Phosphatase Production Research Articles

Microbiological Insights into the Stress-Alleviating Property of an Endophytic Bacillus altitudinis WR10 in Wheat under Low-Phosphorus and High-Salinity Stresses.

An indole–3–acetic acid producing Bacillus altitudinis WR10 was previously isolated from the root of wheat (Triticum aestivum L.). In this study, the strain WR10 was used for relieving abiotic stresses in wheat under low phosphorus and high saline in hydroponic co-culture models. Significantly, strain WR10 improved wheat seed relative germination rate under salinity stress (200/400 mM NaCl) and the root dry weight in wheat seedlings under phosphorus stress (10 μM KH2PO3) when insoluble phosphates are available. To provide insights into its abiotic stress-alleviating properties, the strain was characterized further. WR10 grows well under different culture conditions. Particularly, WR10 resists salt (12% NaCl) and hydrolyzes both inorganic and organic insoluble phosphates. WR10 uses many plant-derived substrates as sole carbon and energy sources. It produces catalase, amylase, phosphatase, phytase, reductase, and 1–aminocyclopropane–1–carboxylate (ACC) deaminase. In addition, WR10 possesses long peritrichous flagella, and its biofilm formation, as well as phytase production, is induced by abiotic stresses. Overall, the salinity-alleviating property of WR10 in wheat can be attributed to its inherent tolerance to NaCl, formation of biofilm, and production of enzymes, like catalase, amylase, and ACC deaminase. Meanwhile, B. altitudinis WR10 reduces low-phosphorus stress in wheat by production of phosphatases and phytases in the presence of insoluble phosphates.

Focal adhesion kinase promotes BMP2-induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways.

Human urine-derived stem cells (hUSCs) serve as favorable candidates for bone transplants due to their efficient proliferative and multipotent differentiation abilities, as well as the capacity to secrete a variety of vasoactive agents to facilitate tissue engineering. The present study aimed to explore the role of focal adhesion kinase (FAK) in bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of hUSCs and to investigate the underlying mechanism. The degree of osteogenic differentiation and the correlated signals, following BMP2 overexpression and siRNA-mediated silencing of FAK, were determined in vitro. Moreover, hUSCs induced bone formation in a rat model with cranial defects, in vivo. Our findings revealed that alkaline phosphatase production, calcium deposits, osteocalcin and osteopontin expression, and bone formation were upregulated in vitro and in vivo following BMP2-induced osteogenic differentiation, and AMPK and Wnt signaling pathway activation by FAK could effectively regulate BMP2-enhanced osteogenic differentiation of hUSCs. Taken together, these findings indicated that FAK could mediate BMP2-enhanced osteogenic differentiation of hUSCs through activating adenosine 5'-monophosphate-activated protein kinase and Wnt signaling pathways.

Bioactive silica nanoparticles with calcium and phosphate for single dose osteogenic differentiation.

The differentiation of adult stem cells is usually performed in vitro, by exposing them to specific factors. Alternatively, one can use nanocarriers containing such factors, to be internalized by the cells. In this work we have reduce the size of those carriers to the nanoscale, developing bioactive silica nanoparticles with diameters under 100 nm, containing calcium and phosphate ions (SiNPs-CaP). These ions, once released inside adult stem cells, induce bone cell proliferation and differentiation, and stimulate the expression of bone-related proteins in a single dose administration. The SiNPs-CaP nanomaterials were prepared through a sol-gel approach, and the ions added with a post-synthesis functionalization method. The synthesized SiNPs-CaP have narrow size distribution, good colloidal stability, and show high levels of ion incorporation. Furthermore, the SiNPs-CaP have good cytocompatibility and promote the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC), with alkaline phosphatase, osteopontin and osteocalcin production levels comparable to the ones obtained in standard osteogenic medium. The novel bioactive SiNPs-CaP are synthesized in a simple and fast manner and show the ability to promote osteogenic differentiation after a single dose administration, independently from external osteogenic inducers, showing great potential as carriers in bone tissue engineering applications.

