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The anti-diabetic drug metformin is one of the most widely prescribed medicines in the world. Together with its degradation product guanylurea, it is a major pharmaceutical pollutant in wastewater treatment plants and surface waters. An operon comprising two genes of the ureohydrolase family in Pseudomonas and Aminobacter species has recently been implicated in metformin degradation. However, the corresponding proteins have not been characterized. Here we show that these genes encode a Ni 2+ -dependent enzyme that efficiently and specifically hydrolyzes metformin to guanylurea and dimethylamine. The active enzyme is a heteromeric complex of α- and β- subunits in which only the α-subunits contain the conserved His and Asp residues for the coordination of two Ni 2+ ions in the active site. A crystal structure of metformin hydrolase reveals an α 2 β 4 stoichiometry of the hexameric complex, which is unprecedented in the ureohydrolase family. By studying a closely related but more widely distributed enzyme, we find that the putative predecessor specifically hydrolyzes dimethylguanidine instead of metformin. Our findings establish the molecular basis for metformin hydrolysis to guanylurea as the primary pathway for metformin biodegradation and provide insight into the recent evolution of ureohydrolase family proteins in response to an anthropogenic compound. The diabetes drug metformin and its degradation product guanylurea are major pharmaceutical contaminants in waste and surface water. Here, a Ni2+-dependent enzyme that hydrolysed metformin to guanylurea and its evolutionary predecessor are presented.

Nitrogen availability is a growth-limiting factor in many habitats 1 , and the global nitrogen cycle involves prokaryotes and eukaryotes competing for this precious resource. Only some bacteria and archaea can fix elementary nitrogen; all other organisms depend on the assimilation of mineral or organic nitrogen. The nitrogen-rich compound guanidine occurs widely in nature 2 – 4 , but its utilization is impeded by pronounced resonance stabilization 5 , and enzymes catalysing hydrolysis of free guanidine have not been identified. Here we describe the arginase family protein GdmH (Sll1077) from Synechocystis sp. PCC 6803 as a Ni 2+ -dependent guanidine hydrolase. GdmH is highly specific for free guanidine. Its activity depends on two accessory proteins that load Ni 2+ instead of the typical Mn 2+ ions into the active site. Crystal structures of GdmH show coordination of the dinuclear metal cluster in a geometry typical for arginase family enzymes and allow modelling of the bound substrate. A unique amino-terminal extension and a tryptophan residue narrow the substrate-binding pocket and identify homologous proteins in further cyanobacteria, several other bacterial taxa and heterokont algae as probable guanidine hydrolases. This broad distribution suggests notable ecological relevance of guanidine hydrolysis in aquatic habitats. A bacterial enzyme is characterized and demonstrated to have Ni 2+ -dependent activity and high specificity for free guanidine enabling the bacteria to use guanidine as the sole nitrogen source for growth.

The effect of increasing seed borne incidence levels (0, 3, 6, 12, 24 and 48 %) of Bipolaris oryzae on brown spot epidemics and crop performance was studied in eleven field trials. These trials were conducted at two sites (Bagé – BA and Cachoeirinha – CA) in the major rice-growing region of Brazil over three seasons (2008 to 2010). Disease variables assessed over time were disease incidence (INC, %) on leaves prior to flowering, and disease severity (SEV, %) on flag leaves after flowering. Kernel infection (KI, %) by B. oryzae was assessed after harvest. Crop-related variables such as plant population density (PD) and yield (YLD) were also assessed. In only three trials, all in the 2009/10 season, which had well above-normal rainfall in the early season, was the disease found at vegetative stages. In those same trials, a significant effect of seed borne inoculum was found for the area under the disease progress curve of INC and SEV. Overall mean SEV at CA (1.67 %) was higher than at BA (0.22 %). Seed borne inoculum levels did not affect final SEV and KI, which was not correlated between each other. PD was significantly reduced with the increase of seed borne inoculum levels in seven out of eight trials and at levels as high as 48 % (2009/10 season). The seed borne inoculum levels did not affect YLD, although significantly reducing PD, which may be due to the rice having a low population compensated through tillering. The risk of yield loss by sowing B. oryzae-infected seeds seems to be low and the early onset of the disease caused by increased levels of seed borne inoculum was dependent on seasonal weather conditions.

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Δ1-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutantrsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS andP5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Δ1-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.

Different screening methods for selection of biological control agents (BCAs), for controlling soil and seed-borne diseases, are discussed. The shortcomings of laboratory methods focused on mechanism of action are discussed and we conclude that these methods should be used with caution if candidates with multifactorial or plant mediated mechanisms of control are to be obtained. In vitro screens may be useful for specific groups of microorganisms, thus, screens for antibiotics may be relevant for Streptomyces spp., and promising results have been obtained using soil plating or precolonized agar methods to screen for mycoparasitism and competitive saprophytic ability. Experience with screening in the Nordic programme 'Biological control of seed borne diseases in cereals' is summarized. Research in the four participating countries - Finland, Sweden, Norway and Denmark - followed the same paradigm: that of obtaining antagonists, well adapted to different Nordic environments, and developing them as effective BCAs. Potential antagonists were isolated from different sources and in planta screening methods were developed in order to optimize selection of antagonists effective against a range of seed borne pathogens. Screens in the laboratory or greenhouse were followed by screening in the field. The different screening procedures are compared and evaluated.[PUBLICATION ABSTRACT]

