Influence of tebuconazole and copper hydroxide on phosphatase and urease activities in red sandy loam and black clay soils.

The efficacy of two selected fungicides i.e., tebuconazole and coppoer hydroxide, was conducted experiments in laboratory and copper hydroxide on the two specific enzymes phosphatase and urease were determined in two different soil samples (red sandy loam and black clay soils) of groundnut (Arachis hypogaea L.) from cultivated fields of Anantapuramu District, Andhra Pradesh. The activities of the selected soil enzymes were determined by incubating the selected fungicides-treated (1.0, 2.5, 5.0, 7.5 and 10.0 kg ha-1) and -untreated groundnut soil samples at 10 day intervals. By determining the effective concentration, the rate of selected enzyme activity was estimated by adding the suitable substrate at 10, 20, 30 and 40 days of soil incubation. Both the enzyme activities were increased up to 5.0 kg ha-1 level of fungicide in both soil samples significantly at 10 days of soil incubation and further enhanced up to 20 days of incubation. The activity of the phosphatase and urease decreased progressively at 30 and 40 days of incubation. From overall studies, higher concentrations (7.5 and 10.0 kg ha-1) of both tebuconazole and copper hydroxide were toxic to phosphatase and urease activities, respectively, in both soil samples.

Influence of the insecticides acetamiprid and carbofuran on arylamidase and myrosinase activities in the tropical black and red clay soils.

The objective of this study was to determine the effects of two insecticides, namely, acetamiprid and carbofuran on the enzymatic activities of arylamidase (as glucose formed from sinigrin) and myrosinase (as ß-naphthylamine formed from L-leucine ß-naphthylamide) in the black and red clay soils collected from a fallow groundnut (Arachis hypogaea L.) fields in the Anantapur District, Andhra Pradesh, India. The study was realized within the framework of the laboratory experiments in which the acetamiprid and carbofuran were applied to the soils at different doses (1.0, 2.5, 5.0, 7.5, 10.0 kg ha(-1)). Initially, the physicochechemical properties of the soil samples were analyzed. After 10 days of pesticide application, the soil samples were analyzed for the enzyme activities. Acetamiprid and carbofuran stimulated the arylamidase and myrosinase activities at lower concentrations after 10 days incubation. Striking stimulation in soil enzyme activities was noticed at 2.5 kg ha(-1), persists for 20 days in both the soils. Overall, higher concentrations (5.0-10.0 kg ha(-1)) of acetamiprid and carbofuran were toxic or innocuous to the arylamidase and myrosinase activities. Nevertheless, the outcomes of the present study clearly indicate that the use of these insecticides (at field application rates) in the groundnut fields (black and red clay soils) stimulated the enzyme (arylamidase and myrosinase) activities.

Influence of insecticides flubendiamide and spinosad on biological activities in tropical black and red clay soils.

A laboratory experiment has been conducted to investigate the ecological toxicity of flubendiamide and spinosad at their recommended field rates and higher rates (1.0, 2.5, 5.0, 7.5, 10.0 kg ha-1) on cellulase, invertase and amylase in black and red clay soils after 10, 20, 30 and 40-day exposure under controlled conditions in groundnut (Arachis hypogaea L.) soils of Anantapur District, Andhra Pradesh, India. Flubendiamide and spinosad were stimulatory to the activities of cellulase, invertase and amylase at lower concentrations at 10-day interval. The striking stimulation in soil enzyme activities noticed at 2.5 kg ha-1, persists for 20 days in both soils. Overall, the higher concentrations (5.0-10.0 kg ha-1) of flubendiamide, and spinosad were toxic or innocuous to cellulase, invertase and amylase activities, respectively. The results of the present study thus, clearly, indicate that application of the insecticides in cultivation of groundnut, at field application rates improved the activities of cellulase, invertase and amylase in soils.

A comparison of fasting plasma glucose and glucose challenge test for screening of gestational diabetes mellitus.

