Aminopeptidases in Caenorhabditis elegans and Panagrellus redivivus: detection using peptide and non-peptide substrates

Aminopeptidase activities were detected in extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus using the aminoacyl substrate L-alanine-4-nitroanilide. The activities exhibited similarities in Km (C. elegans = 2.22 mM; P. redivivus = 2.09 mM) and specific activity (C. elegans = 1.38 +/- 0.43 mAU min(-1) x g(-1); P. redivivus, 1.23 +/- 0.18m AU min(-1) microg(-1). Each is inhibited competitively by amastatin (C. elegans IC50 = 0.46 microM; P. redivivus IC50 = 15.90 microM) and non-competitively by leuhistin (C. elegans IC50 = 3.00 microM; P. redivivus IC50 = 37.35 microM). The bioactive peptides adipokinetic hormone and substance P decrease the apparent aminopeptidase activities of each extract suggesting that the peptides compete with the Ala-pNA as substrates. With each extract, adipokinetic hormone appeared to be the more effective substrate. Digestion of adipokinetic hormone by C. elegans and P. redivivus extracts in the presence and absence of 1 mM amastatin produced distinct chromatographic profiles that suggest different digestion patterns for the two species. However, amastatin had clear effects on chromatographic profiles from each species indicating that an aminopeptidase is involved in the digestion of the peptide substrates. The data presented indicate that extracts of free-living nematodes are capable of metabolizing peptide hormones, and that this metabolism involves substrate-selective aminopeptidases.     

Role of a FMRFamide-like family of neuropeptides in the pharyngeal nervous system of Caenorhabditis elegans

The nervous system of C. elegans has a remarkable abundance of flp genes encoding FMRFamide-like (FLP) neuropeptides. To provide insight into the physiological relevance of this neuropeptide diversity, we have tested more than 30 FLPs (encoded by 23 flps) for bioactivity on C. elegans pharynx. Eleven flp genes encode peptides that inhibit pharyngeal activity, while eight flp genes encode peptides that are excitatory. Three potent peptides (inhibitory, FLP-13A, APEASPFIRFamide; excitatory, FLP-17A, KSAFVRFamide; excitatory, FLP-17B, KSQYIRFamide) are encoded by flp genes, which, according to reporter gene constructs, are expressed in pharyngeal motoneurons. Thus, they may act through receptors localized on the pharyngeal muscle. The two other potent peptides, FLP-8 (excitatory AF1, KNEFIRFamide,) and FLP-11A (inhibitory, AMRNALVRFamide), appear to be expressed in extrapharyngeal neurons and are therefore likely to act either indirectly or as neurohormones. Intriguingly, a single neuron can express peptides that have potent but opposing biological activity in the pharynx. Only five flp genes encode neuropeptides that have no observable effect on the pharynx, but none of these have shown reporter gene expression in the pharyngeal nervous system. To examine the roles of multiple peptides produced from single precursors, a comparison was made between the bioactivity of different neuropeptides for five flp genes (flp-3, flp-13, flp-14, flp-17, and flp-18). For all but one gene (flp-14), the effects of peptides encoded by the same gene were similar. Overall, this study demonstrates the impressive neurochemical complexity of the simple circuit that regulates feeding in the nematode, C. elegans.     

Two FMRFamide-like peptides from the free-living nematode Panagrellus redivivus.

Peptides of the FXRFamide family, where X = M, I or L, are broadly distributed among invertebrates. Two such peptides were purified and sequenced from the free-living nematode, Panagrellus redivivus. Immunohistochemical techniques localized FMRFamide-like material in several regions of these organisms, including the nerve cords and, most prominently, in paired groups of cells located caudally to the base of the pharynx. RIA determinations gave an estimate of 2.8 nmol immunoreactive peptide/g of an acetone extract of P. redivivus. Four sequential HPLC purification steps, followed by sequencing by automated Edman degradation and FAB-MS, led to the identification of Ser-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SDPNFLRFamide) and Ser-Ala-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SADPNFLRFamide) as members of the FXRFamide family in this nematode.     

