Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k_cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K_m) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k_cat = 1.9 × 10² s⁻¹ on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.Aldohexuronate catabolism in bacteria is reported to involve two different pathways, one initiating with an isomerization step and the other with an oxidation step. In the isomerization pathway, aldohexuronate (glucuronate and galacturonate) is isomerized to ketohexuronate by uronate isomerase and ultimately degraded to pyruvate and 3-phosphoglyceraldehyde. The isomerization pathway has been previously reported to occur in bacteria, including Escherichia coli (7), Erwinia carotovora (18), Erwinia chrysanthemi (15), Klebsiella pneumoniae (9, 23), and Serratia marcescens (28). In the oxidation pathway, aldohexuronate is oxidized to aldohexarate by uronate dehydrogenase (Udh) and further catabolized to pyruvate (2, 5, 7, 9, 18, 19, 24). Uronate dehydrogenase, the key enzyme of this pathway, has been investigated in two plant pathogen bacteria, Pseudomonas syringae and Agrobacterium tumefaciens. To date, only limited studies pertaining to the properties of Udh have been reported in the literature (3, 6, 38, 43), and no sequence has yet been identified. Udh is classified as an NAD-linked oxidoreductase (EC 1.1.1.203), with a total molecular weight of about 60,000. It is a homodimer composed of two subunits with molecular weights of about 30,000 each (38). Udh is a thermally unstable, reversible enzyme, with an optimum pH of about 8.0 (3, 6, 38).In E. coli MG1655 that has the isomerization pathway for aldohexuronate catabolism, glucuronate is transported by an aldohexuronate transporter encoded by exuT and converted to fructuronate by uronate isomerase, encoded by uxaC (22, 30) (Fig. ​(Fig.1).1). Fructuronate is transferred to the Entner-Doudoroff pathway to be utilized as an energy source via 2-keto-3-deoxy-6-phospho-gluconate (7, 27, 31, 32). Therefore, E. coli MG1655 with a uxaC deletion cannot use glucuronate as a carbon source. In this strain, glucarate is converted to 5-keto-4-deoxy-d-glucarate by d-glucarate dehydratase, encoded by gudD, and then transferred to glycolysis via pyruvate or 2-phosphoglycerate (27, 33). Recently, a number of bacterial genome sequences have been published, including those of the Udh-containing P. syringae pv. tomato strain DC3000 and A. tumefaciens strain C58 (4, 10). A shotgun library of P. syringae was constructed to identify the gene encoding Udh. Screening for Udh was conducted in E. coli MG1655 ΔuxaC. Since uronate dehydrogenase converts glucuronate to glucarate, uxaC deletion strains of E. coli harboring the shotgun library of P. syringae that can grow in a minimal medium containing glucuronate as a sole carbon source may carry the gene encoding Udh (Fig. ​(Fig.1).1). Once an initial Udh is identified from P. syringae, a BLAST homology search may lead to the identification of Udhs from other bacteria.Open in a separate windowFIG. 1.Catabolism of glucuronate and glucarate in bacteria. Glucuronate consumption is prevented by knockout of the uxaC gene. The presence of uronate dehydrogenase in a uxaC knockout enables growth of E. coli on glucuronate.     

Antifungal Effect and Protective Role of Ursolic Acid and Three Phenolic Derivatives in the Management of Sorghum Grain Mold Under Field Conditions

