Assimilation Pathway Research Articles

Functional genomic analysis of nutrient cycling of plant-soil continuum in the mossy biocrust in the Tengger Desert

The microbiome and metabolic processes occurring in the soil of the moss crust-soil continuum contribute to drive biogeochemical cycles. In this study, soil chemical properties of soil which was attached to bryophyte (BRS) and soil which was dropped from bryophyte (BBS) were analyzed. Differences in microbial species composition, metabolic pathways and related genes of these two different soil types were also analyzed. The results showed that the total organic carbon (TOC), total nitrogen (TN) and ammonium nitrogen (NH4+-N) content of BRS were significantly higher than those of BBS. The dominant phylum in the soil microbiome of the moss crust-soil continuum was Actinobacteria. Genes and metabolic pathways related to the carbon, nitrogen, phosphorus, and sulfur cycle differed between BRS and BBS. The carbon sequestration capacity of BRS was higher than that of BBS. The relative abundance of napA, narH and narI genes involved in denitrification and dissimilatory nitrate reduction in the BRS microbiome was significantly higher than that in the BBS microbiome, while the relative abundance of nasA, NR and nirA genes involved in assimilatory nitrate reduction in the BRS soil microbiome was significantly lower than that in the BBS soil microbiome. The relative abundance of glpQ genes encoding glycerophosphodiester phosphodiesterase in the BRS microbiome was significantly higher than that in the BBS microbime, while the relative abundance of pstS, pstA, pstC, pstB genes responsible for phosphate-specific transport systems in the BRS microbiome was significantly lower than that in the BBS microbiome. The abundance of the assimilatory sulfate-reducing metabolic pathway in the BRS microbiome was significantly higher than that in the BBS microbiome, while the dissimilatory sulfate-reducing metabolic pathway in the BRS microbiome was significantly lower than that in the BBS microbiome. The study found that the microbiome in the soil which was attached to bryophyte plays a more important role in the accumulation of soil nutrients in the Tengger Desert through biodiversity and metabolic activity.

Glutamate dehydrogenase in "Liverworld"-A study in selected species to explore a key enzyme of plant primary metabolism in Marchantiophyta.

In plants, glutamate dehydrogenase (GDH) is an ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate in glutamate. It contributes to both the amino acid homeostasis and the management of intracellular ammonium, and it is regarded as a key player at the junction of carbon and nitrogen assimilation pathways. To date, information about the GDH of terrestrial plants refers to a very few species only. We focused on selected species belonging to the division Marchantiophyta, providing the first panoramic overview of biochemical and functional features of GDH in liverworts. Native electrophoretic analyses showed an isoenzymatic profile less complex than what was reported for Arabidposis thaliana and other angiosperms: the presence of a single isoform corresponding to an α-homohexamer, differently prone to thermal inactivation on a species- and organ-basis, was found. Sequence analysis conducted on amino acid sequences confirmed a high similarity of GDH in modern liverworts with the GDH2 protein of A. thaliana, strengthening the hypothesis that the duplication event that gave origin to GDH1-homolog gene from GDH2 occurred after the evolutionary bifurcation that separated bryophytes and tracheophytes. Experiments conducted on Marchantia polymorpha and Calypogeia fissa grown in vitro and compared to A. thaliana demonstrated through in gel activity detection and monodimensional Western Blot that the aminating activity of GDH resulted in strongly enhanced responses to ammonium excess in liverworts as well, even if at a different extent compared to Arabidopsis and other vascular species. The comparative analysis by bi-dimensional Western Blot suggested that the regulation of the enzyme could be, at least partially, untied from the protein post-translational pattern. Finally, immuno-electron microscopy revealed that the GDH enzyme localizes at the subcellular level in both mitochondria and chloroplasts of parenchyma and is specifically associated to the endomembrane system in liverworts.