Accelerated Bone Regeneration by Gold-Nanoparticle-Loaded Mesoporous Silica through Stimulating Immunomodulation.

Bone repair and regeneration are greatly influenced by the local immune microenvironment. In this regard, the immunomodulatory capability of biomaterials should be considered when evaluating their osteogenic effects. In this study, we investigated the modulatory effects of gold nanoparticle (AuNP)-loaded mesoporous silica nanoparticles (Au-MSNs) on macrophages and the subsequent effects on the behavior of osteoblastic lineage cells. The results demonstrate that Au-MSNs could generate a favorable immune microenvironment by stimulating an anti-inflammatory response and promoting the secretion of osteogenic cytokines by macrophages. As a result, there is an enhancement of osteogenic differentiation in preosteoblastic MC3T3 cells as assessed by the increased expression of osteogenic markers, alkaline phosphatase (ALP) production, and calcium deposition. The immunomodulatory effects and direct osteogenic stimulation by Au-MSNs synergistically increased the osteogenic differentiation capability of MC3T3 cells as a result of crosstalk between Au-MSN-conditioned macrophages and Au-MSN-treated osteoblasts in a coculture system. An in vivo study further revealed that Au-MSNs could accelerate new bone formation in a critical-sized cranial defect site in rats based on computed tomography analysis and histological examination. Together, this novel Au-MSNs could significantly promote osteogenic activity by modulating the immune microenvironment, showing its therapeutic potential for bone tissue repair and regeneration.

Bacterial 16S rDNA and alkaline phosphatase gene diversity in soil applied with composted aquatic plants

The present study aimed to examine the effects of applying composted aquatic plants (CAP) on richness and composition of soil bacterial assemblages responsible for alkaline phosphatase production by pot experiments, as compared to the corresponding effects of inorganic chemical fertilizers (ICF). Soil bacterial assemblages were studied by metabarcoding the 16S region of ribosomal DNA as a universal genetic marker and the phoD alkaline phosphatase gene as a functional marker. The compositions of 16S and phoD assemblages were significantly different among soils applied with CAP, ICF, their mixture (MIX), and control (no application). Furthermore, the richness of phoD assemblages in soils was greater in CAP or MIX application than in ICF or control, although significant difference only existed between MIX and ICF. In conclusion, the present study illustrated that the application of composted aquatic plants affected the richness and composition of soil bacterial assemblages responsible for the production of alkaline phosphatase.

Effect of 6-Shogaol on the Glucose Uptake and Survival of HT1080 Fibrosarcoma Cells.

Ginger is a plant that is native to southern China. In the last decade and research on the components of ginger has significantly increased; of these components, 6-shogaol exhibits the greatest potential antitumor capacity. However, the molecular mechanism through which 6-shogaol exerts its effects has not yet been elucidated. In this study, the effect of 6-shogaol on tumor cells that were derived from human fibrosarcoma (HT1080) was evaluated. Cell viability was determined by a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay testing different concentrations of 6-shogaol (2.5–150 μM). Subsequently, the effect of 6-shogaol on reactive oxygen species (ROS) production, glucose uptake, and protein expression of the signaling pathway phosphatase and tensin homolog/ protein kinase B /mammalian target of rapamycin (PTEN/Akt/mTOR) was measured. 6-Shogaol reduced the viability of the tumor cells and caused an increase in ROS production, which was attenuated with the addition of N-acetylcysteine, and the recovery of cell viability was observed. The increase in ROS production in response to 6-shogaol was associated with cell death. Similarly, glucose uptake decreased with incremental concentrations of 6-shogaol, and an increase in the expression of mTOR-p and Akt-p proteins was observed; PTEN was active in all the treatments with 6-shogaol. Thus, the results suggest that cells activate uncontrolled signaling pathways, such as phosphoinositide 3-kinase (PI3K)/Akt/mTOR, among other alternative mechanisms of metabolic modulation and of survival in order to counteract the pro-oxidant effect of 6-shogaol and the decrease in glucose uptake. Interestingly, a differential response was observed when non-cancerous cells were treated with 6-shogaol.

