Interspecies-cooperations of abutilon theophrasti with root colonizing microorganisms disarm BOA-OH allelochemicals.

A facultative, microbial micro-community colonizing roots of Abutilon theophrasti Medik. supports the plant in detoxifying hydroxylated benzoxazolinones. The root micro-community is composed of several fungi and bacteria with Actinomucor elegans as a dominant species. The yeast Papiliotrema baii and the bacterium Pantoea ananatis are actively involved in the detoxification of hydroxylated benzoxazolinones by generating H2O2. At the root surface, laccases, peroxidases and polyphenol oxidases cooperate for initiating polymerization reactions, whereby enzyme combinations seem to differ depending on the hydroxylation position of BOA-OHs. A glucosyltransferase, able to glucosylate the natural benzoxazolinone detoxification intermediates BOA-5- and BOA-6-OH, is thought to reduce oxidative overshoots by damping BOA-OH induced H2O2 generation. Due to this detoxification network, growth of Abutilon theophrasti seedlings is not suppressed by BOA-OHs. Polymer coats have no negative influence. Alternatively, quickly degradable 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one can be produced by the micro-community member Pantoea ananatis at the root surfaces. The results indicate that Abutilon theophrasti has evolved an efficient strategy by recruiting soil microorganisms with special abilities for different detoxification reactions which are variable and may be triggered by the allelochemical´s structure and by environmental conditions.

OrganoCat pretreatment of perennial plants: Synergies between a biogenic fractionation and valuable feedstocks.

A successful biorefinery needs to align suitable pretreatment with sustainable production of biomasses. Herein, four perennial plants, (Sida, Silphium, Miscanthus and Szarvasi) regarded as promising feedstocks for biorefineries were subjected to the OrganoCat pretreatment. The technology was successfully applied to the different perennial plants revealing that pretreatment of grasses was more efficient than of non-grasses. Thorough analyses of the lignocellulose - before and after fractionation - enabled a detailed description of the fate of cellulosic, non-cellulosic polysaccharides and lignin during the pretreatment. Especially Szarvasi pulp displayed outstanding results in terms of fractionation efficiency and enzymatic digestibility, though in all cases successful lignocellulose fractionation was observed. These insights into the structural composition of different perennial plant species and the impact of the OrganoCat pretreatment on the plant material leads to useful information to strategically adapt such processes to the individual lignocellulosic material aiming for a full valorisation.

6-Hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one-A degradable derivative of natural 6-Hydroxybenzoxazolin-2(3H)-one produced by Pantoea ananatis.

Pantoea ananatis is a bacterium associated with other microorganisms on Abutilon theophrasti Medik. roots. It converts 6-hydroxybenzoxazolin-2(3H)-one (BOA-6-OH), a hydroxylated derivative of the allelochemical benzoxazolin-2(3H)-one, into 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one. The compound was identified by NMR and mass spectrometric methods. In vitro synthesis succeeded with Pantoea protein, with isolated proteins from the Abutilon root surface or with horseradish peroxidase in the presence of nitrite and H2O2. Nitro-BOA-6-OH is completely degraded further by Pantoea ananatis and Abutilon root surface proteins. Under laboratory conditions, 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one inhibits Lepidium sativum seedling growth whereas Abutilon theophrasti is much less affected. Although biodegradable, an agricultural use of 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one is undesirable because of the high toxicity of nitro aromatic compounds to mammals.

Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe(2+) ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis.

A novel isolated Terrabacter-like bacterium can mineralize 2-aminopyrimidine, the principal metabolite of microbial sulfadiazine degradation.

Recently we showed that during the degradation of sulfadiazine (SDZ) by Microbacterium lacus strain SDZm4 the principal metabolite 2-aminopyrimidine (2-AP) accumulated to the same molar amount in the culture as SDZ disappeared (Tappe et al. Appl Environ Microbiol 79:2572-2577, 2013). Although 2-AP is considered a recalcitrant agent, long-term lysimeter experiments with (14)C-pyrimidine labeled SDZ ([(14)C]pyrSDZ) provided indications for substantial degradation of the pyrimidine moiety of the SDZ molecule. Therefore, we aimed to enrich 2-AP degrading bacteria and isolated a pure culture of a Terrabacter-like bacterium, denoted strain 2APm3. When provided with (14)C-labeled SDZ, M. lacus strain SDZm4 degraded [(14)C]pyrSDZ to [(14)C]2-AP. Resting cells of 2APm3 at a concentration of 5 × 10(6) cells ml(-1) degraded 62 µM [(14)C]2-AP to below the detection limit (0.6 µM) within 5 days. Disappearance of 2-AP resulted in the production of at least two transformation products (M1 and M2) with M2 being identified as 2-amino-4-hydroxypyrimidine. After 36 days, the transformation products disappeared and 83 % of the applied [(14)C]2-AP radioactivity was trapped as (14)CO2. From this we conclude that a consortium of two species should be able to almost completely degrade SDZ in soils.