Characterization of simultaneous removal of nitrogen and phosphorus by novel Raoultella ornithinolytica strain YX-4 and application in real farm wastewater treatment

A novel strain having ability to simultaneously remove ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and phosphorus was isolated from swine farm wastewater and was identified as Raoultella ornithinolytica YX-4 (NCBI accession number: OR646540). Nitrogen and phosphorus balance analysis combined with amplification of key enzyme genes of metabolic pathways revealed that the strain possess heterotrophic nitrification, aerobic denitrification, phosphorus accumulation and assimilation pathways. Significant removal of ammonium nitrogen, nitrate nitrogen and nitrite nitrogen were achieved (99%, 97% and 93% respectively) with optimal culture conditions. The transcript level of key enzyme genes was detected at different incubation period, and significant up-regulation of glnA, narI, narH, nirB, nirD, ppk1, and ppk2 was noted. This is the first report of the denitrification of phosphorus accumulating organisms R. ornithinolytica and reveals tangible results of key enzyme gene expression during real wastewater treatment.

Unraveling the molecular mechanisms of selenite reduction: transcriptomic analysis of Bacillus reveals the key role of sulfur assimilation.

Selenite biotransformation by microorganisms is an effective detoxification and assimilation process. However, current knowledge of the molecular mechanisms of selenite reduction remains circumscribed. Here, the reduction of Se(IV) by a highly selenite-resistant Bacillus sp. SL (up to 50mM) was systematically analyzed, and the molecular mechanisms of selenite reduction were investigated. Remarkably, 10mM selenite was entirely transformed by the strain SL within 20h, demonstrating a faster conversion rate compared to other microorganisms. Furthermore, glutathione (GSH) and exopolysaccharides (EPS) changes were also monitored during the process. Transcriptomic analysis revealed that the genes of ferredoxin-sulfite oxidoreductase (6.82) and sulfate adenylyltransferase (6.32) were significantly upregulated, indicating that the sulfur assimilation pathway is the primary reducing pathway involved in selenite reduction by strain SL. Moreover, key genes associated with NAD(P)/FAD-dependent oxidoreductases and thioredoxin were significantly upregulated. The reduction of Se(IV) was mediated by multiple pathways in strain SL. To our knowledge, this is the initial report to identify the involvement of sulfur assimilation pathway in selenite reduction for bacillus, which is rare in aerobic bacteria.

Inhibition of O-acetylserine (thiol) lyase as a promising new mechanism of action for herbicides

Enzymes of the sulfur assimilation pathway of plants have been identified as potential targets for herbicide development, given their crucial role in synthesizing amino acids, coenzymes, and various sulfated compounds. In this pathway, O-acetylserine (thiol) lyase (OAS-TL; EC 2.5.1.47) catalyzes the synthesis of L-cysteine through the incorporation of sulfate into O-acetylserine (OAS). This study used an in silico approach to select seven inhibitors for OAS-TL. The in silico experiments revealed that S-benzyl-L-cysteine (SBC) had a better docking score (−7.0 kcal mol−1) than the substrate OAS (−6.6 kcal mol−1), indicating its suitable interaction with the active site of the enzyme. In vitro experiments showed that SBC is a non-competitive inhibitor of OAS-TL from Arabidopsis thaliana expressed heterologously in Escherichia coli, with a Kic of 4.29 mM and a Kiu of 5.12 mM. When added to the nutrient solution, SBC inhibited the growth of maize and morning glory weed plants due to the reduction of L-cysteine synthesis. Remarkably, morning glory was more sensitive than maize. As proof of its mechanism of action, L-cysteine supplementation to the nutrient solution mitigated the inhibitory effect of SBC on the growth of morning glory. Taken together, our data suggest that reduced L-cysteine synthesis is the primary cause of growth inhibition in maize and morning glory plants exposed to SBC. Furthermore, our findings indicate that inhibiting OAS-TL could potentially be a novel approach for herbicidal action.