Time-series analysis of phosphorus-depleted microbial communities in carbon/nitrogen-amended soils

The mineralization of organic phosphorus (P) in soil is mainly driven by extracellular phosphatases secreted by soil microbes. Previous studies have suggested that microbial phosphatase production is regulated by soil nutrient stoichiometry and, thus, promoted by carbon and nitrogen (CN) amendment. However, the mechanism leading to increased phosphatase activity after nutrient amendment to soil is unclear, and it is not known how long the effect of the nutrient amendment lasts. To address these questions, we performed a 24-day time-series analysis of full-factorial soil microcosms with CN amendment. Phosphatase activity as well as that expressed per unit of β-d-glucosidase activity, increased sharply and significantly in response to the CN amendment. This suggests that the microbial community shifted their resource allocation after the nutrient amendment and preferentially produced phosphatases rather than β-d-glucosidase. This shifted resource allocation pattern was maintained throughout the 24-day incubation period, and copiotrophic microbes dominated soils that received CN amendment. These results indicate that the effect of the CN amendment on soil microbial resource allocation lasted for >24 days. Together, our results highlight the importance of high-resolution time-series observations that complement long-term studies for which frequent observation is often difficult.

The maize mycorrhizosphere as a source for isolation of arbuscular mycorrhizae-compatible phosphate rock-solubilizing bacteria

Phosphate chemical fertilizers are costly and raise concerns about environmental pollution through industrial production. The use of phosphate rock (PRs) emerges as a more sustainable alternative for agriculture. The aim of this work was to isolate phosphate rock-solubilizing bacteria (PRSB) from maize mycorrhizosphere, having growth promoting traits and that will be arbuscular mycorrhizae fungi (AMF) compatible. Bacteria were isolated from the mycorrhizosphere of maize and tested for rock phosphate solubilization, production of organic acids, phosphatases and phytase activities, and growth promotion traits. The capacity of some strains to enhance the dry weight of maize plants was determined in a growth chamber experiment. The compatibility of the selected strains with Rhizophagus irregularis under in vitro conditions was also tested. Out of 118 isolates from maize, eight belonging to Asaia lannaensis, Rahnella sp., Pantoea sp., and Pseudomonas sp. were found to be the best PRSB. On solid media, all strains mobilized P from tricalcium phosphate, hydroxyapatite, and PRs. A. lannaensis was the only PRSB showing visible solubilization of AlPO4 and FePO4. All the PRSBs solubilized PR by producing D-gluconic acid and 2-ketogluconic acid and by lowering the pH. Most strains presented IAA and siderophore production and different biofilm formation and motility capacities. All strains improved the dry weight of maize seedlings compared with non-inoculated plants. The results proved that PRSBs were able to grow on R. irregularis hyphae as the sole in vitro C source. Bacteria isolated from the mycorrhizosphere of maize shows effective solubilization of phosphorus from PR with different reactivity levels. The traits of these bacteria as growth promoters and their biocompatibility with AMF show their potential as inoculants. Improvement of the agronomic effectiveness of PRs is relevant for developing countries that use PRs directly as P-fertilizers (less expensive than soluble P-fertilizers) for sustainable agriculture.