Nitrogen availability is a growth-limiting factor in many habitats , and the global nitrogen cycle involves prokaryotes and eukaryotes competing for this precious resource. Only some bacteria and archaea can fix elementary nitrogen; all other organisms depend on the assimilation of mineral or organic nitrogen. The nitrogen-rich compound guanidine occurs widely in nature , but its utilization is impeded by pronounced resonance stabilization , and enzymes catalysing hydrolysis of free guanidine have not been identified. Here we describe the arginase family protein GdmH (Sll1077) from Synechocystis sp. PCC 6803 as a Ni -dependent guanidine hydrolase. GdmH is highly specific for free guanidine. Its activity depends on two accessory proteins that load Ni instead of the typical Mn ions into the active site. Crystal structures of GdmH show coordination of the dinuclear metal cluster in a geometry typical for arginase family enzymes and allow modelling of the bound substrate. A unique amino-terminal extension and a tryptophan residue narrow the substrate-binding pocket and identify homologous proteins in further cyanobacteria, several other bacterial taxa and heterokont algae as probable guanidine hydrolases. This broad distribution suggests notable ecological relevance of guanidine hydrolysis in aquatic habitats.

A bioassay to screen fungal isolates for endophytic growth and antagonism against Fusarium verticillioides in maize was developed. The method was based on the commonly used toothpick inoculation method followed by measurement of stalk necrosis, and was designed to assure a direct introduction of the endophyte into the plant. Thirty-four fungal endophytes isolated from surface sterilized grass and maize stalks from Costa Rica, and four soil isolates, were tested for antagonism and endophytic growth. Six isolates gave less necrosis (P < 0.05) than the control treated with F. verticillioides alone, but only one isolate, Trichoderma koningii S8, reduced the stalk necrosis when the test was repeated. Reisolations from the stalk showed that none of the isolates were able to completely eliminate F. verticillioides from the maize stalk. It is concluded that F. verticillioides is a very strong competitor that is highly adapted to live in association with maize, and that an effective antagonist against F. verticillioides still remains to be found. The screening assay developed in this study may prove to be a useful tool to study the in vivo interactions between plant pathogens and antagonistic endophytes.

The antagonistic Trichoderma spp. isolates P1 and T3 differed in their ability to colonize and to compete in sphagnum peat moss and on wood chips. In peat supplemented with straw, isolate T3 produced twice as many colony forming units (cfu) as isolate P1. On wood chips, the two isolates formed a similar number of cfu. When the two Trichoderma isolates were cultivated together approximately 85-90% of the cfu were from T3 on both substrates. The presence of Pythium ultimum in peat amended with straw did not influence the number of Trichoderma cfu formed. The two Trichoderma isolates produced different amounts of hydrolytic enzymes both in liquid cultures and in peat. Seven different enzyme activities were tested. Enzyme production by T. harzianum isolate T3 was less influenced by the type of carbon source amendment than that of isolate T. atroviride P1. Culture filtrates of isolate P1 grown on complex carbon sources were high in endochitinase activity, whereas cellulase and endo-1,3-β-glucanase activities were more pronounced in filtrates of isolate T3. There was no significant difference between the two isolates in their ability to protect cucumber seedlings against P. ultimum while the combination of the two fungi resulted in significantly less biocontrol than each isolate alone.[PUBLICATION ABSTRACT]

The most effective magnitude and timing of antiplatelet therapy is important in patients with acute ST-elevation myocardial infarction (STEMI). We investigated whether the results of primary coronary angioplasty (PCI) can be improved by the early administration of the glycoprotein IIb/IIIa blocker tirofiban at first medical contact in the ambulance or referral centre. We undertook a double-blind, randomised, placebo-controlled trial in 24 centres in the Netherlands, Germany, and Belgium. Between June 29, 2006, and Nov 13, 2007, 984 patients with STEMI who were candidates to undergo PCI were randomly assigned to either high-bolus dose tirofiban (n=491) or placebo (N=493) in addition to aspirin (500 mg), heparin (5000 IU), and clopidogrel (600 mg). Randomisation was by blinded sealed kits with study drug, in blocks of four. The primary endpoint was the extent of residual ST-segment deviation 1 h after PCI. Analysis was by intention to treat. The trial is registered, number ISRCTN06195297. 936 (95%) patients were randomly assigned to treatment after a prehospital diagnosis of myocardial infarction in the ambulance. Median time from onset of symptoms to diagnosis was 76 min (IQR 35–150). Mean residual ST deviation before PCI (10·9 mm [SD 9·2] vs 12·1 mm [9·4], p=0·028) and 1 h after PCI (3·6 mm [4·6] vs 4·8 mm [6·3], p=0·003) was significantly lower in patients pretreated with high-bolus dose tirofiban than in those assigned to placebo. The rate of major bleeding did not differ significantly between the two groups (19 [4%] vs 14 [3%]; p=0·36). Our finding that routine prehospital initiation of high-bolus dose tirofiban improved ST-segment resolution and clinical outcome after PCI, emphasises that further platelet aggregation inhibition besides high-dose clopidogrel is mandated in patients with STEMI undergoing PCI. Merck (USA).

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Δ 1-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutant rsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS and P5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Δ 1-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.