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The original version of this Article contained errors where Table S5 and Table S6 were incorrectly cited. As the result, in the Methods section, under the subheading ‘Germline transformation, crossing setups and insertion junction sequencing’, “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S4).” now reads: “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S5).” And, under the subheading ‘Light sheet-based fluorescence microscopy’, “Metadata for the three datasets are provided in Table S5.” now reads: “Metadata for the three datasets are provided in Table S6.” In Data availability section, “Microscopy data can be accessed as described in Table S5.” now reads: “Microscopy data can be accessed as described in Table S6.” Additionally, in the Supplementary Information 8 file, the “Data Access” row was omitted in Table S6. The “Data Access” row now reads: Dataset (DS) DS0001 DS0002 DS0003 Dataset Access DOI: 10.5281/zenodo.4892363 DOI: 10.5281/zenodo.4892373 DOI: 10.5281/zenodo.4892381 The original Supplementary Information 8 file is provided below. Finally, the Supplementary Information 1 and 5 files published with this Article contained tracked changes, these have now been removed. The original Article and accompanying Supplementary Information files have been corrected.
Mosquito breeding sites are complex aquatic environments with wide microbial diversity and physicochemical parameters that can change over time during the development of immature insect stages. Changes in biotic and abiotic conditions in water can alter life-history traits of adult mosquitos but this area remains understudied. Here, using microbial genomic and metabolomics analyses, we explored the metabolites associated with Aedes aegypti breeding sites as well as the potential contribution of Klebsiella sp., symbiotic bacteria highly associated with mosquitoes. We sought to address whether breeding sites have a signature metabolic profile and understand the metabolite contribution of the bacteria in the aquatic niches where Ae. aegypti larvae develop. An analysis of 32 mosquito-associated bacterial genomes, including Klebsiella, allowed us to identify gene clusters involved in primary metabolic pathways. From them, we inferred metabolites that could impact larval development (e.g., spermidine), as well as influence the quality assessment of a breeding site by a gravid female (e.g., putrescine), if produced by bacteria in the water. We also detected significant variance in metabolite presence profiles between water samples representing a decoupled oviposition event (oviposition by single females and manually deposited eggs) versus a control where no mosquito interactions occurred (PERMANOVA: p < 0.05; R2 = 24.64% and R2 = 30.07%). Five Klebsiella metabolites were exclusively linked to water samples where oviposition and development occurred. These data suggest metabolomics can be applied to identify compounds potentially used by female Ae. aegypti to evaluate the quality of a breeding site. Elucidating the physiological mechanisms by which the females could integrate these sensory cues while ovipositing constitutes a growing field of interest, which could benefit from a more depurated list of candidate molecules.
Background: Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results: A fed-batch system with plant biomass-derived sugars D-xylose, L-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and L-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion: This work has identified two additional CAZyme inducers for T. aurantiacus, L-arabinose and cellobiose, along with D-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.
RATIONALE: RBPs (RNA-binding proteins) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP SRSF4 (serine/arginine-rich splicing factor 4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. OBJECTIVE: To investigate the role of SRSF4 in the heart. METHODS AND RESULTS: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long noncoding RNAs, including GAS5 (growth arrest-specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide- resolution cross-linking and immuno-precipitation. GAS5 is a repressor of the GR (glucocorticoid receptor) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. CONCLUSIONS: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in patients with Cushing syndrome.
Aim: The identification of the mechanisms determining spatial variation in biological diversity along elevational gradients is a central objective in ecology and biogeography. Here, we disentangle the direct and indirect effects of abiotic drivers (climatic conditions, and land use) and biotic drivers (vegetation structure and food resources) on functional diversity and composition of bird and bat assemblages along a tropical elevational gradient. Location: Southern slopes of Mt. Kilimanjaro, Tanzania, East Africa. Methods: We counted birds and recorded bat sonotypes on 58 plots distributed in near-natural and anthropogenically modified habitats from 700 to 4,600 m above sea level. For the recorded taxa, we compiled functional traits related to movement, foraging and body size from museum specimens and databases. Further, we recorded mean annual temperature, precipitation, vegetation complexity as well as the number of fruits, flowers, and insect biomass as measures of resource availability on each study site. Results: Using path analyses, we found similar responses of bird and bat functional diversity to the variation in abiotic and biotic drivers along the elevational gradient. In contrast, the functional composition of both taxa showed distinct responses to abiotic and biotic drivers. For both groups, direct temperature effects were most important, followed by resource availability, precipitation and vegetation complexity. Main Conclusions: Our findings indicate that physiological and metabolic constraints imposed by temperature and resource availability determine the functional diversity of bird and bat assemblages, whereas the composition of individual functional traits is driven by taxon-specific processes. Our study illustrates that distinct filtering mechanisms can result in similar patterns of functional diversity along broad environmental gradients. Such differences need to be taken into account when it comes to conserving the functional diversity of flying vertebrates on tropical mountains.