Plant traits and species interactions along gradients of N, P and K availabilities

Abstract Plant performance is driven by nutrient availability. So far, studies on plant nutrient relationships mostly focused on nitrogen (N) and phosphorus (P), whereas potassium (K) received less attention. We evaluated whether plant responses to variation in nutrient availability are similar or different for the nutrients N, P and K and whether the same plant traits are decisive for the competitive ability of species along gradients of N, P and K availabilities. We studied plant trait responses of three temperate perennial grass species (Alopecurus pratensis, Agrostis capillaris and Anthoxanthum odoratum, each dominant under N limitation, P limitation and K limitation, respectively). Each gradient consisted of eight treatments (N1–N8, P1–P8, K1–K8), with N:P:K supply rates being 1:0.8:6 (N1), 128:0.8:6 (N8), 12:0.075:6 (P1), 12:9.6:6 (P8), 12:0.8:0.56 (K1) and 12:0.8:72 (K8) (total supply per plant individual in mg). Plant individuals were kept either in single‐species pots (intraspecific competition) or in mixed‐species pots (interspecific competition). After 2 months, plant biomass, leaf and root traits and root enzyme activity were measured. Most below‐ground traits (e.g. total root length, root diameter) responded most strongly to competition as predictor variable and only secondly to the type of nutrient or their supply rates (unlike RGRTotal and SLA). Leaf chlorophyll content and root enzyme activity responded most strongly to supply rate of nutrients and the interaction of nutrient type and supply rate, respectively. In line with our prediction, A. pratensis was the superior competitor under N limitation and A. odoratum was the superior competitor under K limitation (assessed by relative dominance [RD] and relative competitive strength [RCS]). Both species showed high investment in total root length. Opposite to our expectation, A. capillaris was a bad competitor under P limitation, with low root length but higher root phosphatase activity. Our results indicate that below‐ground plant traits are decisive in nutrient‐related competition between plant species. Further, competitive strength is determined by a high total root length per soil volume both under N and K limitation. Under P limitation, the model species A. capillaris showed a weak competitive performance either because it was lacking colonization with mycorrhiza and/or the high investment in phosphatase activity posed a trade‐off with higher N investment in phosphatase production. A free Plain Language Summary can be found within the Supporting Information of this article.

A new function of the biocontrol bacterium Lysobacter enzymogenes LE16 in the mineralization of soil organic phosphorus

To evaluate the functions of a new biocontrol bacterium, Lysobacter enzymogenes LE16, in the mineralization of soil organic phosphorus (P) and in the stimulation of plant P uptake and plant growth. Liquid culture, soil incubation, and pot experiments were used to study phosphatase production, inorganic P release from lecithin and soil organic P compounds, and the P uptake and growth of lettuce seedlings induced by L. enzymogenes LE16. This bacterium hydrolyzed lecithin by the release of acid, neutral and alkaline phosphatases in culture solutions with a pH ranging from 4.0 to 10.0 at 12–36 °C. The hydrolysis reactions were unaffected by NH4+-N, NO3−-N or urea at equal N concentrations but were promoted by low inorganic P. Bacterial inoculation significantly decreased the organic P but increased the water-soluble P and Olsen P in the soil. Compared to the use of chemical fertilizers alone, the P uptake, biomass, and economic yield of lettuce increased by 16.41%, 11.58%, and 11.30%, respectively, with an increase in soil Olsen P in the L. enzymogenes LE16 plus chemical fertilizer treatment. Environmental adaptation and phosphatase production could be the mechanisms involved in soil P mineralization and plant pathogen suppression by L. enzymogenes LE16. This is the first demonstration of a new use of L. enzymogenes LE16 in agriculture beyond plant protection.

Short phosphate glass fiber - PLLA composite to promote bone mineralization.

The clinical application of composites seeks to exploit the mechanical and chemical properties of materials which make up the composite, and in researching polymer composites for biomedical applications the aim is usually to enhance the bioactivity of the polymer, while maintaining the mechanical properties. To that end, in this study medical grade Poly(L-lactic) acid (PLLA) has been reinforced with short phosphate-based glass fibers (PGF). The materials were initially mixed by melting PLLA granules with the short fibers, before being extruded to form a homogenous filament, which was pelletized and used as feedstock for compression moulding. As made the composite materials had a bending strength of 51 MPa ± 5, and over the course of eight weeks in PBS the average strength of the composite material was in the range 20-50 MPa. Human mesenchymal stromal cells were cultured on the surfaces of scaffolds, and the metabolic activity, alkaline phosphatase production and mineralization monitored over a three week period. The short fiber filler made no significant difference to cell proliferation or differentiation, but had a clear and immediate osteoinductive effect, promoting mineralization by cells at the material surface. It is concluded that the PLLA/PGF composite material offers a material with both the mechanical and biological properties for potential application to bone implants and fixation, particularly where an osteoinductive effect would be valuable.