Development of a low-cost technology for mass production of the free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> as an alternative live food for first feeding fish larvae

The free-living nematode Panagrellus redivivus is a suitable food source for first feeding fish. In the present report, a new method for the mass production of P. redivivus is presented. The technique involves multiplication of the nematode in monoxenic (single microorganism: Saccharomyces cerevisiae) solid culture (fluid media supported by 1- to 4-cm(3) sponge cubes) in autoclavable plastic bags (size range: 50 x 30 cm to 75 x 67 cm). Two growing media were tested: oat-meal medium (OM), which is an oat-based medium (16.7% oat-meal flour in 0.8% saline solution), and purified ingredient medium (PIM), a semi-synthetic medium (1.64% meat peptone, 0.94% yeast extract, 12.6% corn starch, 0.24% glucose, 1.48% sunflower oil, in 0.8% saline solution). The bags were inoculated with 350 nematodes/g medium. After an average period of 12 days (11-13 days) at 25 degrees C, the average yield (number of nematodes/g medium) was 241 x 10(3) for OM and 333 x 10(3) for PIM in 12-l bags (50 x 30 cm). The production scale has currently reached a bag volume of 50 l (75 x 67 cm); using PIM and the conditions described above, it was possible to harvest more than 1.3 x 10(9) nematodes/bag (291 x 10(3) nematodes/g medium). In PIM, when sun flower oil was replaced with the same amount of fish oil or cod liver oil, yields of 259 x 10(3) and 290 x 10(3) nematodes/g medium, respectively, were attained. The technology for mass production and formulation of P. redivivus should enable fish-hatchery operators to rely on a cheap, standardised, and permanently available live food product for first feeding fish larvae.     

In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita)

Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.     

In vitro metabolism of an insect neuropeptide by homogenates of the nematode Caenorhabditis elegans

The cytosolic fraction of homogenates from the free-living soil nematode Caenorhabditis elegans is capable of metabolizing the insect neuropeptide adipokinetic hormone, a decapeptide blocked at the N-terminus by a pGlu residue. Analysis of digests by RP-HPLC and LC-MS revealed that an initial endoproteolytic cleavage step produced a heptapeptide with an unblocked N-terminus that can serve as a substrate for aminopeptidases. The aminopeptidase activity is depressed in the presence of the inhibitor amastatin; the initial product of the endoproteolytic step accumulates during incubation, and expected aminopeptidase product peptides are reduced in amount, as assessed by chromatographic peak size. The absence of some expected peptide fragments in the reaction mixtures suggests that multiple proteases contribute to short peptide half-lives. Comparison of the adipokinetic hormone digestion in C. elegans to that reported previously for insects reveals the same general pattern of peptide fragment production.     

Stereumin A-E, sesquiterpenoids from the fungus Stereum sp. CCTCC AF 207024

Five cadinane sesquiterpenoids, named stereumin A (1), B (2), C (3), D (4) and E (5) were isolated from the CHCl(3) extract of the culture broth of the fungal strain CCTCC AF 207024. Based on the sequences at the internal transcribed spacer (ITS) region and partial 28S rDNA, this fungus was identified as a Stereum sp. The structures of the five compounds were elucidated using spectroscopic data from 1D, 2D NMR and HRESIMS experiments, and the structures of 1 and 2 were further confirmed by single-crystal X-ray diffraction analysis. Compounds 1-5 showed nematicidal activities against the nematode Panagrellus redivivus at 400 mg l(-1). Among these five compounds, compounds 3 and 4 killed 84.4% and 94.9% of P. redivivus, respectively in 48 h.     

Responses of Heterodera glycines and Meloidogyne incognita to exogenously applied neuromodulators