In this study, investigation was carried out under in vitro as well as field conditions to explore inhibitors of sorghum grain mold. Phytochemicals, viz., methyl trans‐p‐coumarate (AIC‐1), methyl caffeate (AIC‐2), syringic acid (AIC‐3), and ursolic acid (UA), at different concentrations (500, 750, and 1000 ppm) were tested on spore germination of Alternaria alternata, Curvularia lunata, Fusarium moniliforme, F. pallidoroseum, and Helminthosporium sp. Significant growth inhibition (P < 0.001) was observed against all fungi except A. alternata which was found to be resistant to AIC‐3. Further, two separate sets of field experiments involving spraying of water and F. moniliforme suspension over chemicals treated (1000 ppm) sorghum panicles were done. The levels of protection varied with different treatments which were graded using a standard 1 – 9 rating scale. The Fusarium‐challenged panicles (FCP) showed lesser susceptibility and decreased the rate of infection of grain mold (grade 7.0), compared to simple UA, AIC‐2, and AIC‐1 treatments (7.4, 7.6, and 8.0 grade, resp.). The HPLC quantification of differentially induced phenolic acids in treated sorghum grains substantiated this effect disclosing the higher accumulation of chlorogenic, vanillic, and salicylic acids in FCP. This might be due to defensive induction of these acids by the plants. Although mold control by examined chemicals were lesser than the standard Tilt (grade 5.9), they were found to be nontoxic to mammalian cells under cytotoxicity assay.     

AXanthomonas maltophilia isolate tolerating up to 1% sodium azide in Tris/HCl buffer

A bacterium tolerating up to 1% NaN₃ found as a contaminant of Sephadex colums being run with Tris/HCl buffer, was identified asXanthomonas maltophilia. It had low nutrient requirements, was strongly proteolytic and interfered with Sephadex columns run with Tris/HCl buffers.The authors are with the Department of Plant Pathology, Swedish University of Agricultural Sciences, Box 7044, S-750 07 Uppsala, Sweden;     

Antiviral Activity of Faramea hyacinthina and Faramea truncata Leaves on Dengue Virus Type‐2 and Their Major Compounds

The defatted fractions of the Faramea hyacinthina and F. truncata (Rubiaceae) leaf MeOH extracts showed in vitro non‐cytotoxic and anti‐dengue virus serotype 2 (DENV2) activity in human hepatocarcinoma cell lineage (HepG2). Submitting these fractions to the developed RP‐SPE method allowed isolating the antiviral flavanone (2S)‐isosakuranetin‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 1 ) from both species and yielded less active sub‐fractions. The new diastereoisomeric epimer pair (2S) + (2R) of 5,3′,5′‐trihydroxyflavanone‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 2a / 2b ) from F. hyacinthina; the known narigenin‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 3 ) from both species; rutin ( 4 ) and quercetin‐4′‐β‐d ‐O‐glucopyranosyl‐3‐O‐rutinoside ( 5 ) from F. hyacinthina, and kaempferol‐3‐O‐rutinoside ( 6 ), erythroxyloside A ( 7 ) and asperuloside ( 8 ) from F. truncata have been isolated from these sub‐fractions. Compounds 4  –  8 are reported for the first time in Faramea spp.     

Pellet forming and fragmentation in liquid culture of Streptomyces griseus

Pellets of Streptomyces griseus formed during submerged liquid culture were effectively dispersed in Tris/HCl buffer at 37 °C. However, inclusion of Tris/HCl in the culture medium did not lead to dispersed growth. In order to decrease the pellet formation in batch culture, a large inoculum of spores was required and dispersed growth was further favoured by culture at 37 °C. This method was also used to achieve dispersed culture of S. coelicolor A3(2). Autolytic enzyme activity was presumed to be responsible for fragmentation of pellets.     

Agroinfiltration Reduces ABA Levels and Suppresses Pseudomonas syringae-Elicited Salicylic Acid Production in Nicotiana tabacum

Background

Agrobacterium tumefaciens strain GV3101 (pMP90) is widely used in transient gene expression assays, including assays to study pathogen effectors and plant disease resistance mechanisms. However, inoculation of A. tumefaciens GV3101 into Nicotiana tabacum (tobacco) leaves prior to infiltration with pathogenic and non-host strains of Pseudomonas syringae results in suppression of macroscopic symptoms when compared with leaves pre-treated with a buffer control.

Methodology/Findings

To gain further insight into the mechanistic basis of symptom suppression by A. tumefaciens we examined the effect of pre-treatment with A. tumefaciens on the growth of P. syringae, the production of the plant signalling molecules salicylic acid (SA) and abscisic acid (ABA), and the presence of callose deposits. Pre-treatment with A. tumefaciens reduced ABA levels, P. syringae multiplication and P. syringae-elicited SA and ABA production, but promoted increased callose deposition. However, pre-treatment with A. tumefaciens did not suppress necrosis or SA production in leaves inoculated with the elicitor HrpZ.