Screening of perfused combinatorial 3D microenvironments for cell culture

Biomaterials combining biochemical and biophysical cues to establish close-to-extracellular matrix (ECM) models have been explored for cell expansion and differentiation purposes. Multivariate arrays are used as material-saving and rapid-to-analyze platforms, which enable selecting hit-spotted formulations targeting specific cellular responses. However, these systems often lack the ability to emulate dynamic mechanical aspects that occur in specific biological milieus and affect physiological phenomena including stem cells differentiation, tumor progression, or matrix modulation. We report a tailor-made strategy to address the combined effect of flow and biochemical composition of three-dimensional (3D) biomaterials on cellular response. We suggest a simple-to-implement device comprising (i) a perforated platform accommodating miniaturized 3D biomaterials and (ii) a bioreactor that enables the incorporation of the biomaterial-containing array into a disposable perfusion chamber. The system was upscaled to parallelizable setups, increasing the number of analyzed platforms per independent experiment. As a proof-of-concept, porous chitosan scaffolds with 1 mm diameter were functionalized with combinations of 5 ECM and cell-cell contact-mediating proteins, relevant for bone and dental regeneration, corresponding to 32 protein combinatorial formulations. Mesenchymal stem cells adhesion and production of an early osteogenic marker were assessed on-chip on static and under-flow dynamic perfusion conditions. Different hit-spotted biomaterial formulations were detected for the different flow regimes using direct image analysis. Cell-binding proteins still poorly explored as biomaterials components – amelogenin and E-cadherin – were here shown as relevant cell response modulators. Their combination with ECM cell-binding proteins – fibronectin, vitronectin, and type 1 collagen – rendered specific biomaterial combinations with high cell adhesion and ALP production under flow. The developed versatile system may be targeted at widespread tissue regeneration applications, and as a disease model/drug screening platform. Statement of SignificanceA perfusion system that enables cell culture in arrays of three-dimensional biomaterials under dynamic flow is reported. The effect of 31 cell-binding protein combinations in the adhesion and alkaline phosphatase (ALP) production of mesenchymal stem cells was assessed using a single bioreactor chamber. Flow perfusion was not only assessed as a classical enhancer/accelerator of cell growth and early osteogenic differentiation. We hypothesized that flow may affect cell-protein interactions, and that key components driving cell response may differ under static or dynamic regimes. Indeed, hit-spotted formulations that elicited highest cell attachment and ALP production on static cell culture differed from the ones detected for dynamic flow assays. The impacting role of poorly studied proteins as E-cadherin and amelogenin as biomaterial components was highlighted.

Benzylideneacetone Derivatives Inhibit Osteoclastogenesis and Activate Osteoblastogenesis Independently Based on Specific Structure-Activity Relationship.