Biogenic amines regulate important behaviours in nematodes and are associated with pharyngeal activity in plant-parasitic nematodes. A robust behavioural assay based upon nematode body movements was developed to expand the study of these and other neuroregulators in plant-parasitic nematodes. Dopamine, octopamine and serotonin each had significant but differing effects on the behaviour of soybean cyst nematode Heterodera glycines and root-knot nematode Meloidogyne incognita juveniles. Body movement frequency was increased twofold in H. glycines by 5 mM dopamine (P = 0.0001), but decreased by 50 mM dopamine in H. glycines (88%) and M. incognita (53%) (P < 0.0001). Movement frequency in both species was increased by 50-70% (P < 0.0001) by 50 mM octopamine, and 5 mM octopamine increased M. incognita movement frequency more than twofold (P < 0.0001). Movement frequency in each species was reduced by more than 90% by 5 mM serotonin (P < 0.0001). While amplitude of body movement in H. glycines was unaffected by any amine, it was significantly reduced in M. incognita by all amines (P < 0.0006). Stylet pulsing frequencies in either species were unaffected by dopamine or octopamine, but 5 mM serotonin stimulated pulsing in H. glycines by nearly 13-fold (P < 0.0001) and in M. incognita by more than 14-fold (P < 0.0001). The invertebrate neuropeptide FLRFamide (N-Phe-Leu-Arg-Phe) increased M. incognita body movement frequency 45% (P = 0.02) at 1 mM but did not affect stylet activity. Finally, H. glycines egg hatch was completely suppressed by 50 mM serotonin, and partially suppressed by 50 mM dopamine (75%; P < 0.0001) and 50 mM octopamine (55%; P < 0.0001).     

Coprinus comatus: A basidiomycete fungus forms novel spiny structures and infects nematode

Nematophagous basidiomycete fungi kill nematodes by trapping, endoparasitizing and producing toxin. In our studies Coprinus comatus (O.F.Müll. : Fr.) Pers. is found to be a nematode-destroying fungus; this fungus immobilizes, kills and uses free-living nematode Panagrellus redivivus Goodey and root-knot nematode Meloidogyne arenaria Neal. C. comatus produces an unusual structure designated spiny ball. Set on a sporophore-like branch, the spiny ball is a burr-like structure assembled with a large number of tiny tubes. Purified spiny balls exhibit moderate nematicidal activity. Experiments show that spiny balls are not chlamydospores because of the absence of nuclei in the structures and quick formation within 3 d in a young colony. Nematodes added to C. comatus cultures on potato-dextrose agar (PDA) and cornmeal agar (CMA) become inactive in hours. Infection of nematodes by the fungus occurs only after the nematodes are immobilized (feeble or dead), probably by a toxin. Electron micrographs illustrate that C. comatus infect P. redivivus by producing penetration pegs with which hyphae colonize nematode bodies. An infected nematode is digested and consumed within days and hyphae grow out of the nematode.     

The possible absence of cytochrome P-450 linked xenobiotic metabolism in helminths

The distribution of cytochrome P-450, b5 and associated oxidations, aniline hydroxylase, 7-ethoxycoumarin O-deethylase and 4-nitroanisole O-demethylase were studied in the parasitic nematode Heligmosomoides polygyrus, its mammalian host, Mus musculus and the free-living nematode Panagrellus redivivus. Cytochrome P-450, its associated oxidations and cytochrome b5 could not be detected in whole homogenates or subcellular fractions (mitochondrial, microsomal and soluble fractions) of either nematode under a variety of assay conditions which included attempted induction with sodium phenobarbital. P. redivivus was able to reduce 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene and azobenzene, which is predominantly microsomal. The implications of these results in terms of chemotherapy are discussed.     

Structure of the flavin site of Chromatium flavocytochrome c -552

Two flavin peptides have been isolated from $\text{Chromatium}$ cytochrome $\text{c}{}_{552}$ by digestion with pepsin and with trypsin-chymotrypsin, respectively. Acid hydrolysis and aminopeptidase digestion of the peptic peptide shows the presence of 1 mole each of threonine and cysteine and 2 of tyrosine, per mole of FAD. Edman degradation gives the sequence: tyr-thr-cys (flavin)-tyr. Tryptic-chymotryptic digestion yields a flavin tripeptide of the structure: thr-cys (flavin)-tyr. The N-terminal tyrosine in the peptic tetrapeptide shows a strong interaction with the flavin, as judged by CD spectra, and this may account for the resistance of the bridge sulfur to oxidation by cold performic acid. Both peptides show abnormally low fluorescence in the pure state (1 to 5% relative to riboflavin) and positive iodoplatinic test; the peptic peptide yields a positive, the trypticchymotryptic peptide negative ninhydrin reaction. The electrophoretic mobility of the major product of aminopeptidase digestion (presumably the thiazolidine form of cysteinyl-8α-FAD thiohemiacetal) and other properties of the two peptides are in accord with previous suggestions that the linkage of the flavin to the peptide is a thiohemiacetal.     

Enzymatic properties of the glycine d-alanine aminopeptidase of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji)

The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9.     