Conclusions/Significance

Collectively, these results show that inoculation of N. tabacum leaves with A. tumefaciens alters plant hormone levels and plant defence responses to P. syringae, and demonstrate that researchers should consider the impact of A. tumefaciens on plant signal transduction when using A. tumefaciens-mediated transient expression assays to investigate ABA-regulated processes or pathogenicity and plant defence mechanisms.     

Solvent selection and optimization of α‐chymotrypsin‐catalyzed synthesis of N‐Ac‐Phe‐Tyr‐NH2 using mixture design and response surface methodology

A peptide, N‐Ac‐Phe‐Tyr‐NH₂, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH₂). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH₂ could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH₂, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

F1FO ATP Synthase Is Expressed at the Surface of Embryonic Rat Heart‐Derived H9c2 Cells and Is Affected by Cardiac‐Like Differentiation

Taking advantage from the peculiar features of the embryonic rat heart‐derived myoblast cell line H9c2, the present study is the first to provide evidence for the expression of F₁F_O ATP synthase and of ATPase Inhibitory Factor 1 (IF₁) on the surface of cells of cardiac origin, together documenting that they were affected through cardiac‐like differentiation. Subunits of both the catalytic F₁ sector of the complex (ATP synthase‐β) and of the peripheral stalk, responsible for the correct F₁‐F_O assembly/coupling, (OSCP, b, F6) were detected by immunofluorescence, together with IF₁. The expression of ATP synthase‐β, ATP synthase‐b and F6 were similar for parental and differentiated H9c2, while the levels of OSCP increased noticeably in differentiated cells, where the results of in situ Proximity Ligation Assay were consistent with OSCP interaction within ecto‐F₁F_O complexes. An opposite trend was shown by IF₁ whose ectopic expression appeared greater in the parental H9c2. Here, evidence for the IF₁ interaction with ecto‐F₁F_O complexes was provided. Functional analyses corroborate both sets of data. i) An F₁F_O ATP synthase contribution to the exATP production by differentiated cells suggests an augmented expression of holo‐F₁F_O ATP synthase on plasma membrane, in line with the increase of OSCP expression and interaction considered as a requirement for favoring the F₁‐F_O coupling. ii) The absence of exATP generation by the enzyme, and the finding that exATP hydrolysis was largely oligomycin‐insensitive, are in line in parental cells with the deficit of OSCP and suggest the occurrence of sub‐assemblies together evoking more regulation by IF₁. J. Cell. Biochem. 9999: 1–13, 2015. © 2015 Wiley Periodicals, Inc. J. Cell. Biochem. 117: 470–482, 2016. © 2015 Wiley Periodicals, Inc.     

Artificial Agrobacterium tumefaciens strains exhibit diverse mechanisms to repress Xanthomonas oryzae pv. oryzae‐induced hypersensitive response and non‐host resistance in Nicotiana benthamiana

Xanthomonas oryzae pv. oryzae (Xoo) rapidly triggers a hypersensitive response (HR) and non‐host resistance in its non‐host plant Nicotiana benthamiana. Here, we report that Agrobacterium tumefaciens strain GV3101 blocks Xoo‐induced HR in N. benthamiana when pre‐infiltrated or co‐infiltrated, but not when post‐infiltrated at 4 h after Xoo inoculation. This suppression by A. tumefaciens is local and highly efficient to Xoo. The HR‐inhibiting efficiency of A. tumefaciens is strain dependent. Strain C58C1 has almost no effect on Xoo‐induced HR, whereas strains GV3101, EHA105 and LBA4404 nearly completely block HR formation. Intriguingly, these three HR‐inhibiting strains employ different strategies to repress HR. Strain GV3101 displays strong antibiotic activity and thus suppresses Xoo growth. Comparison of the genotype and Xoo antibiosis activity of wild‐type A. tumefaciens strain C58 and a set of C58‐derived strains reveals that this Xoo antibiosis activity of A. tumefaciens is negatively, but not solely, regulated by the transferred‐DNA (T‐DNA) of the Ti plasmid pTiC58. Unlike GV3101, strains LBA4404 and EHA105 exhibit no significant antibiotic effect on Xoo, but rather abolish hydrogen peroxide accumulation. In addition, expression assays indicate that strains LBA4404 and EHA105 may inhibit Xoo‐induced HR by suppression of the expression of Xoo type III secretion system (T3SS) effector genes hpa1 and hrpD6. Collectively, our results unveil the multiple levels of effects of A. tumefaciens on Xoo in N. benthamiana and provide insights into the molecular mechanisms underlying the bacterial antibiosis of A. tumefaciens and the non‐host resistance induced by Xoo.     