(E)-3,4-Dihydroxybenzylideneacetone (compound 1) inhibited receptor activator of NF-κB ligand-induced osteoclastogenesis of C57BL/6 bone marrow monocyte/macrophages with IC50 of 7.8 μM (IC50 of alendronate, 3.7 μM) while stimulating the differentiation of MC3T3-E1 osteoblastic cells, accompanied by the induction of Runt-related transcription factor 2, alkaline phosphatase, and osteocalcin. (E)-4-(3-Hydroxy-4-methoxyphenyl)-3-buten-2-one (compound 2c) showed a dramatically increased osteoclast-inhibitory potency with IC50 of 0.11 μM while sustaining osteoblast-stimulatory activity. (E)-4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one (compound 2g) stimulated alkaline phosphatase production 2-fold at 50 μM without changing osteoclast-inhibitory activity, compared with compound 1. Oral administration of compounds 1, 2c, and 2g prevented ovariectomy-induced osteoporosis in ddY mice to a degree proportional to their osteoclastogenesis-inhibitory potencies. The administration of 1 (mg/kg)/d compound 2c ameliorated histomorphometry of osteoporotic bone to a degree comparable with 10 (mg/kg)/d alendronate. Conclusively, the in vitro capacity of a few benzylideneacetone derivatives to inhibit osteoclastogenesis supported by independent osteoblastogenesis activation was convincingly reflected in in vivo management of osteoporosis, suggesting a potential novel therapeutics for osteopenic diseases.

Extracellular Alkaline Phosphatase from Mangrove Soil Yeast

Alkaline phosphatase is a widely used enzyme in research and industry. Yeast is one of a microbial group that has the potency to produce alkaline phosphatase. This study aims to screen yeast from mangrove in Surabaya’s East Coast with the potential of alkaline phosphatase and to optimize the production of these enzymes. Screening test includes the measurement of phosphate solubilization index and concentration of soluble phosphate. Yeast with potential of phosphate solubilization will be used to measured the enzyme activity and also further optimized for extracellular alkaline phosphatase production. Optimization was done by determining the incubation time and a variety of combinations of phosphate source and temperature. The incubation time was adjusted to 7 days. Ca 3 (PO 4 ) 2 and KH 2 PO 4 as the phosphate source and incubation temperature were adjusted to 29° C, 45° C, and 55° C. The results showed that isolates with the highest phosphate solubilization index were W1.1 and G3.2 (1.18 for W1.1 and 1.15 for G3.2). Yeast with the ability to solubilize phosphate was assumed to be Candida genus. W1.1 and G3.2 produced soluble phosphate in a concentration of 0.50 ppm and 0.77 ppm at the 7th day respectively. Candida G3.2 has higher enzyme activity than Candida W1.1. Candida G3.2 has optimal production for 3 days incubation time with a combination of temperature 29 °C and Ca 3 (PO4) 2 as the phosphate source which caused 97.8 U/mL enzyme activity.

Bioremoval of toxic substances in synthetic wastewater using Trichoderma pubescens (NPK2), isolated from mangrove soil

Eutrophication drives water contamination, and the primary responsible pollutant is phosphorus in hydric systems. Based on phosphatase (EC 3.1.3.2) capacity to convert organic phosphorus into inorganic compounds, the power of phosphatase production in four fungal isolates and the potential of these organisms trapped in polyurethane foams to remove phosphorus from domestic effluents were investigated. The fungal biomass was grown in a minimal medium plus potassium (1 and 4 g L−1) containing blocks (0.71 mm3) of commercial polyurethane foam. A 32 factorial design was set up to verify the influence of immobilization time and foam supply on phosphatase production by the fungi. Thereafter, the effects of reaction time and volume of domestic effluent were simultaneously evaluated. All fungi produced more phosphatase after immobilization by entrapment. The isolates Aspergillus niger, and A. flavus had higher phosphatase activity (229 µmol h−1) after 192 and 96 h of growth, respectively. Based on the rotational central composite planning, the two independent variables were identified as significant factors (p<0.05) to produce phosphatases, and the maximum activity was obtained with 96 h of immobilization and 9 foam blocks for A. flavus, 72 h of immobilization and 12 blocks for Talaromyces pinophilus and Trichoderma asperellum, and 72 h of immobilization and 15 blocks for A. niger. The immobilized system of A. flavus, A. niger, and T. asperellum could remove 99.9% of the phosphorus in 1 h 10 m, while that of T. pinophilus removed phosphorus at 2 h after incubation with the domestic effluent. Overall, all fungal isolates are potential bioremediators of phosphorus in domestic effluents.