Unlocking the potential of elemental sulfur autotrophic denitrification through inorganic nutrient optimization

Elemental sulfur packed-bed (S0PB) bioreactors are increasingly employed to treat secondary effluents from wastewater treatment plants. Limited availability of inorganic nutrients, such as ammonium, phosphate, and inorganic carbon, significantly determines the growth of autotrophic microorganisms and the denitrification efficiency. This study investigated the constraining thresholds of inorganic nutrients on the elemental sulfur autotrophic denitrification process. In the absence of ammonium, phosphate, and bicarbonate, the denitrification rate decreased. Specifically, the ammonium assimilation process could be replaced by nitrate assimilation pathways, resulting in a 29.5 % decline in denitrification efficiency when the ammonium-N feed was reduced from 2.0 to 0 mg-N/L. However, the absence of phosphate-P and bicarbonate-C led to substantial drops in denitrification efficiency by 66.7 % and 77.5 %, respectively. This was primarily attributed to diminished biomass yield, as evidenced by reductions of 77.8–82.6 % in ATP and 35.7–44.3 % in protein content, along with a 12.4 %–14.7 % decrease in the relative abundances of dominant sulfur-oxidizing bacteria. Correlation analyses established specific thresholds for desired nitrate removal amounts: 0.131 for ammonium-N, 0.053 for phosphate-P, and 0.597 for bicarbonate-C. These thresholds provided valuable guidance for adjusting real-life wastewater through external supplementation when employing a pilot-scale S0PB bioreactor, leading to a significant enhancement of denitrification efficiency. This study emphasizes the significance of considering the concentrations of inorganic nutrients in wastewater when implementing S0PB bioreactors, which is a context that frequently neglects potential negative consequences.

Overexpressing plant ferredoxin-like protein enhances photosynthetic efficiency and carbohydrates accumulation in Phalaenopsis.

Crassulacean acid metabolism (CAM) is one of three major models of carbon dioxide assimilation pathway with better water-use efficiency and slower photosynthetic efficiency in photosynthesis. Previous studies indicated that the gene of sweet pepper plant ferredoxin-like protein (PFLP) shows high homology to the ferredoxin-1(Fd-1) family that belongs to photosynthetic type Fd and involves in photosystem I. It is speculated that overexpressing pflp in the transgenic plant may enhance photosynthetic efficiency through the electron transport chain (ETC). To reveal the function of PFLP in photosynthetic efficiency, pflp transgenic Phalaenopsis, a CAM plant, was generated to analyze photosynthetic markers. Transgenic plants exhibited 1.2-folds of electron transport rate than that of wild type (WT), and higher CO2 assimilation rates up to 1.6 and 1.5-folds samples at 4pm and 10pm respectively. Enzyme activity of phosphoenolpyruvate carboxylase (PEPC) was increased to 5.9-folds in Phase III, and NAD+-linked malic enzyme (NAD+-ME) activity increased 1.4-folds in Phase IV in transgenic plants. The photosynthesis products were analyzed between transgenic plants and WT. Soluble sugars contents such as glucose, fructose, and sucrose were found to significantly increase to 1.2, 1.8, and 1.3-folds higher in transgenic plants. The starch grains were also accumulated up to 1.4-folds in transgenic plants than that of WT. These results indicated that overexpressing pflp in transgenic plants increases carbohydrates accumulation by enhancing electron transport flow during photosynthesis. This is the first evidence for the PFLP function in CAM plants. Taken altogether, we suggest that pflp is an applicable gene for agriculture application that enhances electron transport chain efficiency during photosynthesis.

Microbial degradation and assimilation of veratric acid in oxic and anoxic groundwaters.

Microbial communities are key players in groundwater ecosystems. In this dark environment, heterotrophic microbes rely on biomass produced by the activity of lithoautotrophs or on the degradation of organic matter seeping from the surface. Most studies on bacterial diversity in groundwater habitats are based on 16S gene sequencing and full genome reconstructions showing potential metabolic pathways used in these habitats. However, molecular-based studies do not allow for the assessment of population dynamics over time or the assimilation of specific compounds and their biochemical transformation by microbial communities. Therefore, in this study, we combined DNA-, phospholipid fatty acid-, and metabolomic-stable isotope probing to target and identify heterotrophic bacteria in the groundwater setting of the Hainich Critical Zone Exploratory (CZE), focusing on 2 aquifers with different physico-chemical conditions (oxic and anoxic). We incubated groundwater from 4 different wells using either 13C-labeled veratric acid (a lignin-derived compound) (single labeling) or a combination of 13CO2 and D-labeled veratric acid (dual labeling). Our results show that heterotrophic activities dominate all groundwater sites. We identified bacteria with the potential to break down veratric acid (Sphingobium or Microbacterium). We observed differences in heterotrophic activities between the oxic and anoxic aquifers, indicating local adaptations of bacterial populations. The dual labeling experiments suggested that the serine pathway is an important carbon assimilation pathway and that organic matter was an important source of hydrogen in the newly produced lipids. These experiments also yielded different labeled taxa compared to the single labeling experiments, showing that there exists a complex interaction network in the groundwater habitats.