Methods of assessment of transfer of a gentamycin-resistance gene (aacC1) from genetically engineered microorganisms into agriculturally important soil bacteria

In order to assess the risk associated with the deliberate release of genetically engineered microorganisms (GEMs) into the agricultural environment, the transfer of plasmids between bacterial strains was investigated under laboratory conditions. Genetically modified Rhizobium leguminosarum and Agrobacterium tumefaciens strains carrying the gentamycin acetyltransferase resistance gene (aacC1) on various plasmids were investigated for their ability to transfer the aacC1 gene to their wild-type (w.t.) counterparts, as well as to Pseudomonas syringae. Conjugation experiments between the various strains, were carried out after the relevant characteristics and conditions for selective growth of each bacterial strain had been ascertained. After conjugations on filters had been completed, the putative transconjugants were grown in media containing antibiotics and assessed for the presence of aacC1 gene by: (a) DNA plasmid profile; (b) expression of AAC(3)-I enzyme activity; (c) colony hybridization using a ³²P-labelled DNA probe complementary to the aacC1 gene. The results obtained indicate that transfer of the aacC1 gene from genetically modified strains of R. leguminosarum into a plasmid-free strain of A. tumefaciens occurred via self-transmissible plasmids. Alternatively, genetically modified A. tumefaciens bearing the aacC1 gene on plasmids acquired from R. leguminosarum strains, transferred it ineffectively to a hardly detectable frequency. No transfer of the aacC1 gene from genetically modified R. leguminosarum or A. tumefaciens strains into P. syringae has been observed. These data indicate that in the absence of the RP4 element, genetically modified A. tumefaciens is not able to efficiently transfer aacC1 into w.t. R. leguminosarum and P. syringae. Correspondence to: A. S. Tsiftsoglou     

Effects of elevated ozone concentration on methane emission from a rice paddy in Yangtze River Delta,China

Few investigations have been made on the impact of elevated ozone (O₃) concentration on methane (CH₄) emission from rice paddies. Using open‐top chambers in situ with different O₃ treatments, CH₄ emissions were measured in a rice paddy in Yangtze River Delta, China in 2007 and 2008. There were four treatments applied: charcoal‐filtered air (CF), nonfiltered air (NF), and charcoal‐filtered air with different O₃ additions (O₃‐1 and O₃‐2). The mean O₃ concentrations during the O₃ fumigation were 19.7, 22.6, 69.6 and 118.6 ppb in 2007 and 7.0, 17.4, 82.2 and 138.3 ppb in 2008 for treatments CF, NF, O₃‐1 and O₃‐2, respectively. The rice yields, as compared with CF, were reduced by 32.8% and 37.1%, 58.3% and 52.1% in treatments O₃‐1 and O₃‐2 in 2007 and 2008, respectively. The diurnal patterns of CH₄ emission varied temporally with treatments and there was inconsistence in diurnal variations in CH₄ emissions from the paddy field. The daily mean CH₄ emissions were significantly lower in treatments O₃‐1 and O₃‐2 than those in treatments CF and NF. Compared with CF treatment, CH₄ emissions from the paddy field were decreased to 46.5% and 38.3%, 50.6% and 46.8% under treatments O₃‐1 and O₃‐2 in the whole growing seasons of 2007 and 2008, respectively. The seasonal mean CH₄ emissions were negatively related with AOT40 (accumulative O₃ concentration above 40 ppb; P < 0.01 in both years), but positively related to the relative rice yield (reference to CF; P < 0.01 in 2007 and P < 0.001 in 2008), aboveground biomass (P < 0.01 in both years) and underground biomass (P < 0.01 in 2007 and P < 0.05 in 2008). The decreased CH₄ emission from the rice paddy due to an increased O₃ exposure might partially mitigate the global warming potential induced by soil carbon loss under elevated O₃ concentrations.     