Glucose challenge test (GCT) has been used as an effective screening test for gestational diabetes mellitus (GDM), though it has its own limitations. Hence, we assessed the effectiveness of fasting plasma glucose (FPG) as a simpler alternative procedure. A prospective study was done in 500 pregnant women with gestational age between 22 and 37 weeks. FPG, GCT and GTT were performed in all patients using the glucose oxidase/peroxidase method. The overall sensitivity and specificity of GCT were 75.0% and 92.0%, respectively and the corresponding values for FPG were 88.8% and 95.2%. The positive predictive value and negative predictive value were 42.2% and 97.9% for GCT and 59.2% and 99.1% for FPG, respectively. We conclude that FPG can be used as an effective screening tool for gestational diabetes mellitus.

Effect of insecticides alone and in combination with fungicides on nitrification and phosphatase activity in two groundnut (Arachis hypogeae L.) soils.

The effect of selected pesticides, monocrotophos, chlorpyrifos alone and in combination with mancozeb and carbendazim, respectively, was tested on nitrification and phosphatase activity in two groundnut (Arachis hypogeae L.) soils. The oxidation of ammonical nitrogen was significantly enhanced under the impact of selected pesticides alone and in combinations at 2.5 kg ha(-1) in black soil, and furthermore, increase in concentration of pesticides decreased the rate of nitrification, whereas in the case of red soil, the nitrification was increased up to 5.0 kg ha(-1) after 4 weeks, and then decline phase was started gradually from 6 to 8 weeks of incubation. The activity of phosphatase was increased in soils, which received the monocrotophos alone and in combination with mancozeb up to 2.5 and 5.0 kg ha(-1), whereas the application of chlorpyrifos singly and in combination with carbendazim at 2.5 kg ha(-1) profoundly increased the phosphatase activity after 20 days of incubation, in both soils. But higher concentrations of pesticides were either innocuous or inhibitory to the phosphatase activity.

Impact of Fungicides Chlorothalonil and Propiconazole on Microbial Activities in Groundnut (Arachis hypogaea L.) Soils.

Introduction of agrochemicals (fungicides) into soil may have lasting effects on soil microbial activities and thus affect soil health. In order to determine the changes in microbial activity in a black clay and red sandy loam soils of groundnut (Arachis hypogaea L.) cultivated fields, a case study was conducted with propiconazole and chlorothalonil to evaluate its effects on soil enzymes (cellulase and invertase) throughout 40 days of incubation under laboratory conditions with different concentrations (1.0, 2.5, 5.0, 7.5, and 10.0 kg ha(-1)). Individual application of the two fungicides at 1.0, 2.5, and 5.0 kg ha(-1) to the soil distinctly enhanced the activities of cellulase and invertase but at higher concentrations of 7.5 and 10 kg ha(-1) was toxic or innocuous to both cellulase and invertase activities. In soil samples receiving 2.5-5.0 kg ha(-1) of the fungicides, the accumulation of reducing sugar was pronounced more at 20 days, and the activity of the cellulase and invertase was drastically decreased with increasing period of incubation up to 30 and 40 days.

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor.

AIMS: An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated. METHODS AND RESULTS: Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l(-1) h(-1) over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity. CONCLUSIONS: Polyurethane foams offer an excellent support for biofilm formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The system was very robust and could be operated for prolonged period at a volumetric productivity of 4-6 g l(-1) h(-1).

Influence of selected insecticides on phosphatase activity in groundnut (Arachis hypogeae L.) soils.

Selected insecticides, Chloropyrifos, Dichlorovos, Methyl parathion, Phorate and Methomyl, at concentrations ranging from 0 to 10 kg ha(-1) were tested for their non-target effects towards activity of phosphatases in two soils. In soil samples receiving 2.5 kg ha(-1) of the insecticides Dichlorovos, Phorate and Methomyl and also in soil samples receiving 5.0 kg ha(-1) of the insecticides, Chloropyrifos and Methyl parathion, the activity of phosphatase was significantly more at 20 days period of incubation and decreased progressively with increasing period of incubation.

Analysis of the enzymatic domains in the modular portion of the coronafacic acid polyketide synthase.