Macrogenomic analysis of the effects of aqueous-phase from hydrothermal carbonation of sewage sludge on nitrogen metabolism pathways and associated bacterial communities during composting

The effects of aqueous phases (AP) formed from hydrothermal carbonation of sewage sludge (with or without rice husk) as moisture regulators of nitrogen metabolism pathways during composting are currently unclear. Macrogenomic analyses revealed that both APs resulted in notably changes in bacterial communities during composting; increased levels of nitrogen assimilation, nitrification, and denitrification metabolic pathways; and decreased levels of nitrogen mineralization metabolic pathways. Genes associated with nitrogen assimilation and mineralization accounted for 34–41% and 32–40% of the annotated reads related to nitrogen cycling during composting, respectively, representing them as the most abundant nitrogen metabolism processes. The gudB and norB were identified as key genes for nitrogen mineralization and nitrous oxide emission, respectively. This research offers a better understanding of the effects of additional nitrogen sources on nitrogen metabolism pathways during composting.

Pseudophaeobacter profundi sp. nov., isolated from the Western Pacific Ocean.

A novel Gram-stain-negative, facultatively anaerobic and heterotrophic bacterium, designated strain ZH257T, was isolated from in situ enrichment samples incubated on the seamount floor of the Western Pacific Ocean. Cells were rod-shaped, oxidase- and catalase- positive, and motile by means of polar flagella. Strain ZH257T grew at 4-37 °C (optimum, 28-32 °C), pH 6.0-9.0 (optimum, pH 7.0) and with 2.0-9.0 % (w/v) NaCl (optimum, 3.0-4.0 %). Strain ZH257T was most closely related to members of the genus Pseudophaeobacter, sharing 99.13, 98.27 and 96.89 % 16S rRNA gene sequence identities with Pseudophaeobacter flagellatus GDMCC 1.2988T, Pseudophaeobacter arcticus DSM 23566T and Pseudophaeobacter leonis DSM 25627T, respectively. The DNA G+C content was 59.2 mol%. The estimated average nucleotide identity and digital DNA-DNA hybridization values between strain ZH257T and its closely related species were 79.61-93.04 % and 23.10-50.20 %, respectively. Strain ZH257T harboured complete denitrification and nitrate assimilation pathways. Strain ZH257T contained summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) as major fatty acids (>5 %), and Q-10 as the major respiratory quinone. The polar lipid profile contained phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, an unidentified phospholipid, an unidentified aminolipid and four unidentified lipids. The combined phenotypic, genotypic and chemotaxonomic data showed that strain ZH257T represents a novel species of the genus Pseudophaeobacter, for which the name Pseudophaeobacter profundi sp. nov. is proposed, with the type strain ZH257T (=MCCC M29024T=KACC 23147T).

Rhizosphere and detritusphere habitats modulate expression of soil N-cycling genes during plant development.