携带cip基因的酿酒酵母对自由生活线虫Panagrellus redivivus生长繁殖的影响

Photorhabdus luminescens细菌与昆虫病原异小杆属Heterorhabditis线虫专性共生。初生型共生细菌产生两种胞内晶体蛋白CipA and CipB,为共生线虫提供营养。为探索Cip蛋白是否对自由生活的全齿复活线虫Panagrellus redivivus具有类似的营养功能,建立了Cip蛋白的重组酿酒酵母表达体系,并用于饲喂无菌的P. redivivus线虫J1幼虫。重组酿酒酵母表达的Cip蛋白能为线虫所利用,表现为营养支持作用,体现为线虫生长发育速度的加快以及繁殖能力的提高,说明Cip蛋白能为此种自由生活线虫提供营养来源。     

Coprinus comatus damages nematode cuticles mechanically with spiny balls and produces potent toxins to immobilize nematodes

We reported recently a unique fungal structure, called the spiny ball, on the vegetative hyphae of Coprinus comatus (O. F. Müll.:Fr.) Pers. Although some observations regarding the role of this structure were presented, its function remained largely unknown. In this study, we showed that purified (isolated and washed) spiny balls could immobilize and kill the free-living nematode Panagrellus redivivus Goodey highly efficiently. Scanning electron microscopy studies illustrated that the spiny structure damaged the nematode cuticle, suggesting the presence of a mechanical force during the process of nematode immobilization. Severe injuries on nematode cuticles caused the leakage of inner materials of the nematodes. When these structures were ground in liquid nitrogen, their killing efficacy against nematodes was lost, indicating that the shape and the complete structure of the spiny balls are indispensable for their function. However, extraction with organic solvents never lowered their activity against P. redivivus, and the extracts showed no obvious effect on the nematode. We also investigated whether C. comatus was able to produce toxins which would aid in the immobilization of nematodes. In total, we identified seven toxins from C. comatus that showed activity to immobilize the nematodes P. redivivus and Meloidogyne incognita (Kofoid et White) Chitwood. The chemical structures of these toxins were identified with nuclear magnetic resonance, mass spectrometry, infrared, and UV spectrum analysis. Two compounds were found to be novel. The toxins found in C. comatus are O-containing heterocyclic compounds.     

Loss of parietal cell superoxide dismutase leads to gastric oxidative stress and increased injury susceptibility in mice

Mitochondrial superoxide dismutase (SOD2) prevents accumulation of the superoxide that arises as a consequence of oxidative phosphorylation. However, SOD2 is a target of oxidative/nitrosative inactivation, and reduced SOD2 activity has been demonstrated to contribute to portal hypertensive gastropathy. We investigated the consequences of gastric parietal cell-specific SOD2 deficiency on mitochondrial function and gastric injury susceptibility. Mice expressing Cre recombinase under control of the parietal cell Atpase4b gene promoter were crossed with mice harboring loxP sequences flanking the sod2 gene (SOD2 floxed mice). Cre-positive mice and Cre-negative littermates (controls) were used in studies of SOD2 expression, parietal cell function (ATP synthesis, acid secretion, and mitochondrial enzymatic activity), increased oxidative/nitrosative stress, and gastric susceptibility to acute injury. Parietal cell SOD2 deficiency was accompanied by a 20% (P < 0.05) reduction in total gastric SOD activity and a 93% (P < 0.001) reduction in gastric SOD2 activity. In SOD2-deficient mice, mitochondrial aconitase and ATP synthase activities were impaired by 36% (P < 0.0001) and 44% (P < 0.005), respectively. Gastric tissue ATP content was reduced by 34% (P < 0.002). Basal acid secretion and peak secretagogue (histamine)-induced acid secretion were reduced by 43% (P < 0.0001) and 40% (P < 0.0005), respectively. There was a fourfold (P < 0.02) increase in gastric mucosal apoptosis and 41% (P < 0.001) greater alcohol-induced gastric damage in the parietal cell SOD2-deficient mice. Our findings indicate that loss of parietal cell SOD2 leads to mitochondrial dysfunction, resulting in perturbed energy metabolism, impaired parietal cell function, and increased gastric mucosal oxidative stress. These alterations render the gastric mucosa significantly more susceptible to acute injury.     