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen Pseudomonas syringae. The CFA polyketide synthase (PKS) consists of two open reading frames (ORFs) that encode type I multifunctional proteins and several ORFs that encode monofunctional proteins. Sequence comparisons of the modular portions of the CFA PKS with other prokaryotic, modular PKSs elucidated the boundaries of the domains that are involved in the individual stages of polyketide assembly. The two beta-ketoacyl:acyl carrier protein synthase (KS) domains in the modular portion of the CFA PKS exhibit a high degree of similarity to each other (53%), but are even more similar to the KS domains of DEBS, RAPS, and RIF. Cfa6 possesses two acyltransferases- AT0, which is associated with a loading domain, and AT1, which uses ethylmalonyl-CoA (eMCoA) as a substrate for chain extension. Cfa7 contains an AT that uses malonyl-CoA as a substrate for chain extension. The Cfa6 AT0 shows 35 and 32% similarity to the DEBS1 and NidA1 AT0s, respectively, and 32 and 36% similarity to the Cfa6 and Cfa7 AT1s. Sequence motifs have previously been identified that correlate with AT substrates. The motifs in Cfa6 AT1 were found to correlate reasonably well with those predicted for methylmalonyl-CoA (mMCoA) ATs. The motifs in the AT of Cfa7 correlated more poorly with those predicted for MCoA ATs. Three ACP domains occur in the modular proteins of the COR PKS. The loading domain-associated ACP0 showed 38% similarity to the loading domain ACP0s of DEBS1 and NidA1 and 32-36% similarity to the two module-associated ACPs of the COR PKS. It exhibited a higher degree of similarity to the module-associated ACPs of RAPS. The two module-associated ACPs show 39% similarity to each other, but appear more closely related to module-associated ACP domains in RAPS and RIFS. Furthermore, the DH and KR domains of Cfa6 and Cfa7 show greater similarity to DH and KR domains in RAPS and RIFS than to each other. The CFA PKS includes a thioesterase domain (TE I) that resides at the C-terminus of Cfa7 and a second thioesterase, which exists as a separate ORF (Cfa9, a TE II). Analysis of a Cfa7 thioesterase mutant demonstrated that the TE domain is required for the production of CFA. The co-existence of TE domains within modular PKSs along with physically separated, monofunctional TEs (TE IIs) has been reported for a number of modular polyketide and non-ribosomal peptide synthases (NRPS). An analysis of the two types of thioesterases using Clustal X yielded a dendrogram showing that TE IIs from PKSs and NRPSs are more closely related to each other than to domain TEs from either PKSs or NRPSs. Furthermore, the dendrogram indicates that both types of TE IIs are more closely related to TE domains associated with PKSs than to TE domains in NRPSs. Finally, the overall % G+C content and the % G+C content at the third codon for all of the PKS genes in the COR cluster suggest that these genes may have been recruited from a gram-positive bacterium.

Phosphorylation of CorS and CorR, regulatory proteins that modulate production of the phytotoxin coronatine in Pseudomonas syringae.

Production of the phytotoxin coronatine (COR) in Pseudomonas syringae pv. glycinea PG4180 is controlled by a modified two-component regulatory system consisting of three genes, corR, corP, and corS. CorR and CorP show similarity to response regulators, and CorS is related to histidine protein kinases that function as environmental sensors. In this study, CorR, CorP and the cytoplasmic portion of CorS, designated CorSDelta, were overproduced in P. syringae as translational fusions to the maltose-binding protein and purified by affinity chromatography. Autophosphorylation of CorSDelta was demonstrated when [gamma-(32)P]ATP was used as a phosphodonor. Phosphorylated CorSDelta (CorSDelta approximately P) was stable under basic conditions, but extremely sensitive when the pH was reduced, which is consistent with phosphorylation at a histidine residue. CorSDelta approximately P transferred its phosphoryl group to CorR but not to CorP, which correlates with the presence of a receiver aspartate residue in the former but not the latter protein. These results indicate that CorS functions as a histidine protein kinase and transphosphorylates CorR, a positive activator of cor gene transcription.

POLYKETIDE PRODUCTION BY PLANT-ASSOCIATED PSEUDOMONADS.

Polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes; however, plant-associated pseudomonads also produce a variety of compounds via the polyketide pathway including the phytotoxin coronatine, the antibiotic mupirocin, and the antifungal compounds pyoluteorin and 2,4-diacetylphloroglucinol. This review focuses on the mode of action, regulation, biosynthesis, and genetics of these four compounds and the potential use of Pseudomonas-derived polyketide synthases in the generation of novel compounds with unique activities.