Toxicogenomic differentiation of functional responses to fipronil and imidacloprid in Daphnia magna
(2021)
Active substances of pesticides, biocides or pharmaceuticals can induce adverse side effects in the aquatic ecosystem, necessitating environmental hazard and risk assessment prior to substance registration. The freshwater crustacean Daphnia magna is a model organism for acute and chronic toxicity assessment representing aquatic invertebrates. However, standardized tests involving daphnia are restricted to the endpoints immobility and reproduction and thus provide only limited insights into the underlying modes-of-action. Here, we applied transcriptome profiling to a modified D. magna Acute Immobilization test to analyze and compare gene expression profiles induced by the GABA-gated chloride channel blocker fipronil and the nicotinic acetylcholine receptor (nAChR) agonist imidacloprid. Daphnids were expose to two low effect concentrations of each substance followed by RNA sequencing and functional classification of affected gene ontologies and pathways. For both insecticides, we observed a concentration-dependent increase in the number of differentially expressed genes, whose expression changes were highly significantly positively correlated when comparing both test concentrations. These gene expression fingerprints showed virtually no overlap between the test substances and they related well to previous data of diazepam and carbaryl, two substances targeting similar molecular key events. While, based on our results, fipronil predominantly interfered with molecular functions involved in ATPase-coupled transmembrane transport and transcription regulation, imidacloprid primarily affected oxidase and oxidoreductase activity. These findings provide evidence that systems biology approaches can be utilized to identify and differentiate modes-of-action of chemical stressors in D. magna as an invertebrate aquatic non-target organism. The mechanistic knowledge extracted from such data will in future contribute to the development of Adverse Outcome Pathways (AOPs) for read-across and prediction of population effects.
Recently, the potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and its potential transformation products 4-methyl-7-ethylaminocoumarin (C47T1) and 4-methyl-7-aminocoumarin (C47T2) were identified as novel environmental contaminants. We assessed for the first time the sources, distribution, and fate of these compounds in aquatic systems using the Holtemme River (Saxony-Anhalt, Germany), which is a hotspot for these contaminants. To this end, wastewater-treatment plant (WWTP) influent and effluent samples, surface water samples over 3 years, and the longitudinal profiles in water, sediment, and gammarids were analyzed. From the longitudinal profile of the river stretch, the WWTP of Silstedt was identified as the sole point source for these compounds in the River Holtemme, and exposure concentrations in the low micrograms per liter range could be recorded continuously over 3 years. Analysis of WWTP influent and effluent showed a transformation of approximately half of the C47 into C47T1 and C47T2 but no complete removal. A further attenuation of the three coumarins after discharge into the river could be largely attributed to dilution, while transformation was only approximately 20%, thus suggesting a significant persistence in aquatic systems. Experimentally derived partitioning coefficients between water and sediment organic carbon exceeded those predicted using the OPERA quantitative structure–activity relationship tools and polyparameter linear free-energy relationships by up to 93-fold, suggesting cation binding as a significant factor for their sorption behavior. Near-equilibrium conditions between water and sediment were not observed close to the emitting WWTP but farther downstream in the river. Experimental and predicted bioaccumulation factors for gammarids were closely matching, and the concentrations in field-sampled gammarids were close to steady state with exposure concentrations in the water phase of the river. Environ Toxicol Chem 2021;40:3078–3091. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
Background: The industrial production of various alcohols from organic carbon compounds may be performed at high rates and with a low risk of contamination using thermophilic microorganisms as whole-cell catalysts. Thermoanaerobacter species that thrive around 50–75 °C not only perform fermentation of sugars to alcohols, but some also utilize different organic acids as electron acceptors, reducing them to their corresponding alcohols. Results: We purified AdhE as the major NADH- and AdhB as the major NADPH-dependent alcohol dehydrogenase (ADH) from the cell extract of the organic acid-reducing Thermoanaerobacter sp. strain X514. Both enzymes were present in high amounts during growth on glucose with and without isobutyrate, had broad substrate spectra including different aldehydes, with high affinities (< 1 mM) for acetaldehyde and for NADH (AdhE) or NADPH (AdhB). Both enzymes were highly thermostable at the physiological temperature of alcohol production. In addition to AdhE and AdhB, we identified two abundant AdhA-type ADHs based on their genes, which were recombinantly produced and biochemically characterized. The other five ADHs encoded in the genome were only expressed at low levels. Conclusions: According to their biochemical and kinetic properties, AdhE and AdhB are most important for ethanol formation from sugar and reduction of organic acids to alcohols, while the role of the two AdhA-type enzymes is less clear. AdhE is the only abundant aldehyde dehydrogenase for the acetyl-CoA reduction to aldehydes, however, acid reduction may also proceed directly by aldehyde:ferredoxin oxidoreductase. The role of the latter in bio-alcohol formation from sugar and in organic acid reduction needs to be elucidated in future studies.
Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains (N/CDDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of “designer” NRPS from proteins that normally do not interact. The multiprotein “peptide-antimicrobial-Xenorhabdus” (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small CDD of PaxA cooperates with its preceding thiolation (T1) domain to bind the NDD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.
The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.