatomophthora), others are within the Leptomitales of the Saprolegniomycetes (Ectrogella, Lagenisma) and some have been described in the Peronosporomycetes (Aphanomycopsis, Lagenidium). Even though some species have been recently described and two new genera were introduced (Miracula and Diatomophthora), the phylogeny and taxonomy of most of these organisms remain unresolved. This is contrasted by the high number of sequences from unclassified species, as recently revealed from environmental sequencing, suggesting the presence of several undiscovered species. In this study, a new species of Miracula is reported from a marine centric diatom (Minidiscus sp.) isolated from Skagaströnd harbor in Northwest Iceland. The morphology and life cycle traits of this novel oomycete parasite are described herein, and its taxonomic placement within the genus Miracula is confirmed by molecular phylogeny. As it cannot be assigned to any previously described species, it is introduced as Miracula islandica in this study. The genus Miracula thus contains three described holocarpic species (M. helgolandica, M. islandica, M. moenusica) to which likely additional species will need to be added in the future, considering the presence of several lineages known only from environmental sequencing that clustered within the Miracula clade.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Acinetobacter baumannii is an opportunistic pathogen, which has become a rising threat in healthcare facilities worldwide due to increasing antibiotic resistances and optimal adaptation to clinical environments and the human host. We reported in a former publication on the identification of three phopholipases of the phospholipase D (PLD) superfamily in A. baumannii ATCC 19606T acting in concerted manner as virulence factors in Galleria mellonella infection and lung epithelial cell invasion. This study focussed on the function of the three PLDs. A Δpld1-3 mutant was defect in biosynthesis of the phospholipids cardiolipin (CL) and monolysocardiolipin (MLCL), whereas the deletion of pld2 and pld3 abolished the production of MLCL. Complementation of the Δpld1-3 mutant with pld1 restored CL biosynthesis demonstrating that the PLD1 is implicated in CL biosynthesis. Complementation of the Δpld1-3 mutant with either pld2 or pld3 restored MLCL and CL production leading to the conclusion that PLD2 and PLD3 are implicated in CL and MLCL production. Mutant studies revealed that two catalytic motifs are essential for the PLD3-mediated biosynthesis of CL and MLCL. The Δpld1-3 mutant exhibited a decreased colistin and polymyxin B resistance indicating a role of CL in cationic antimicrobial peptides (CAMPs) resistance.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K+, in osmostress response. Growth of A. baumannii was K+-dependent and the K+-dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K+ and K+ accumulation increased with the salinity of the medium. K+ uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K+ concentration: mannitol and glutamate concentrations increased with increasing K+ concentrations whereas trehalose was highest at low K+. After osmotic upshock, cells first accumulated K+ followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K+ and glutamate. These experiments demonstrate K+ uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K+ and different organic solutes.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities and therefore its adaptation mechanism to osmotic stress. Here we report on the identification and characterization of five different secondary active compatible solute transporters, belonging to the betaine-choline-carnitine transporter (BCCT) family. Our studies revealed two choline-specific and three glycine betaine-specific BCCTs. Activity of the BCCTs was differentially dependent to the osmolality: one choline and one betaine transporter were osmostress-independent. Addition of choline to resting cells of Acinetobacter grown in the presence of the co-substrate choline or with phosphatidylcholine as sole carbon source led to ATP synthesis in the wild type but not in the BCCT quadruple mutant. This indicates that the BCCTs are essential to transport the energy substrate choline. The role of the different BCCTs in osmostress resistance and in metabolic adaptation of A. baumannii to the human host is discussed.
A1AO ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg−1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na+)-binding site. Indeed, ATP hydrolysis was strictly Na+-dependent. N,N′-dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+. Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD-4 for a common binding site. After incorporation of the A1AO ATP synthase into liposomes, ATP-dependent primary transport of 22Na+ as well as ΔµNa+-driven ATP synthesis could be demonstrated. The Na+ A1AO ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases.
