Aging of nanosized titanium dioxide modulates the effects of dietary copper exposure on Daphnia magna - an assessment over two generations.

Nanosized titanium dioxide (nTiO2) is widely used in products, warranting its discharge from various sources into surface water bodies. However, nTiO2 co-occurs in surface waters with other contaminants, such as metals. Studies with nTiO2 and metals have indicated that the presence of natural organic matter (NOM) can mitigate their toxicity to aquatic organisms. In addition, "aging" of nTiO2 can affect toxicity. However, it is a research challenge, particularly when addressing sublethal responses from dietary exposure over multiple generations. We, therefore exposed the alga Desmodesmus subspicatus to nTiO2 (at concentrations of 0.0, 0.6 and 3.0 mg nTiO2/L) in nutrient medium aged for 0 or 3 days with copper (Cu) at concentrations of 0 and 116 µg Cu/L and with NOM at concentrations equivalent to 0 and 8 mg total organic carbon (TOC) per litre. Subsequently, the exposed alga was fed to Daphnia magna for 23 days over two generations and survival, reproduction and body length were assessed as endpoints of toxicity. In parallel, Cu accumulation and depuration from D. magna were measured. The results indicate that the reproduction of D. magna was the most sensitive parameter in this study, being reduced by 30% (at both parental (F0) and filial (F1) generations) and 50% (at F0 but not F1) due to the dietary Cu exposure in combination with nTiO2 for 0 and 3 days aging, respectively. There was no relationship between the effects observed on reproduction and Cu body burden in D. magna. Moreover, D. magna from the F1 generation showed an adaptive response to Cu in the treatment with 3.0 mg nTiO2/L aged for 3 days, potentially due to epigenetic inheritance. Unexpectedly, the presence of NOM hardly changed the observed effects, pointing towards the function of algal exopolymeric substances or intracellular organic matter, rendering the NOM irrelevant. Ultimately, the results indicate that the transferability of the impacts observed during the F0 to the responses in the F1 generation is challenging due to opposite effect directions. Additional mechanistic studies are needed to unravel this inconsistency in the responses between generations and to support the development of reliable effect models.

Reduction of Pesticide Toxicity Under Field-Relevant Conditions? The Interaction of Titanium Dioxide Nanoparticles, Ultraviolet, and Natural Organic Matter.

In surface waters, the illumination of photoactive engineered nanomaterials (ENMs) with ultraviolet (UV) light triggers the formation of reactive intermediates, consequently altering the ecotoxicological potential of co-occurring organic micropollutants including pesticides due to catalytic degradation. Simultaneously, omnipresent natural organic matter (NOM) adsorbs onto ENM surfaces, altering the ENM surface properties. Also, NOM absorbs light, reducing the photo(cata)lytic transformation of pesticides. Interactions between these environmental factors impact 1) directly the ecotoxicity of photoactive ENMs, and 2) indirectly the degradation of pesticides. We assessed the impact of field-relevant UV radiation (up to 2.6 W UVA/m²), NOM (4 mg TOC/L), and photoactive ENM (nTiO2, 50 µg/L) on the acute toxicity of 6 pesticides in Daphnia magna. We selected azoxystrobin, dimethoate, malathion, parathion, permethrin, and pirimicarb because of their varying photo- and hydrolytic stabilities. Increasing UVA alone partially reduced pesticide toxicity, seemingly due to enhanced degradation. Even at 50 µg/L, nano-sized titanium dioxide (nTiO2) reduced but also increased pesticide toxicity (depending on the applied pesticide), which is attributable to 1) more efficient degradation and potentially 2) photocatalytically induced formation of toxic by-products. Natural organic matter 1) partially reduced pesticide toxicity, not evidently accompanied by enhanced pesticide degradation, but also 2) inhibited pesticide degradation, effectively increasing the pesticide toxicity. Predicting the ecotoxicological potential of pesticides based on their interaction with UV light or interaction with NOM was hardly possible, which was even more difficult in the presence of nTiO2. Environ Toxicol Chem 2020;39:2237-2246. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway in Bacillus subtilis for the production of pyridoxine.

Vitamin B6 is a designation for the vitamers pyridoxine, pyridoxal, pyridoxamine, and their respective 5'-phosphates. Pyridoxal 5'-phosphate, the biologically most-important vitamer, serves as a cofactor for many enzymes, mainly active in amino acid metabolism. While microorganisms and plants are capable of synthesizing vitamin B6, other organisms have to ingest it. The vitamer pyridoxine, which is used as a dietary supplement for animals and humans is commercially produced by chemical processes. The development of potentially more cost-effective and more sustainable fermentation processes for pyridoxine production is of interest for the biotech industry. We describe the generation and characterization of a Bacillus subtilis pyridoxine production strain overexpressing five genes of a non-native deoxyxylulose 5'-phosphate-dependent vitamin B6 pathway. The genes, derived from Escherichia coli and Sinorhizobium meliloti, were assembled to two expression cassettes and introduced into the B. subtilis chromosome. in vivo complementation assays revealed that the enzymes of this pathway were functionally expressed and active. The resulting strain produced 14mg/l pyridoxine in a small-scale production assay. By optimizing the growth conditions and co-feeding of 4-hydroxy-threonine and deoxyxylulose the productivity was increased to 54mg/l. Although relative protein quantification revealed bottlenecks in the heterologous pathway that remain to be eliminated, the final strain provides a promising basis to further enhance the production of pyridoxine using B. subtilis.

Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites.