Interactions between plant roots and rhizosphere bacteria modulate nitrogen (N)-cycling processes and create habitats rich in low molecular weight compounds (exudates) and complex organic molecules (decaying root litter) compared to those of bulk soil. Microbial N-cycling is regulated by edaphic conditions and genes from many interconnected metabolic pathways, but most studies of soil N-cycling gene expression have focused on single pathways. Currently, we lack a comprehensive understanding of the interplay between soil N-cycling gene regulation, spatial habitat, and time. We present results from a replicated time series of soil metatranscriptomes; we followed gene expression of multiple N transformations in four soil habitats (rhizosphere, detritusphere, rhizo-detritusphere, and bulk soil) during active root growth for the annual grass, Avena fatua. The presence of root litter and living roots significantly altered the trajectories of N-cycling gene expression. Upregulation of assimilatory nitrate reduction in the rhizosphere suggests that rhizosphere bacteria were actively competing with roots for nitrate. Simultaneously, ammonium assimilatory pathways were upregulated in both rhizosphere and detritusphere soil, which could have limited N availability to plants. The detritusphere supported dissimilatory processes DNRA and denitrification. Expression of nitrification genes was dominated by three phylotypes of Thaumarchaeota and was upregulated in bulk soil. Unidirectional ammonium assimilation and its regulatory genes (GS/GOGAT) were upregulated near relatively young roots and highly decayed root litter, suggesting N may have been limiting in these habitats (GS/GOGAT is typically activated under N limitation). Our comprehensive analysis indicates that differences in carbon and inorganic N availability control contemporaneous transcription of N-cycling pathways in soil habitats. IMPORTANCE Plant roots modulate microbial nitrogen (N) cycling by regulating the supply of root-derived carbon and nitrogen uptake. These differences in resource availability cause distinct micro-habitats to develop: soil near living roots, decaying roots, near both, or outside the direct influence of roots. While many environmental factors and genes control the microbial processes involved in the nitrogen cycle, most research has focused on single genes and pathways, neglecting the interactive effects these pathways have on each other. The processes controlled by these pathways determine consumption and production of N by soil microorganisms. We followed the expression of N-cycling genes in four soil microhabitats over a period of active root growth for an annual grass. We found that the presence of root litter and living roots significantly altered gene expression involved in multiple nitrogen pathways, as well as tradeoffs between pathways, which ultimately regulate N availability to plants.

Nitrogen removal capability and mechanism of a novel heterotrophic nitrifying-aerobic denitrifying strain H1 as a potential candidate in mariculture wastewater treatment.

The nitrogen removal performance and mechanisms of Bacillus subtilis H1 isolated from a mariculture environment were investigated. Strain H1 efficiently removed NH4+-N, NO2--N, and NO3--N in simulated wastewater with removal efficiencies of 85.61%, 90.58%, and 57.82%, respectively. Strain H1 also efficiently degraded mixed nitrogen (NH4+-N mixed with NO2--N and/or NO3--N) and had removal efficiencies ranging from 82.39 to 89.54%. Nitrogen balance analysis revealed that inorganic nitrogen was degraded by heterotrophic nitrification-aerobic denitrification (HN-AD) and assimilation. 15N isotope tracing indicated that N2O was the product of the HN-AD process, while N2 as the final product was only detected during the reduction of 15NO2--N. The nitrogen assimilation and dissimilation pathways by strain H1 were further clarified using complete genome sequencing, nitrification inhibitor addition, and enzymatic activity measurement, and the ammonium oxidation process was speculated as NH4+ → NH2OH → NO → N2O. These results showed the application prospect of B. subtilis H1 in treating mariculture wastewater.

Transcription factor OxyR regulates sulfane sulfur removal and L-cysteine biosynthesis in Corynebacterium glutamicum.