Response of two Antarctic bryophytes to stratospheric ozone depletion

We report a study which measured changes to the radiative environment arising from stratospheric O₃ depletion at Rothera Point on the western Antarctic Peninsula (67°S, 68°W) and subsequent associations between these changes and the pigmentation and maximum quantum yield of photochemistry (F_v/F_m) of two Antarctic bryophytes, the liverwort Cephaloziella varians and the moss Sanionia uncinata. We found a strong relationship between O₃ column depth and the ratio of UV‐B to PAR irradiance (F_UV‐B/F_PAR) recorded at ground level. Weaker, but significant, associations were also found between O₃ column depth and noon irradiances and daily doses of unweighted and biologically effective UV‐B radiation received at ground level. Regression analyses indicated that F_UV‐B/F_PAR and daily dose of unweighted UV‐B were best predictors for concentrations of total carotenoids and UV‐B screening pigments extracted from bryophyte tissues. Concentrations of these pigments were loosely but significantly positively associated with O₃‐dependent irradiance parameters. HPLC analyses of carotenoids also suggested that both species increased the synthesis of neoxanthin during periods of O₃ depletion. Violaxanthin, lutein, zeaxanthin and b,bββ‐carotene concentrations were also apparently influenced by O₃ reduction, but not consistently across both bryophyte species. Concentrations of chlorophylls a and b were apparently unaffected by O₃ depletion. No direct associations between F_v/F_m and O₃‐dependent irradiance parameters were found. However stepwise multiple regression analyses suggested that the production of UV‐B screening pigments conferred protection from elevated F_UV‐B/F_PAR on F_v/F_m in both species and that carotenoids conferred protection on F_v/F_m in Sanionia. Our data suggest that changes to the radiative environment associated with stratospheric O₃ depletion influence the pigmentation of two Antarctic bryophytes, but that F_v/F_m is unaffected, at least in part because of rapid synthesis of protective pigments.     

Molecular Identification and Genetic Diversity of Fusarium species Associated with Onion Fields in Turkey

To identify Fusarium species associated with diseases of root and basal plate of onion, surveys were conducted in seven provinces of Turkey in 2007. Samplings were performed in 223 fields, and 332 isolates belonging to 7 Fusarium spp. were obtained. The isolates were identified as F. oxysporum, F. solani, F. acuminatum, F. equiseti, F. proliferatum, F. redolens, and F. culmorum based on morphological and cultural characteristics. Also, species‐specific primers were used to confirm the identity of Fusarium species. F. oxysporum was the most commonly isolated species, comprising 66.57% of the total Fusarium species. F. redolens was identified for the first time in onion‐growing areas of Turkey. Selected isolates of each species were evaluated for their aggressiveness on onion plant. F. oxysporum, F. solani, F. acuminatum, F. proliferatum, and F. redolens were highly pathogenic, causing severe damping‐off on onion plants cv. Texas Early Grano. Inter‐simple sequence repeats (ISSR) markers revealed a high degree of intra‐ and interspecific polymorphisms among Fusarium spp.     