In Bacillus subtilis, recent in vivo studies revealed that particular enzymes of the tricarboxylic acid cycle form complexes that allow an efficient transfer of metabolites. Remarkably, a complex of the malate dehydrogenase (Mdh) (EC 1.1.1.37) with isocitrate dehydrogenase (Icd) (EC 1.1.1.42) was identified, although both enzymes do not catalyze subsequent reactions. In the present study, the interactions between these enzymes were characterized in vitro by surface plasmon resonance in the absence and presence of their substrates and cofactors. These analyses revealed a weak but specific interaction between Mdh and Icd, which was specifically stimulated by a mixture of substrates and cofactors of Icd: isocitrate, NADP(+) and Mg(2+). Wild-type Icd converted these substrates too fast, preventing any valid quantitative analysis of the interaction with Mdh. Therefore, binding of the IcdS104P mutant to Mdh was quantified because the mutation reduced the enzymatic activity by 174-fold but did not affect the stimulatory effect of substrates and cofactors on Icd-Mdh complex formation. The analysis of the unstimulated Mdh-IcdS104P interaction revealed kinetic constants of k(a) = 2.0 ± 0.2 × 10(2) m(-1) ·s(-1) and k(d) = 1.0 ± 0.1 × 10(-3) ·s(-1) and a K(D) value of 5.0 ± 0.1 µm. Addition of isocitrate, NADP(+) and Mg(2+) stimulated the affinity of IcdS104P to Mdh by 33-fold (K(D) = 0.15 ± 0.01 µm, k(a) = 1.7 ± 0.7 × 10(3) m(-1) ·s(-1), k(d) = 2.6 ± 0.6 × 10(-4) ·s(-1)). Analyses of the enzymatic activities of wild-type Icd and Mdh showed that Icd activity doubles in the presence of Mdh, whereas Mdh activity was slightly reduced by Icd. In summary, these data indicate substrate control of complex formation in the tricarboxylic acid cycle metabolon assembly and maintenance of the α-ketoglutarate supply for amino acid anabolism in vivo.

Two roles for aconitase in the regulation of tricarboxylic acid branch gene expression in Bacillus subtilis.

Previously, it was shown that an aconitase (citB) null mutation results in a vast overaccumulation of citrate in the culture fluid of growing Bacillus subtilis cells, a phenotype that causes secondary effects, including the hyperexpression of the citB promoter. B. subtilis aconitase is a bifunctional protein; to determine if either or both activities of aconitase were responsible for this phenotype, two strains producing different mutant forms of aconitase were constructed, one designed to be enzymatically inactive (C450S [citB2]) and the other designed to be defective in RNA binding (R741E [citB7]). The citB2 mutant was a glutamate auxotroph and accumulated citrate, while the citB7 mutant was a glutamate prototroph. Unexpectedly, the citB7 strain also accumulated citrate. Both mutant strains exhibited overexpression of the citB promoter and accumulated high levels of aconitase protein. These strains and the citB null mutant also exhibited increased levels of citrate synthase protein and enzyme activity in cell extracts, and the major citrate synthase (citZ) transcript was present at higher-than-normal levels in the citB null mutant, due at least in part to a >3-fold increase in the stability of the citZ transcript compared to the wild type. Purified B. subtilis aconitase bound to the citZ 5' leader RNA in vitro, but the mutant proteins did not. Together, these data suggest that wild-type aconitase binds to and destabilizes the citZ transcript in order to maintain proper cell homeostasis by preventing the overaccumulation of citrate.

Malate metabolism in Bacillus subtilis: distinct roles for three classes of malate-oxidizing enzymes.

The Gram-positive soil bacterium Bacillus subtilis uses glucose and malate as the preferred carbon sources. In the presence of either glucose or malate, the expression of genes and operons for the utilization of secondary carbon sources is subject to carbon catabolite repression. While glucose is a preferred substrate in many organisms from bacteria to man, the factors that contribute to the preference for malate have so far remained elusive. In this work, we have studied the contribution of the different malate-metabolizing enzymes in B. subtilis, and we have elucidated their distinct functions. The malate dehydrogenase and the phosphoenolpyruvate carboxykinase are both essential for malate utilization; they introduce malate into gluconeogenesis. The NADPH-generating malic enzyme YtsJ is important to establish the cellular pools of NADPH for anabolic reactions. Finally, the NADH-generating malic enzymes MaeA, MalS, and MleA are involved in keeping the ATP levels high. Together, this unique array of distinct activities makes malate a preferred carbon source for B. subtilis.

Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway.

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate.

Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon.

The majority of all proteins of a living cell is active in complexes rather than in an isolated way. These protein-protein interactions are of high relevance for many biological functions. In addition to many well established protein complexes an increasing number of protein-protein interactions, which form rather transient complexes has recently been discovered. The formation of such complexes seems to be a common feature especially for metabolic pathways. In the Gram-positive model organism Bacillus subtilis, we identified a protein complex of three citric acid cycle enzymes. This complex consists of the citrate synthase, the isocitrate dehydrogenase, and the malate dehydrogenase. Moreover, fumarase and aconitase interact with malate dehydrogenase and with each other. These five enzymes catalyze sequential reaction of the TCA cycle. Thus, this interaction might be important for a direct transfer of intermediates of the TCA cycle and thus for elevated metabolic fluxes via substrate channeling. In addition, we discovered a link between the TCA cycle and gluconeogenesis through a flexible interaction of two proteins: the association between the malate dehydrogenase and phosphoenolpyruvate carboxykinase is directly controlled by the metabolic flux. The phosphoenolpyruvate carboxykinase links the TCA cycle with gluconeogenesis and is essential for B. subtilis growing on gluconeogenic carbon sources. Only under gluconeogenic growth conditions an interaction of these two proteins is detectable and disappears under glycolytic growth conditions.