Salt stress and hydroxyectoine enhance phosphate solubilisation and plant colonisation capacity of Kosakonia radicincitans

Gram-negative bacterial endophytes have attracted research interest caused by their advantageous over epiphytic bacteria in plant nutrition and protection. However, research on these typically Gram-negative endophytes has deficiencies concerning the role of cultivation and pre-formulation strategies on further plant colonisation capabilities. Besides, the influence of cultivation conditions and osmotic stress within bacterial endophytes on their phosphate solubilising ability has not yet been addressed. By pre-conditioning cells with an osmoadaptation and a hydroxyectoine accumulation approach, this research aimed at enhancing the capability of the plant growth promoting bacterium Kosakonia radicincitans strain DSM 16656T to both solubilise phosphate and colonise plant seedlings. The results showed that halotolerant bacterial phenotypes increased the root-colonising capability by approximately 3-fold and presented growth-promoting effects in radish plants. Interestingly, findings also demonstrated that salt stress in the culture media along with the accumulation of hydroxyectoine led to an increase in the in vitro phosphate-solubilising ability by affecting the production of acid phosphatases, from 1.24 to 3.34 U mg-1 for non-salt stressed cells and hydroxyectoine-added cells respectively. Thus, this approach provides a useful knowledge upon which the salt stress and compatible solutes in bacteria endophytes can confer phenotypic adaptations to support the eco-physiological performance concerning phosphate-solubilising abilities and endosphere establishment.

Nutrient regeneration mediated by extracellular enzymes in water column and interstitial water through a microcosm experiment

In coastal lakes the role of microorganisms in driving nutrients regeneration at different water depths and in sediments is not yet fully understood. The dynamics of microbial (algal and bacterial) abundance and bacterial activities involved in organic matter transformation were measured, together with nutrient concentrations, through a microcosm experiment set up using the oligotrophic Faro lake as a study model over a total period of 15 days and with a four-day frequency. Water column at different depths (surface, middle and bottom) and interstitial water obtained by sediment centrifugation were used in appropriate ratios (mixed 1:1 with surface waters) to fill 21-Litre plastic aquaria in order to simulate processes occurring in natural conditions. At early experimental period, the sharp decrease of dissolved organic nutrients and the abundant production of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) in correspondence with high phytoplankton abundance in bottom and interstitial water reflected the relevance of organic nutrients for inorganic nutrients regeneration and phytoplankton growth. Size fractionation of LAP and AP as well as the positive relationship between microbial compartments suggested that bacteria and phytoplankton worked in close reciprocal synergy, and coupling of nitrogen and phosphorus regeneration, especially in bottom and interstitial waters, was observed. At later experimental period, the change in bacterial community, especially the increase of filamentous shaped cells, together with a simultaneous increase of protozoan abundance indicated that nutrient replenishment made the microbial loop structure more competitive. In oligotrophic conditions, such as those in Faro lake, organic nutrient enrichment of bottom and interstitial waters was associated with changes in the bacterial community, with consequent stimulation of extracellular enzymes to support phytoplankton growth. Nutrient availability from microbial regeneration resulted in an increased complexity of the microbial loop structure, with bacteria and phytoplankton adopting specific strategies to respond to the changing environment.

Multiple phosphorus acquisition strategies adopted by fine roots in low-fertility soils in Central Amazonia