A critical role for Arabidopsis MILDEW RESISTANCE LOCUS O2 in systemic acquired resistance

Members of the MILDEW RESISTANCE LOCUS O (MLO) gene family confer susceptibility to powdery mildews in different plant species, and their existence therefore seems to be disadvantageous for the plant. We recognized that expression of the Arabidopsis MLO2 gene is induced after inoculation with the bacterial pathogen Pseudomonas syringae, promoted by salicylic acid (SA) signaling, and systemically enhanced in the foliage of plants exhibiting systemic acquired resistance (SAR). Importantly, distinct mlo2 mutant lines were unable to systemically increase resistance to bacterial infection after inoculation with P. syringae, indicating that the function of MLO2 is necessary for biologically induced SAR in Arabidopsis. Our data also suggest that the close homolog MLO6 has a supportive but less critical role in SAR. In contrast to SAR, basal resistance to bacterial infection was not affected in mlo2. Remarkably, SAR‐defective mlo2 mutants were still competent in systemically increasing the levels of the SAR‐activating metabolites pipecolic acid (Pip) and SA after inoculation, and to enhance SAR‐related gene expression in distal plant parts. Furthermore, although MLO2 was not required for SA‐ or Pip‐inducible defense gene expression, it was essential for the proper induction of disease resistance by both SAR signals. We conclude that MLO2 acts as a critical downstream component in the execution of SAR to bacterial infection, being required for the translation of elevated defense responses into disease resistance. Moreover, our data suggest a function for MLO2 in the activation of plant defense priming during challenge by P. syringae.     

Activité néformatrice d'acides ribonucléiques extraits de tumeurs végétales

RNAs extracted from crown-gall tumors, induced by Agrobacterium tumefaciens (strain B6) on stems of Datura stramonium L., have been isolated by the phenol method and purified through Biogel P 60 columns. These RNAs have been transformed into complexes with l -amino acids by incubation in a medium containing: Tris HCI buffer, pH 7.6, ATP, MgCl₂, a mixture of l -amino acids and a polypeptide synthetase purified from Alcaligenes faecalis. The inoculation of stems of Datura plants with these complexes induces the development of nodular outgrowths, whereas other complexes made from RNAs isolated from healthy Datura plants, from Agrobacterium tumefaciens, Escherichia coli and Alcaligenes faecalis do not cause any hyperplasia under our experimental conditions. The analysis of the results obtained, supported by histological studies of the outgrowths, suggests that these neoformations should be of tumoral nature.     

Agrobacterium tumefaciens ferritins play an important role in full virulence through regulating iron homeostasis and oxidative stress survival

Ferritins are a large family of iron storage proteins, which are used by bacteria and other organisms to avoid iron toxicity and as a safe iron source in the cytosol. Agrobacterium tumefaciens, a phytopathogen, has two ferritin-encoding genes: atu2771 and atu2477. Atu2771 is annotated as a Bfr-encoding gene (Bacterioferritin, Bfr) and atu2477 as a Dps-encoding gene (D NA binding p rotein from s tarved cells, Dps). Three deletion mutants (Δbfr, Δdps, and bfr-dps double-deletion mutant ΔbdF) of these two ferritin-encoding genes were constructed to investigate the effects of ferritin deficiency on the iron homeostasis, oxidative stress resistance, and pathogenicity of A. tumefaciens. Deficiency of two ferritins affects the growth of A. tumefaciens under iron starvation and excess. When supplied with moderate iron, the growth of A. tumefaciens is not affected by the deficiency of ferritin. Deficiency of ferritin significantly reduces iron accumulation in the cells of A. tumefaciens, but the effect of Bfr deficiency on iron accumulation is severer than Dps deficiency and the double mutant ΔbdF has the least intracellular iron content. All three ferritin-deficient mutants showed a decreased tolerance to 3 mM H₂O₂ in comparison with the wild type. The tumour induced by each of three ferritin-deficient mutants is less than that of the wild type. Complementation reversed the effects of ferritin deficiency on the growth, iron homeostasis, oxidative stress resistance, and tumorigenicity of A. tumefaciens. Therefore, ferritin plays an important role in the pathogenesis of A. tumefaciens through regulating iron homeostasis and oxidative stress survival.     