Sulfane sulfur, a collective term for hydrogen polysulfide and organic persulfide, often damages cells at high concentrations. Cells can regulate intracellular sulfane sulfur levels through specific mechanisms, but these mechanisms are unclear in Corynebacterium glutamicum. OxyR is a transcription factor capable of sensing oxidative stress and is also responsive to sulfane sulfur. In this study, we found that OxyR functioned directly in regulating sulfane sulfur in C. glutamicum. OxyR binds to the promoter of katA and nrdH and regulates its expression, as revealed via in vitro electrophoretic mobility shift assay analysis, real-time quantitative PCR, and reporting systems. Overexpression of katA and nrdH reduced intracellular sulfane sulfur levels by over 30% and 20% in C. glutamicum, respectively. RNA-sequencing analysis showed that the lack of OxyR downregulated the expression of sulfur assimilation pathway genes and/or sulfur transcription factors, which may reduce the rate of sulfur assimilation. In addition, OxyR also affected the biosynthesis of L-cysteine in C. glutamicum. OxyR overexpression strain Cg-2 accumulated 183 mg/L of L-cysteine, increased by approximately 30% compared with the control (142 mg/L). In summary, OxyR not only regulated sulfane sulfur levels by controlling the expression of katA and nrdH in C. glutamicum but also facilitated the sulfur assimilation and L-cysteine synthesis pathways, providing a potential target for constructing robust cell factories of sulfur-containing amino acids and their derivatives. IMPORTANCE C. glutamicum is an important industrial microorganism used to produce various amino acids. In the production of sulfur-containing amino acids, cells inevitably accumulate a large amount of sulfane sulfur. However, few studies have focused on sulfane sulfur removal in C. glutamicum. In this study, we not only revealed the regulatory mechanism of OxyR on intracellular sulfane sulfur removal but also explored the effects of OxyR on the sulfur assimilation and L-cysteine synthesis pathways in C. glutamicum. This is the first study on the removal of sulfane sulfur in C. glutamicum. These results contribute to the understanding of sulfur regulatory mechanisms and may aid in the future optimization of C. glutamicum for biosynthesis of sulfur-containing amino acids.

Characterization of the Nitrogen Removal Potential of Two Newly Isolated Acinetobacter Strains under Low Temperature

Excess nitrogen and phosphorus in the water causes several ecological problems for nutrients. Biological nitrogen removal is an economical and efficient way to prevent excessive nitrogen in the environment. For most areas of China, temperatures are usually lower than 20 °C except during the summertime. It is necessary to discover microbes that can efficiently remove nitrogen at low temperatures. In this study, two Acinetobacter strains were isolated from a sample in a wastewater tank in Taizhou for their capabilities to remove NO3−–N and NO2−–N at 15 °C. Heterotrophic nitrification experiments showed that both strains could efficiently remove nitrogen from the culture medium. The maximum removal rates of NH4+–N were 3.15 mg/L·h and 4.74 mg/L·h for heterotrophic nitrification by the strains F and H, respectively. Strain H grew faster and removed both nitrite and nitrate more efficiently than strain F. Genome sequencing showed that strains F and H could be classified into Acinetobacter johnsonii and Acinetobacter bereziniae, respectively. NO2−–N (100 mg/L) was completely removed in 3 days by strain H. The maximum NO3−–N removal rate was 3.53 mg/L·h for strain F. When strain H was cultured in a broth with 200 mg/L NO3−–N, 97.46% of NH4+–N (200 mg/L) was removed in 5 days, and the maximum NH4+–N removal rate was 4.04 mg/L·h. Genomic sequence analysis showed that both the strains lacked genes involved in the denitrification pathway that transforms NO3− into N2. This implies that nitrate or nitrite is removed through the nitrogen assimilation pathway. Genes responsible for nitrate assimilation are clustered together with molybdopterin cofactor biosynthesis genes. Strain H contains fewer resistance genes and transfer elements. All the above data demonstrate that strain H is a promising candidate for nitrogen removal at lower temperatures. But there is still a lot to be done to systematically evaluate the potential of A. bereziniae strain H in treating wastewater at a pilot scale. These include the long-term performance, environmental tolerance, and nitrogen removal efficiency in wastewater. And the application of these Acinetobacter strains in diverse wastewater treatment settings might require careful optimization and real-time monitoring.

The effect of glutathione on glucosinolate biosynthesis through the sulfur assimilation pathway in pakchoi associated with the growth conditions