Biosynthesis of the Pseudomonas polyketide coronafacic acid requires monofunctional and multifunctional polyketide synthase proteins.

Coronafacic acid (CFA) is the polyketide component of the phytotoxin coronatine, a virulence factor of the plant pathogen Pseudomonas syringae. Our current knowledge of polyketide biosynthesis largely is based on the analysis of polyketide synthases (PKSs) in actinomycetes and other Gram-positive bacteria. Consequently, the cloning and characterization of the CFA biosynthetic gene cluster will contribute significantly to our knowledge of polyketide synthesis in Pseudomonas. In this report, we describe two genes in the CFA biosynthetic gene cluster that encode PKSs that are structurally and functionally similar to the multifunctional modular PKSs, which catalyze the synthesis of macrolide antibiotics. The CFA PKS genes were overproduced in Escherichia coli and shown to cross-react with antisera made to a modular PKS involved in erythromycin synthesis. A scheme for CFA biosynthesis is presented that incorporates the activities of all proteins in the CFA PKS. In this report a gene cluster encoding a pseudomonad polyketide has been completely sequenced and the deduced gene functions have been used to develop a biosynthetic scheme.

Analysis of genes involved in biosynthesis of coronafacic acid, the polyketide component of the phytotoxin coronatine.

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3' end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated cfa8, cfa9, and tnp1; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of cfa8 and cfa9 were overproduced in Escherichia coli BL21; however, tnp1 was not translated in these experiments. Mutagenesis and complementation analysis indicated that cfa8 is required for the production of CFA and COR. Analysis of a cfa9 mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tnp1, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a P. syringae strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.

Biosynthesis and regulation of coronatine, a non-host-specific phytotoxin produced by Pseudomonas syringae.

Many P. syringae pathovars are known to produce low-molecular-weight, diffusible toxins in infected host plants. These phytotoxins reproduce some of the symptoms of the relevant bacterial disease and are effective at very low concentrations. Phytotoxins generally enhance the virulence of the P. syringae pathovar which produces them, but are not required for pathogenesis. Genes encoding phytotoxin production have been identified and cloned from several P. syringae pathovars. With the exception of coronatine, toxin biosynthetic gene clusters are generally chromosomally encoded. In several pathovars, the toxin biosynthetic gene cluster also contains a resistance gene which functions to protect the producing strain from the biocidal effects of the toxin. In the case of phaseolotoxin, a resistance gene (argK) has been utilized to engineer phaseolotoxin-resistant tobacco plants. Although P. syringae phytotoxins can induce very similar effects in plants (chlorosis and necrosis), their biosynthesis and mode of action can be quite different. Knowledge of the biosynthetic pathways to these toxins and the cloning of the structural genes for their biosynthesis has relevance to the development of new bioactive compounds with altered specificity. For example, polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes. It is also important to note that it is now possible to utilize a genetic rather than synthetic approach to biosynthesize novel polyketides with altered biological properties (Hutchinson and Fujii, 1995; Kao et al., 1994; Donadio et al., 1993; Katz and Donadio, 1993). Most of the reprogramming or engineering of novel polyketides has been done using actinomycete PKSs, but much of this technology could also be applied to polyketides synthesized by Pseudomonas when sufficient sequence information is available. It is important to note that Pseudomonas produces a variety of antimicrobial compounds from the polyketide pathway, including mupirocin (pseudomonic acid) (Feline et al., 1977), pyoluteorin (Cuppels et al., 1986), and 2-4 diacetylphloroglucinol (Phl) (Bangera and Thomashow, 1996). Pseudomonic acid is valued for its pharmaceutical properties as an antibiotic (Aldridge, 1992), whereas pyoluteorin and Phl have antifungal properties (Howell and Stipanovic, 1980; Keel et al., 1992). A thorough understanding of the biosynthetic pathway to polyketide phytotoxins such as coronatine may ultimately lead to the development of novel compounds with altered biological properties. Thus, specific genes in the biosynthetic pathways of P. syringae phytotoxins could be deployed in other systems to develop new compounds with a wide range of activities.