Production and Application of Syringomycin E as an Organic Fungicide Seed Protectant against Pythium Damping‐off

Syringomycin E (SRE) is a cyclic lipodepsinonapeptide with potent antifungal activity and is produced by certain strains of Pseudomonas syringae pv. syringae. In this study, its potential as an organic‐compatible agrofungicide and vegetable seed treatment against the soilborne pathogen Pythium ultimum var. ultimum was examined. A variant of P. syringae pv. syringae strain B301D with enhanced SRE‐producing capabilities was isolated and grown in a bioreactor with SRE yields averaging 50 mg/l in 40 h. SRE was extracted and purified through a large‐scale chromatography system using organic‐compatible processes and reagents. The minimum concentrations of the purified product required to inhibit 50 and 90% of P. ultimum oospore germination were determined as 31.3 and 250 μg/ml, respectively. Drench treatment of cucumber seeds in P. ultimum‐infested potting medium (500 oospores/g) with 50 μg/ml SRE or water with no SRE resulted in 90.2 ± 4.5% and 65.7 ± 4.6% germination rates, respectively. Seed coating with 0.03% (w/w) SRE allowed 65.7 ± 4.6% seedlings to germinate on naturally infested soil while 100.0 ± 0.0% of non‐coated seeds were unable to germinate due to Pythium infection. Organic‐compatible and scalably produced SRE is potentially a novel organic fungicide seed protectant.     

Determination of phenol using an enhanced chemiluminescent assay

Enhanced chemiluminescence (ECL) describes the phenomenon of increased light output in the luminol oxidation reaction catalysed by horseradish peroxidase (HRP) in the presence of certain compounds, such as para‐iodophenol. In this work, the effects of phenol on the para‐iodophenol‐enhanced HRP‐catalysed chemiluninescent reaction intensity in an aqueous buffer (Tris–HCl buffer, pH 8.5) and in a surfactant–water–octane mixture were compared. Preincubation of HRP at low phenol concentrations stimulated the chemiluminescent intensity in the assay performed in an aqueous buffer, but did not have significant effect in the sodium bis(2‐ethylhexyl)sulphosuccinate) (Aerosol OT, AOT) applied system. It was also observed that HRP preincubation with phenol concentration higher than 0.003 mg/mL produced an inhibitory effect on the enzyme activity for both assay systems. Only an inhibitory effect of phenol on the chemiluminescent intensity in the surfactant system in octane (as organic solvent) was observed. Three assays were developed to determine phenol concentration in water and in an organic solvent mixture. The detection limits were 0.006, 0.003 and 0.0005 mg/mL, respectively, for the buffer‐containing system, the AOT‐applied system with phenol standard solutions in water and for the AOT‐applied system with phenol standard solutions in octane. Copyright © 2002 John Wiley & Sons, Ltd.     

Epiphytic Rotifer Abundance and Diversity in Moss Patches in Bogs and Fens in the Polesie National Park (Eastern Poland)

Moss‐inhabiting rotifers were studied in 2 raised bogs, 2 poor fens (i.e. intermediate between raised bogs and typical fens), 1 typical fen, and 1 base‐rich fen, located in the Polesie National Park (eastern Poland). Moss samples were collected from April till November 2007 in monthly intervals. In total, 42 taxa of Monogononta and 26 of Bdelloidea were found. Analysis of variance (ANOVA) detected highly significant differences between the habitats in conductivity (F = 37.19, P < 0.001), total organic content (F = 9.34, P < 0.001), total P (F = 51.71, P < 0.001), and nitrate N (F = 48.98, P < 0.001). The mean density of rotifers was the highest in a bog dominated by Sphagnum angustifolium Jensen (Durne Bagno), and the lowest in the base‐rich fen (Bubnów). In all the studied habitats, the Bdelloidea were the most abundant, accounting for 56 to 85% of the total rotifer density. Canonical correspondence analysis (CCA), made for selected physicochemical parameters and taxa, confirms strong relationships between abiotic conditions and selected rotifer taxa: Habrotrocha angusticolis, Dicranophorus capucinus, Keratella serrulata, and Lepadella elliptica. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)