Glucosinolates (GSLs) are a group of nitrogen- and sulfur-containing secondary metabolites, synthesized primarily in members of the Brassicaceae family, that play an important role in food flavor, plant antimicrobial activity, resistance to insect attack, stress tolerance, and human anti-cancer effects. As a sulfur-containing compound, glutathione has a strong connection with GSLs biosynthesis as a sulfur donor or redox system, and exists in reduced (glutathione; GSH) and oxidized (glutathione disulfide; GSSG) forms. However, the mechanism of GSH regulating GSLs biosynthesis remainds unclear. Hence, the exogenous therapy to pakchoi under normal growth condition and sulfur deficiency condition were conducted in this work to explore the relevant mechanism. The results showed that exogenous application of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased the transcript levels of GSLs synthesis-related genes and transcription factors, as well as sulfur assimilation-related genes under the normal growth condition. Application of exogenous GSH inhibited the expression of GSLs synthesis- and sulfur assimilation-related genes under the normal condition, while the GSLs biosynthesis and the sulfur assimilation pathway were activated by exogenous application of GSH when the content of GSH in vivo of plants decreased owing to sulfur deficiency. Moreover, exogenous application of GSSG increased the transcript levels of GSLs synthesis- and sulfur assimilation-related genes under the normal growth condition and under sulfur deficiency. The present work provides new insights into the molecular mechanisms of GSLs biosynthesis underlying glutathione regulation.

Development of polymer films and biological matrices standards for selenium, mercury and endogenous elements quantitative LA-ICP MS imaging in entire rainbow trout fry

The quantitative imaging of trace elements in biological samples can bring information on their assimilation pathways and help understanding their toxicity or essentiality. This study presents the development of two quantitative LA-ICP MS methodologies and their comparison for Se, Hg, Cu, Zn and Mo imaging in rainbow trout. The first method is based on spiked polymer films (dextran) allowing internal standardization with Ge and Te. The second method relies on the matrix-matched standards (MMS) methodology, with homogenized and spiked trout muscle and calf liver. For Se, Cu, Zn and Mo, linear regressions with correlation coefficient above 0.994 were obtained by LA-ICP MS analysis for both methods. For Hg, only MMS calibration provided linear regression as polymer films exhibited Hg unstable signals during LA-ICP MS analysis. Element concentrations spiked in muscle and liver MMS were also confirmed by ICP MS and used to validate the polymer films method as values obtained for Se, Hg, Mo and Cu were in good agreement. While LODs of polymer film method were in the range of 0.06–0.6 µg.g−1 for Cu, Se, Mo, and 5 µg.g−1 for Zn, values were higher for MMS method (from 0.06 (Mo) to 2.2 (Cu) µg.g−1, and 30 of µg.g−1 for Zn). Particular attention has been paid to sample embedding and water was found to minimize internal standard response bias. Both methods were then applied to the quantitative mapping of these five elements in rainbow trout fry supplemented or not with organic forms of Se and Hg through parental and direct feeding. Calibrations were analysed before and after sample imaging evidencing a lower bias between the two quantifications with polymer films calibration than with MMS calibration. For the first time, elements concentrations were determined at 60 × 60 µm spatial resolution in specific tissues or organs of entire fry. Depending on the element, polymer films quantification was in agreement with trout muscle (Cu, Se) or with calf liver MMS quantification (Zn).

The Isolation of Anaerobic and Facultative Anaerobic Sulfate-Reducing Bacteria (SRB) and a Comparison of Related Enzymes in Their Sulfate Reduction Pathways.

Sulfate-reducing bacteria (SRB) are an important group of microorganisms that cause microbial corrosion. In this study, culturable SRB were isolated and identified from the inner rust layer of three kinds of steel and from sediments, and a comparison of amino acid sequences encoding related enzymes in the sulfate reduction pathway between anaerobic and facultative anaerobic SRB strains was carried out. The main results are as follows. (1) Seventy-seven strains were isolated, belonging to five genera and seven species, with the majority being Desulfovibrio marinisediminis. For the first time, Holodesulfovibrio spirochaetisodalis and Acinetobacter bereziniae were separated from the inner rust layer of metal, and sulfate reduction by A. bereziniae, Virgibacillus dokdonensis, and Virgibacillus chiguensis, etc., was also demonstrated for the first time. (2) Three strains of strictly anaerobic bacteria and four strains of facultative anaerobic bacteria were screened from seven bacterial strains. (3) Most of the anaerobic SRB only contained enzymes for the dissimilatory sulfate reduction pathway, while those of facultative anaerobic bacteria capable of producing hydrogen sulfide included two possible ways: containing the related enzymes from the dissimilatory pathway only, or containing enzymes from both dissimilatory and assimilation pathways. This study newly discovered that some bacterial genera exhibit sulfate reduction ability and found that there are differences in the distribution of enzymes related to the sulfate reduction pathway between anaerobic and facultative anaerobic SRB type trains, providing a basis for the development and utilization of sulfate-reducing bacterial resources and furthering our understanding of the metabolic mechanisms of SRB.