Background and aimsAncient Amazon soils are characterised by low concentrations of soil phosphorus (P). Therefore, it is hypothesised that plants may invest a substantial proportion of their resources belowground to adjust their P-uptake strategies, including root morphological, physiological (phosphatase enzyme activities) and biotic (arbuscular mycorrhizal (AM) associations) adaptations. Since these strategies are energy demanding, we hypothesise that trade-offs between morphological traits and root phosphatase exudation and symbiotic associations would occur. Specifically, we expected that plants which invest in finer roots, and therefore have greater ability to explore large soil volumes, would have a high investment in physiological adaptations such as enhanced phosphatase production. In contrast, we expected that plants with predominantly thicker roots would invest more in symbiotic associations, in which carbon is traded for P acquired from AM fungal communities.MethodsWe collected absorptive roots (<2 mm diameter) from a lowland Central Amazon forest near Manaus, Brazil. We measured fine root diameter, specific root length (SRL), specific root area (SRA), root tissue density (RTD), root phosphatase activity (APase) and arbuscular mycorrhizal (AM) fungi colonisation.ResultsRoot morphological traits were related to APase activity, with higher APase activity in roots with higher SRL and SRA but lower RTD. However, the degree of AM colonisation was not related to any measured root morphological trait.ConclusionsFine absorptive roots likely benefit from having low RTD, high SRL, SRA and APase exudation to acquire P efficiently. However, because AM colonisation was not related to root morphology, we suggest that investment in multiple P-uptake strategies is required for maintaining productivity in Central Amazon forests.

Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels

Escherichia coli have developed one of the most efficient regulatory response mechanisms to phosphate starvation. The machinery involves a cascade with a two-component system (TCS) that relays the external signal to the genetic circuit, resulting in a feedback response. Achieving a quantitative understanding of this system has implications in synthetic biology and biotechnology, for example, in applications for wastewater treatment. To this aim, we present a computational model and experimental results with a detailed description of the TCS, consisting of PhoR and PhoB, together with the mechanisms of gene expression. The model is parameterised within the feasible range, and fitted to the dynamic response of our experimental data on PhoB as well as PhoA, the product of this network that is used in alkaline phosphatase production. Deterministic and stochastic simulations with our model predict the regulation dynamics in higher external phosphate concentrations while reproducing the experimental observations. In a cycle of simulations and experimental verification, our model predicts and explores phenotypes with various synthetic promoter designs that can optimise the inorganic phosphate intake in E. coli. Sensitivity analysis demonstrates that the Pho-controlled genes have a significant influence over the phosphate response. Together with experimental findings, our model should thus provide insights for the investigations on engineering new sensors and regulators for living technologies.

Sialyllactose and Galactooligosaccharides Promote Epithelial Barrier Functioning and Distinctly Modulate Microbiota Composition and Short Chain Fatty Acid Production In Vitro.

Human milk oligosaccharides (HMO) and prebiotic oligosaccharides are proposed to confer several health benefits to the infant. They shape the microbiota, have anti-inflammatory properties, and support epithelial barrier functioning. However, in order to select the best oligosaccharides for inclusion in infant formulas, there is a need to increase our understanding of the specific effects of HMO and prebiotics on the host immune system. Therefore, we investigated the effects of the HMO sialyllactose (SL), and galactooligosaccharides (GOS) on epithelial barrier functioning, microbiota composition, and SCFA production. The effect of GOS and SL on epithelial barrier functioning and microbiota composition was investigated using in vitro models. Epithelial barrier function was investigated by transcriptome analysis of fully polarized Caco-2 cells exposed for 6 h to SL or GOS. In addition, epithelial cell growth, alkaline phosphatase production, and re-epithelization was studied. Further, we investigated the effect of SL and GOS on microbiota composition and SCFA production using in vitro fecal batch cultures. Transcriptome analysis showed that SL and GOS both induced pathways that regulate cell cycle control. This gene-expression profile translated to a phenotype of halted proliferation and included the induction of alkaline phosphatase activity, a marker of epithelial cell differentiation. SL and GOS also promoted re-epithelialization in an in vitro epithelial wound repair assay. SL and GOS did show distinct modulation of microbiota composition, promoting the outgrowth of Bacteroides and bifidobacteria, respectively, which resulted in distinct changes in SCFA production profiles. Our results show that SL and GOS can both modulate epithelial barrier function by inducing differentiation and epithelial wound repair, but differentially promote the growth of specific genera in the microbiota, which is associated with differential changes in SCFA profiles.