Phosphorus deficiencies invoke optimal allocation of exoenzymes by ectomycorrhizas.
ABSTRACT: Ectomycorrhizal (EM) fungi can acquire phosphorus (P) through the production of extracellular hydrolytic enzymes (exoenzymes), but it is unclear as to the manner and extent native EM fungal communities respond to declining soil P availability. We examined the activity of six exoenzymes (xylosidase, N-acetyl glucosaminidase, β-glucosidase, acid phosphomonoesterase, acid phosphodiesterase [APD], laccase) from EM roots of Pseudotsuga menzesii across a soil podzolization gradient of coastal British Columbia. We found that APD activity increased fourfold in a curvilinear association with declining inorganic P. Exoenzyme activity was not related to organic P content, but at a finer resolution using 31P-NMR, there was a strong positive relationship between APD activity and the ratio of phosphodiesters to orthophosphate of surface organic horizons (forest floors). Substantial increases (two- to fivefold) in most exoenzymes were aligned with declining foliar P concentrations of P. menzesii, but responses were statistically better in relation to foliar nitrogen (N):P ratios. EM fungal species with consistently high production of key exoenzymes were exclusive to Podzol plots. Phosphorus deficiencies in relation to N limitations may provide the best predictor of exoenzyme investment, reflecting an optimal allocation strategy for EM fungi. Resource constraints contribute to species turnover and the assembly of distinct, well-adapted EM fungal communities.
Project description:Microbial heterotopic metabolism in the ocean is fueled by a supply of essential nutrients acquired via exoenzymes catalyzing depolymerization of high-molecular-weight compounds. Although the rates of activity for a variety of exoenzymes across various marine environments are well established, the factors regulating the production of these exoenzymes, and to some extent their correlation with microbial community composition, are less known. This study focuses on addressing these challenges using a mesocosm experiment that compared a natural seawater microbial community (control) and exposed (to oil) treatment. Exoenzyme activities for ?-glucosidase, leucine aminopeptidase (LAP), and lipase were significantly correlated with dissolved nutrient concentrations. We measured correlations between carbon- and nitrogen-acquiring enzymes (?-glucosidase/lipase versus LAP) and found that the correlation of carbon-acquiring enzymes varies with the chemical nature of the available primary carbon source. Notably, a strong correlation between particulate organic carbon and ?-glucosidase activity demonstrates their polysaccharide depolymerization in providing the carbon for microbial growth. Last, we show that exoenzyme activity patterns are not necessarily correlated with prokaryotic community composition, suggesting a redundancy of exoenzyme functions among the marine microbial community and substrate availability. This study provides foundational work for linking exoenzyme function with dissolved organic substrate and downstream processes in marine systems.IMPORTANCE Microbes release exoenzymes into the environment to break down complex organic matter and nutrients into simpler forms that can be assimilated and utilized, thereby addressing their cellular carbon, nitrogen, and phosphorus requirements. Despite its importance, the factors associated with the synthesis of exoenzymes are not clearly defined, especially for the marine environment. Here, we found that exoenzymes associated with nitrogen and phosphorus acquisition were strongly correlated with inorganic nutrient levels, while those associated with carbon acquisition depended on the type of organic carbon available. We also show a linear relationship between carbon- and nitrogen-acquiring exoenzymes and a strong correlation between microbial biomass and exoenzymes, highlighting their significance to microbial productivity. Last, we show that changes in microbial community composition are not strongly associated with changes in exoenzyme activity profiles, a finding which reveals a redundancy of exoenzyme activity functions among microbial community. These findings advance our understanding of previously unknown factors associated with exoenzyme production in the marine environment.