Microbial electrolysis cell as an effective tool for mitigating the toxic effects of Cu2+ on microorganisms in the acid mine drainage treatment: Efficacy and mechanisms

It is poorly understood how the mechanism of heavy metals affect sulfate-reducing bacteria (SRB) in microbial electrolysis cell (MEC) system, however, this is essential to improve the effectiveness of sulfate reduction in acid mine drainage (AMD). This study evaluated the impact of Cu2+ on MEC performance using SRBs enriched biocathode. The sulfate reduction rate and pathway, the fate of Cu, as well as the succession of microbial community were investigated. Results showed that MEC performance was optimized at influent Cu2+ concentration of 20 mg/L, while slight inhibition was observed at 40 mg/L. The removal of Cu2+ was accomplished through bioprecipitation and biosorption, forming CuS and Cu(OH)2. However, MEC performance was inhibited at higher Cu2+ concentrations (60 mg/L) due to damaged cell structure, increased charge transfer internal resistance, and altered microbial communities. Specifically, the relative abundance of Firmicutes and Bacteroidota decreased, and functional genes related to sulfate reduction metabolism were reduced, leading to impairment of the assimilatory and dissimilatory sulfate reduction pathways. This knowledge provides revealing insights and theoretical support for developing a feasible approach to realize the treatment of wastewater containing multiple pollutants.

The Role of Serine-Glyoxylate Aminotransferase and Malyl-CoA Lyase in the Metabolism of Methylococcus capsulatus Bath.

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin-Benson-Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation. The Δsga mutant obtained by double homologous recombination showed a prolonged lag phase, and after the lag period, the growth rate became similar to that of the wild type strain. The elevated intracellular levels of glutamate, serine, glycine, alanine, methionine, leucine, and succinate suggested significant metabolic changes in the mutant cells. Deletion of the mcl gene resulted in very poor growth and glycine only partially improved growth of the mutant strain. Cells of Δmcl mutant possess lower content of histidine, but enhanced level of alanine, leucine, and lysine than those of the wild type strain. Our data imply the importance of the serine cycle enzymes in metabolism of the methanotroph as well as relationships of the three C1 assimilation pathways in the gammaproteobacterial methanotrophs.

Exogenous thiocyanate inhibits sulfurtransferase pathway and induces β-cyanoalanine synthase pathway to enhance exogenous cyanide assimilation in rice plants

Cyanide (CN−) assimilation in plants takes place by β-cyanoalanine synthase (β-CAS) and sulfurtransferase (ST), in which the ST pathway converts CN− into thiocyanate (SCN−). Both chemicals (CN− and SCN−) are frequently detected in the effluent of gold mining operations. In this connection, exogenous SCN− was applied to rice plants with CN− and compared with CN− alone to investigate its effects on CN− assimilation and degradation pathways. Interestingly, the CN− and SCN− content in both roots and shoots were increased with the increase in “CN−” treatments, but surprisingly their content under “SCN−+CN−” treatments did not show the similar trend. The increasing trend remained the same for CN− but the SCN− content was constant with increasing CN− concentrations in comparison with the control (SCN− alone). Additionally, the assimilation rates of CN− in rice plants under “SCN−+CN−” treatments were significantly higher than “CN−” treatments. The application of SCN− with CN− mostly alters the expression of both β-CAS and ST-associated genes. On one side, the application of SCN− significantly repressed the expression of genes encoded with ST in rice plants, but on the other side, it significantly up-regulated the expression of the β-CAS gene located in mitochondria. These results reveal that the application of exogenous SCN− increases CN− assimilation rates by inhibiting the ST pathway and stimulating the β-CAS pathway. This study would provide new insight into the positive effects of exogenous SCN− in increasing CN− assimilation by altering the degradation pathways in rice plants.