Relationship of fat mass and serum estradiol with lower extremity bone in persons with chronic spinal cord injury

In the spinal cord injury (SCI) population, a relationship between adiposity and leg bone has not been reported, nor one between serum estradiol and leg bone mass. A cross-sectional, comparative study of 10 male pairs of monozygotic twins discordant for SCI was performed. Relationships were determined among bone mineral density (BMD), bone mineral content (BMC), lean mass, fat mass, and serum sex steroids. In the twins with SCI, significant relationships were evident between leg BMD or BMC with total body percent fat (r2= 0.49, P < 0.05; r2= 0.45, P = 0.05), leg fat mass (r2 = 0.76, P < 0.0005; r2= 0.69, P = 0.005), and serum estradiol (r2= 0.40, P = 0.05; r2= 0.37, P = 0.05). By stepwise regression analysis, in the twins with SCI, leg fat mass was found to be the single most significant predictor of leg BMD or BMC (F = 12.01, r2= 0.76, P = 0.008; F = 50.87, r2= 0.86, P < 0.0001). In the able-bodied twins, leg lean mass correlated with leg BMD and BMC (r2= 0.58, P = 0.01; r2= 0.87, P = 0.0001). By use of within-pair differences, significant correlations were found for leg lean mass loss with leg BMD loss (r2= 0.56, P = 0.01) or leg BMC loss (r2= 0.64, P = 0.0005). In conclusion, in twins with SCI, significant correlations were observed between fat mass and leg BMD or BMC as well as between serum estradiol values and leg BMD. The magnitude of the leg muscle mass loss was correlated with the magnitude of bone loss.     

Deciphering the Role of EGL-3 for Neuropeptides Processing in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Using High-Resolution Quadrupole–Orbitrap Mass Spectrometry

Neuropeptides are derived from large and inactive proteins which require endoproteolytic processing for the biosynthesis of the bioactive peptides. The maturation of pro-neuropeptide to neuropeptide is believed to be performed by ortholog pro-protein convertase EGL-3 in Caenorhabditis elegans (C. elegans). Furthermore, ortholog of Cathepsin L, CPL-1 are found in C. elegans and can potentially cleave paired basic amino acids at the N-terminal suggesting the presence of both pathways. The objective of this study was to decipher the role of EGL-3 in the proteolysis of FMRF amide-related peptides (FLPs) or neuropeptide-like proteins (NLPs) using synthetic surrogate peptides based on a universal enzymatic cleavage pattern published by Schechter and Berger and used widely in enzymology. The results show evidence that proteolysis controls FLP-21 and NLP-8 related neuropeptide levels in C. elegans. Surrogate peptides were degraded rapidly when exposed to C. elegans S9 fractions leading to the formation of specific peptide fragments related to EGL-3 and CPL-1 pathway. The results suggest that CPL-1 pathway does not compensate for the loss of the EGL-3 pathway. Proteolysis of pro-neuropeptides associated to FLP-21 and NLP-8 in elg-3 mutants are severely hampered leading to a lack of mature bioactive neuropeptides.     

The nematode Panagrellus redivivus is susceptible to killing by human pathogens at 37 degrees C

Caenorhabditis elegans has been used as a host for the study of bacteria that cause disease in mammals. However, a significant limitation of the model is that C. elegans is not viable at 37 degrees C. We report that the gonochoristic nematode Panagrellus redivivus survives at 37 degrees C and maintains its life cycle at temperatures up to and including 31.5 degrees C. The C. elegans pathogens Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, but not Yersinia pseudotuberculosis, reduced P. redivivus lifespan. Of four strains of Burkholderia multivorans tested, one reduced P. redivivus lifespan at both temperatures, one was avirulent at both temperatures and two strains reduced P. redivivus lifespan only at 37 degrees C. The mechanism by which one of these strains killed P. redivivus at 37 degrees C, but not at 25 degrees C, was investigated further. Killing required viable bacteria, did not involve bacterial invasion of tissues, is unlikely to be due to a diffusible, bacterial toxin and was not associated with increased numbers of live bacteria within the intestine of the worm. We believe B. multivorans may kill P. redivivus by a temperature-regulated mechanism similar to B. pseudomallei killing of C. elegans.     

Acanthocytes of Stropharia rugosoannulata function as a nematode-attacking device

Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.