Abutilon theophrasti's defense against the allelochemical benzoxazolin-2(3H)-one: support by Actinomucor elegans.

Abutilon theophrasti Medik., previously found to be rather insensitive to benzoxazinoid containing rye mulch and the allelochemical benzoxazolin-2(3H)-one (BOA), can be associated with the zygomycete Actinomucor elegans, whereby the fungus colonizes the root relatively superficially and mainly in the maturation zone. The fungus mitigates necrosis of the cotyledons when seedlings are incubated with 2 mM BOA, in contrast to those that lack the fungus. In liquid cultures of the fungus, tryptophan was identified. The accumulation of tryptophan is increased in presence of BOA. This amino acid seems to be important in protecting Abutilon against BOA and its derivatives since it suppressed the accumulation of BOA derived, highly toxic 2-aminophen-oxazin-3-one (APO) in the medium and on the root surface during BOA incubations of Abutilon seedlings. Although A. elegans is insensitive to BOA and APO, the fungus is not able to protect the plant against harmful effects of APO, when seedlings are treated with the compound. Abutilon can detoxify BOA via BOA-6-OH glucosylation probably by a cell wall associated glucosyltransferase, but only low amounts of the product accumulate. Low tryptophan concentrations can contribute to a degradation of the toxic intermediate BOA-6-OH by Fenton reactions, whereby the amino acid is oxidized. One of the oxidation products was identified as 4(1H)-quinolinone, which is the core substructure of the quorum sensing molecule 2-heptyl-3-hydroxy-4-quinolone. The mutualistic association of Abutilon theophrasti with Actinomucor elegans is considered as opportunistic and facultative. Such plant-fungus associations depend rather likely on environmental conditions, such as the mode of fertilization.

Chlordecone fate and mineralisation in a tropical soil (andosol) microcosm under aerobic conditions.

Chlordecone is a persistent organochlorine insecticide that, even decades after its ban, poses a threat to the environment and human health. Nevertheless, its environmental fate in soils has scarcely been investigated, and elementary data on its degradation and behaviour in soil are lacking. The mineralisation and sorption of chlordecone and the formation of possible metabolites were evaluated in a tropical agricultural andosol. Soil microcosms with two different soil horizons (S-A and S-B) were incubated for 215 days with 14C-chlordecone. At five different times (1, 33, 88, 150 and 215 days) the extractability of 14C-chlordecone was analysed. Mineralisation was monitored using 14CO2 traps of NaOH. The appearance of metabolites was studied using thin layer and gas chromatography techniques. At the end of the experiment, the water soluble 14C-activity was 2% of the remaining 14C-chlordecone for S-A and 8% for S-B. Only 12% of the remaining activity was non extractable and more than 80% remained extractable with organic solvents. For the first time to our knowledge, a significant mineralisation of chlordecone was measured in a microcosm under aerobic conditions (4.9% for S-A and 3.2% for S-B of the initial 14C-activity). The drastically lower emission of 14CO2 in sterilised microcosms indicated the biological origin of chlordecone mineralisation in the non-sterilised microcosms. No metabolites could be detected in the soil extracts. The mineralisation rate of chlordecone decreased by one order of magnitude throughout the incubation period. Thus, the chlordecone content in the soil remained large. This study confirms the existence of chlordecone degrading organisms in a tropical andosol. The reasons why their activity is restricted should be elucidated to allow the development of bioremediation approaches. Possible reasons are a heterogeneous distribution a chlordecone between sub-compartments with different microbial activities or a degradation of chlordecone by co-metabolic processes controlled by a limited supply of nutrients.

Structure elucidation of the thermal degradation products of the nucleotide cofactors NADH and NADPH by nano-ESI-FTICR-MS and HPLC-MS.

Redox cofactors like NADH and NADPH are essential for the catalytic activity of several oxidoreductases. Here, we describe a comparative study of the thermal degradation products of both cofactors in the dry and liquid states. The degradation products were first separated, detected, and quantified by high-performance liquid chromatography (HPLC). Subsequently, selected main fractions were investigated by nanoelectrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (MS). Additionally, HPLC-MS was used to elucidate the structure of all degradation products. From these data, degradation pathways for both the liquid and the solid states were elucidated. Thermal degradation in water is significantly faster compared to degradation in the solid state. Hydrolysis and oxidative ring opening of the reduced nicotinamide adenine dinucleotide (phosphate) were shown to be the main reaction paths. Surprisingly, no significant differences were observed between the degradation of both cofactors in solution and in the solid state. Our results demonstrate that the stability of both cofactors is not limiting at moderate temperatures if they are used in the dry state (e.g., solid/gas catalysis). Significant degradation of dry cofactors was only observed under conditions, which are usually not appropriate for biocatalysis (>95 °C). Besides, the situation is completely different in solution where degradation is already observed at moderate temperatures.