Project description:C3-like exoenzymes are ADP-ribosyltransferases that specifically modify some Rho GTPase proteins, leading to their sequestration in the cytoplasm, and thus inhibiting their regulatory activity on the actin cytoskeleton. This modification process goes through three sequential steps involving NAD-hydrolysis, Rho recognition, and binding, leading to Rho ADP-ribosylation. Independently, three distinct residues within the ARTT loop of the C3 exoenzymes are critical for each of these steps. Supporting the critical role of the ARTT loop, we have shown previously that it adopts a distinct conformation upon NAD binding. Here, we present seven wild-type and ARTT loop-mutant structures of C3 exoenzyme of Clostridium botulinum free and bound to its true substrate, NAD, and to its NAD-hydrolysis product, nicotinamide. Altogether, these structures expand our understanding of the conformational diversity of the C3 exoenzyme, mainly within the ARTT loop.
Project description:C3 exoenzymes (members of the ADP-ribosyltranferase family) are produced by Clostridium botulinum (C3bot1 and -2), Clostridium limosum (C3lim), Bacillus cereus (C3cer), and Staphylococcus aureus (C3stau1-3). These exoenzymes lack a translocation domain but are known to specifically inactivate Rho GTPases in host target cells. Here, we report the crystal structure of C3bot1 in complex with RalA (a GTPase of the Ras subfamily) and GDP at a resolution of 2.66 A. RalA is not ADP-ribosylated by C3 exoenzymes but inhibits ADP-ribosylation of RhoA by C3bot1, C3lim, and C3cer to different extents. The structure provides an insight into the molecular interactions between C3bot1 and RalA involving the catalytic ADP-ribosylating turn-turn (ARTT) loop from C3bot1 and helix alpha4 and strand beta6 (which are not part of the GDP-binding pocket) from RalA. The structure also suggests a molecular explanation for the different levels of C3-exoenzyme inhibition by RalA and why RhoA does not bind C3bot1 in this manner.
Project description:Ectomycorrhizal (EM) fungi play vital roles in ensuring host plants' health, plant diversity, and the functionality of the ecosystem. However, EM fungal diversity, community composition, and underlying assembly processes in Inner Mongolia, China, where forests are typically semiarid and cold-temperate zones, attract less attention. In this study, we investigated EM fungal communities from 63 root samples of five common pine plants in Inner Mongolia across 1,900 km using Illumina Miseq sequencing of the fungal internal transcribed spacer 2 region. We evaluated the impact of host plant phylogeny, soil, climatic, and spatial variables on EM fungal diversity and community turnover. Deterministic vs. stochastic processes for EM fungal community assembly were quantified using β-nearest taxon index scores. In total, we identified 288 EM fungal operational taxonomic units (OTUs) belonging to 31 lineages, of which the most abundant lineages were <i>Tomentella-Thelephora</i>, <i>Wilcoxina</i>, <i>Tricholoma</i>, and <i>Suillus-Rhizopogon</i>. Variations in EM fungal OTU richness and community composition were significantly predicted by host phylogeny, soil (total nitrogen, phosphorus, nitrogen-phosphorus ratio, and magnesium), climate, and spatial distance, with the host plant being the most important factor. β-nearest taxon index demonstrated that both deterministic and stochastic processes jointly determined the community assembly of EM fungi, with the predominance of stochastic processes. At the Saihanwula site selected for preference analysis, all plant species (100%) presented significant preferences for EM fungi, 54% of abundant EM fungal OTUs showed significant preferences for host plants, and 26% of pairs of plant species and abundant fungal OTUs exhibited remarkably strong preferences. Overall, we inferred that the high diversity and distinctive community composition of EM fungi associated with natural pine species in Inner Mongolia and the stochastic processes prevailed in determining the community assembly of EM fungi. Our study shed light on the diversity and community assembly of EM fungi associated with common pine species in semiarid and cold temperate forests in Inner Mongolia, China, for the first time and provided a better understanding of the ecological processes underlying the community assembly of mutualistic fungi.
Project description:Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression (quality/quantity) of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w/v of dead intact American cockroach (Periplaneta americana) or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion.
Project description:C3 exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix-loop-helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix alpha3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.