Understanding hominin expansions requires the comprehension of movement processes at different scales. In many models of hominin expansion these processes are viewed as being determined by large-scale effects, such as changes in climate and vegetation spanning continents and thousands or even millions of years. However, these large-scale patterns of expansions also need to be considered as possibly resulting from the accumulation of small-scale decisions of individual hominins. Moving on a continental scale may for instance involve crossing a water barrier. We present a generalized agent-based model for simulating the crossing of a water barrier where the agents represent the hominin individuals. The model can be configured to represent a variety of movement modes across water. Here, we compare four different behavioral scenarios in conjunction with a set of water barrier configurations, in which agents move in water by either paddling, drifting, swimming or rafting. We introduce the crossing-success-rate (CSR) to quantify the performance in water crossing. Our study suggests that more focus should be directed towards the exploration of behavioral models for hominins, as directionality may be a more powerful factor for crossing a barrier than environmental opportunities alone. A prerequisite for this is to perceive the opposite shore. Furthermore, to provide a comprehensive understanding of hominin expansions, the CSR allows for the integration of results obtained from small-scale simulations into large-scale models for hominin expansion.
Young trees of deciduous Quercus robur and Q. petraea and evergreen Q. ilex were grown together in a competition lysimeter experiment to assess i) – whether the observed growth differences between evergreen Q. ilex and the deciduous Q. robur and Q. petraea on sandy soil in the field and ii) – whether the different natural distribution of Q. robur and Q. petraea could be attributed to physiological differences between the species under experimental drought stress (DS). Half of the plants were subjected to long-term DS in two consecutive years and monitored for physiological and growth parameters. In the first year, water withholding for more than three months did not lead to significant drought stress, probably because of a sufficient residual water volume in the lysimeter for the relatively small plants. However, in the second year, 2018, which was warmer, the bigger plants now competed for the residual water and clear drought stress symptoms developed for more than two months in all trees in the DS lysimeter basin. Growth was only moderately (and mostly not significantly) affected by the DS in the second year, except for a smaller total leaf area in DS Q. ilex as compared to DS Q. robur and Q. petraea and smaller root collar diameter in DS Q. ilex compared to DS Q. robur. Under DS, the deciduous species revealed significant decreases in ΔVIP, indicating a negative effect on electron transport through PS I. Pn, PIabs and water relations parameters (ΨPD and LWC) all decreased to various extents under DS in all three species, leading to clear separation of the deciduous from the evergreen species by PCA. However, PCA did not separate the two deciduous species from each other. It is concluded that longer root growth in the two deciduous species as compared to Q. ilex ameliorates DS effects in Q. robur and Q. petraea and may be the key to understand the better performance of deciduous oaks on sandy soil in the field.
The Southern Ocean (SO) continental shelf and deep sea are environments characterised by different benthic communities. Their structure and composition are driven and shaped by different variables: whilst on the continental shelf physical environmental variables are the main drivers shaping faunal abundance, structure and composition, the deep-sea fauna is most problably driven by biological variables such as predation and competition. Among shelf and deep-sea benthic communities, peracarids (e.g. amphipods and isopods) are one of the most dominant groups, showing high levels of abundance and diversity in both environments. Knowledge on their assemblage structure and composition in the SO remains limited, as well as the knowledge of the environmental variables that influence them. Therefore, the aim of our study was to investigate peracarid assemblages from the SO continental shelf and deep sea and to assess the main drivers shaping their assemblage structure along a wide bathymetric gradient (from 160 m to about 6000 m depth) and at a large geographic scale. We analysed the spatial distribution of 183,606 peracarids sampled using an epibenthic sledge (EBS) during nine different expeditions in the SO, covering a latitudinal range of 77° to 41° South. Depth was identified as the main driver shaping peracarid abundance pattern, their assemblage structure from the continental shelf (<1499 m) was dissimilar to that from the deep sea (>1500 m). Also, depth was differently correlated with different peracarid orders: while isopod abundances increased with depth, amphipods and mysids were negatively correlated; no correlation was found with cumaceans and tanaidaceans. The dissimilar peracarid assemblage structure between the SO continental shelf and the SO deep sea can be due to the assumption that there are different driving forces shaping benthic assemblages from these two environments (physical variables on the continental shelf, biological interactions in the deep sea). As a result, we also suggest that environmental changes due to climate change (e.g. temperature, ice coverage, productivity) would have different consequences depending on the bathymetric range considered.
Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, partial and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and their severe discomfort.
FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms—that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3′ region of FAD1 mRNA was analyzed by 3′RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3′UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3′UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3′UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.