Expression and analysis of coronafacate ligase, a thermoregulated gene required for production of the phytotoxin coronatine in Pseudomonas syringae.

Coronafacic acid, the polyketide component of the phytotoxin coronatine, is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the coronafacate ligase (cfl) gene product. In the present study, cfl was fused to the carboxy terminus of malE, which encodes the maltose-binding protein (MBP), and overexpressed in Escherichia coli. Immunoblot analysis indicated that Cfl contained an ATP-binding region, a motif conserved in enzymes which activate their substrates by adenylation. MBP-Cfl was overproduced and purified from Pseudomonas syringae and the protein fusion was used to generate antisera. Anti-MBP-Cfl antibodies and a transcriptional fusion of the cfl promoter to a promoterless glucuronidase gene were used to follow the temporal expression of coronafacate ligase. The results indicated that transcription of cfl is temperature-sensitive. Furthermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.

Use of translational fusions to the maltose-binding protein to produce and purify proteins in Pseudomonas syringae and assess their activity in vivo.

A simple approach is described for the production and purification of proteins in Pseudomonas syringae. The strategy involves the use of the tac promoter, the maltose-binding protein, and the broad-host-range vector, pRK415. This approach was used to partially purify two proteins involved in coronatine biosynthesis from P. syringae. The activity of the fusions was demonstrated in vivo in complementation experiments using the appropriate mutants.

Ketohexokinase (ATP:D-fructose 1-phosphotransferase) from a halophilic archaebacterium, Haloarcula vallismortis: purification and properties.

Ketohexokinase (ATP:D-fructose 1-phosphotransferase [EC 2.7.1.3]), detected for the first time in a prokaryote, i.e., the extreme halophile Haloarcula vallismortis, was isolated and characterized from the same archaebacterium. This enzyme was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity, and kinetic properties. Gel filtration and sucrose density gradient centrifugation revealed a native molecular mass of 100 kDa for halobacterial ketohexokinase, which is larger than its mammalian counterpart. The enzyme could be labeled by UV irradiation in the presence of [ gamma-32P]ATP, suggesting the involvement of a phosphoenzyme intermediate. Other catalytic features of the enzyme were similar to those of its mammalian counterparts. No antigenic cross-reactivity could be detected between the H. vallismortis ketohexokinase and the ketohexokinases from different rat tissues.

Characterization of 1-phosphofructokinase from halophilic archaebacterium Haloarcula vallismortis.

1-Phosphofructokinase (EC 2.7.1.56) (1PFK) was purified and characterized for the first time from an archaebacterial halophile Haloarcula vallismortis. The purification procedure involving (NH4)2SO4 fractionation, (NH4)2SO4-mediated chromatography on Sepharose 4B, CM-cellulose chromatography, hydrophobic chromatography on phenyl Sepharose and adsorption chromatography on hydroxylapatite yielded a preparation with a specific activity of 128 and 100-fold purification. From gel filtration and sucrose density gradient ultracentrifugation, the apparent molecular mass of halobacterial 1PFK was found as 76 +/- 5 kDa. The halobacterial 1PFK appears to be monomeric and the possibility of an unstable phosphoenzyme intermediate during its catalysis could not be ruled out. As in the case of many halobacterial enzymes, the 1PFK was found to be halophilic and thermostable. Other catalytic features of halobacterial 1PFK were similar to its counterparts from eubacterial sources.

Stimulation of ammonification and nitrification in soils by the insecticides monocrotophos and quinalphos.

The application (up to 5 kg ha-1) of monocrotophos or quinalphos to four types of agricultural soils significantly stimulated the mineralization of peptone-nitrogen and the oxidation of ammonium-nitrogen. In soils treated with 2.5 kg ha-1 of either insecticide, the rate of ammonification and nitrification was fairly rapid after 2 and 4 weeks of incubation. The enhancement of both transformations, mediated by microorganisms, was significantly more in the quinalphostreated soils. The results suggest that a balance between the effect of insecticides on insect pests and their impact on beneficial microbial activities in soil must be determined.