Project description:Pseudomonas aeruginosa (PA) expresses the type III secretion system (T3SS) and effector exoenzymes that interfere with intracellular pathways. Natural killer (NK) cells play a key role in antibacterial immunity and their activation is highly dependent on IL-12 produced by myeloid cells. We studied PA and NK cell interactions and the role of IL-12 using human peripheral blood mononuclear cells, sorted human NK cells, and a human NK cell line (NK92). We used a wild-type (WT) strain of PA (PAO1) or isogenic PA-deleted strains to delineate the role of T3SS and exoenzymes. Our hypotheses were tested in vivo in a PA-pneumonia mouse model. Human NK cells or NK92 cell line produced low levels of IFN-? in response to PA without IL-12 stimulation, whereas PA significantly increased IFN-? after IL-12 priming. The modulation of IFN-? production by PA required bacteria-to-cell contact. Among T3SS effectors, exoenzyme T (ExoT) upregulates IFN-? production and control ERK activation. In vivo, ExoT also increases IFN-? levels and the percentage of IFN-?+ NK cells in lungs during PA pneumonia, confirming in vitro data. In conclusion, our results suggest that T3SS could modulate the production of IFN-? by NK cells after PA infection through ERK activation.
Project description:Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum.
Project description:In renal failure, hyperphosphatemia is common and correlates with increased mortality making phosphate removal a key priority for dialysis therapy. We investigated phosphate clearance, removal and serum level, and factors associated with phosphate control in patients undergoing continuous ambulatory (CAPD), continuous cyclic (CCPD) and automated (APD) peritoneal dialysis (PD). In 154 prevalent PD patients (mean age 53.2?±?17.6 year, 59% men, 47% anuric), 196 daily collections of urine and 368 collections of dialysate were evaluated in terms of renal, peritoneal and total (renal plus peritoneal) phosphorus removal (g/week), phosphate and creatinine clearances (L/week) and urea KT/V. Dialytic removal of phosphorus was lower in APD (1.34?±?0.62 g/week) than in CAPD (1.89?±?0.73 g/week) and CCPD (1.91?±?0.63 g/week) patients; concomitantly, serum phosphorus was higher in APD than in CAPD (5.55?±?1.61 vs. 4.84?±?1.23 mg/dL; p?<?0.05). Peritoneal and total phosphate clearances correlated with peritoneal (rho?=?0.93) and total (rho?=?0.85) creatinine clearances (p?<?0.001) but less with peritoneal and total urea KT/V (rho?=?0.60 and rho?=?0.65, respectively, p?<?0.001). Phosphate removal, clearance and serum levels differed between PD modalities. CAPD was associated with higher peritoneal removal and lower serum level of phosphate than APD.
Project description:Transformation of Fonsecaea pedrosoi into muriform cells enhances the resistance against phagocytosis and elimination by host immune cells, and links to the chronicity of chromoblastomycosis. Here, we aim to determine whether the muriform cells can reproduce in tissue without reverse transformation into hyphal form by using an experimental nu/nu-BALB/c mouse model of chromoblastomycosis due to F. pedrosoi. During the whole 81-day observation period, most of the hyphal inocula had transformed into muriform cells at 75 days postinoculation and maintained as this parasitic morphology till 81 days postinoculation simultaneously with increased fungal loads in tissue and the worsening of footpad lesion. Scanning and transmitting electronic microscope examinations showed that the muriform cells obtained in tissue or induced in vitro can reproduce daughter cells by dividing, and, meanwhile, the daughter cells had the potential to produce buds and grow into hyphae reversely. Furthermore, exoenzyme examination suggested that the profile of exoenzymes constituted by muriform cells was quite different from that constituted by hyphae although the assay showed both of them had obvious metabolic activity. By contrast, most muriform cells in the footpad gradually transformed into the elongated hyphae without obvious infiltration of inflammatory cells during repeated intraperitoneal administration of cyclophosphamide (50 mg/kg, per every other day) from 50 to 80 days postinoculation. Therefore, we infer that F. pedrosoi can reproduce by dividing as muriform cells in mouse tissue, and the morphological transformation between hyphal form and muriform cells is possibly associated with the host immune status.