Isolation and characterization of phorate degrading soil bacteria of environmental and agronomic significance

Phorate [O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate] degrading bacteria were isolated from agricultural soil and characterized based on their morphological and biochemical characteristics. The selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteous species, respectively. The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days. The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 days-1. Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid, and siderophore production. The isolate PS-3 also showed anti-fungal activity against a phytopathogen Fusarium oxysporum. As a result of the multifarious biological properties, the isolates have been suggested to be important bioresource for efficient bioinoculant development.     

Episodic positive selection during the evolution of naphthalene dioxygenase to nitroarene dioxygenase

Using different maximum-likelihood models of adaptive evolution, signatures of natural selective pressure, operating across the naphthalene family of dioxygenases, were examined. A lineage- and branch-site specific combined analysis revealed that purifying selection pressure dominated the evolutionary history of the enzyme family. Specifically, episodic positive Darwinian selection pressure, affecting only a few sites in a subset of lineages, was found to be responsible for the evolution of nitroarene dioxygenases (NArDO) from naphthalene dioxygenase (NDO). Site-specific analysis confirmed the absence of diversifying selection pressure at any particular site. Different sets of positively selected residues, obtained from branch-site specific analysis, were detected for the evolution of each NArDO. They were mainly located around the active site, the catalytic pocket and their adjacent regions, when mapped onto the 3D structure of the α-subunit of NDO. The present analysis enriches the current understanding of adaptive evolution and also broadens the scope for rational alteration of substrate specificity of enzyme by directed evolution.     

Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity

AIM: To screen for novel antagonistic Pseudomonas strains producing both phenazines and biosurfactants that are as effective as Pseudomonas aeruginosa PNA1 in the biocontrol of cocoyam root rot caused by Pythium myriotylum. MATERIAL AND RESULTS: Forty pseudomonads were isolated from the rhizosphere of healthy white and red cocoyam plants appearing in natural, heavily infested fields in Cameroon. In vitro tests demonstrated that Py. myriotylum antagonists could be retrieved from the red cocoyam rhizosphere. Except for one isolate, all antagonistic isolates produced phenazines. Results from whole-cell protein profiling showed that the antagonistic isolates are different from other isolated pseudomonads, while BOX-PCR revealed high genomic similarity among them. 16S rDNA sequencing of two representative strains within this group of antagonists confirmed their relatively low similarity with validly described Pseudomonas species. These antagonists are thus provisionally labelled as unidentified Pseudomonas strains. Among the antagonists, Pseudomonas CMR5c and CMR12a were selected because of their combined production of phenazines and biosurfactants. For strain CMR5c also, production of pyrrolnitrin and pyoluteorin was demonstrated. Both CMR5c and CMR12a showed excellent in vivo biocontrol activity against Py. myriotylum to a similar level as Ps. aeruginosa PNA1. CONCLUSION: Pseudomonas CMR5c and CMR12a were identified as novel and promising biocontrol agents of Py. myriotylum on cocoyam, producing an arsenal of antagonistic metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the identification of two newly isolated fluorescent Pseudomonas strains that can replace the opportunistic human pathogen Ps. aeruginosa PNA1 in the biocontrol of cocoyam root rot and could be taken into account for the suppression of many plant pathogens.     

Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity

AIM: This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil-borne phytopathogenic fungi from the avocado rhizoplane. METHODS AND RESULTS: A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram-positive isolates. From these gram-positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil-borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis-lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities. CONCLUSIONS: Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil-borne phytopathogenic fungi F. oxysporum f.sp. radicis-lycopersici and R. necatrix.     

Saposin C is required for normal resistance of acid beta-glucosidase to proteolytic degradation

Saposins (A, B, C, and D) are small sphingolipid activator proteins that are derived by proteolytic processing of a common precursor, prosaposin. In the lysosomal sphingolipid degradation pathway, acid beta-glucosidase (GCase) requires saposin C for optimal in vitro and in vivo hydrolysis of glucocerebroside. The deficiency of prosaposin/saposins (PS-/-) in humans and mice leads to a decrease of GCase activity in selected tissues. Concordant decreases (>50%) of GCase protein and in vitro activity were detected in extracts of cultured fibroblasts and hepatocytes from PS-/- mice and human prosaposin-deficient fibroblasts. GCase RNA in the PS-/- cells was at wild-type levels. Compared with that in wild-type cells (t(1/2) >24 h), the GCase protein in the PS-/- cells had a faster disappearance rate (t(1/2) approximately 1 h in mouse and approximately 8 h in human) as determined by metabolic labeling and immunoprecipitation with anti-GCase antibodies. Treatment of PS-/- cells with leupeptin, an inhibitor of cysteine proteases, led to significant increases (approximately 2-fold) in GCase protein and in vitro activity. Loading saposin C to human PS-/- fibroblasts resulted in an enhancement of GCase protein and in vitro activity. Saposin D loading had no effect. These data indicate that saposin C is required for GCase resistance to proteolytic degradation in the cell. Thus, diminished in vivo GCase activity would be greater than expected only from the lack of GCase activation by saposin C. These results indicate a new property for saposin C, an anti-proteolytic protective function toward GCase.     

Evaluation of potential biocontrol rhizobacteria from different host plants of Verticillium dahliae Kleb

AIMS: A screening approach was developed to assess the potential of rhizobacterial strains to control Verticillium wilt caused by Verticillium dahliae Kleb. METHODS AND RESULTS: Sixty randomly chosen antagonistic bacterial strains originally isolated from rhizosphere of three different host plants of V. dahliae--strawberry, potato and oilseed rape--were evaluated for biocontrol and plant growth promotion by analysing in vitro antagonism towards V. dahliae and other plant pathogenic fungi, production of fungal cell wall-degrading enzymes and plant growth-promoting effects on strawberry seedlings. To test the plant growth-promoting effect, a microplate assay with strawberry seedlings was developed. Although the rhizobacterial strains were isolated from different plants they showed effects on the growth of strawberry seedlings. According to the in vitro biocontrol and plant growth-promoting activity, the three best candidates Pseudomonas putida B E2 (strawberry rhizosphere), Ps. chlororaphis K15 (potato rhizosphere) and Serratia plymuthica R12 (oilseed rape rhizosphere) were selected for greenhouse experiments to verify the in vitro screening results. Under greenhouse conditions the isolates selected according to this strategy were as effective, or more effective than commercial biocontrol agents and may therefore possibly be valuable as antagonists of V. dahliae. CONCLUSIONS: In this study, the screening strategy resulted in a selection of three interesting biocontrol candidates against Verticillium: Ps. putida B E2 (strawberry rhizosphere), Ps. chlororaphis K15 (potato rhizosphere) and Ser. plymuthica R12 (oilseed rape rhizosphere). SIGNIFICANCE AND IMPACT OF THE STUDY: A new combination of in vitro screening methods including a microplate assay with strawberry seedlings to test the plant growth promoting effect which allow to more efficiently select potential biological control agents was developed successfully.     

Molecular and genetic analyses of geographic variation in isolates of Phoma macrostoma used for biological weed control

Molecular and genetic approaches were used to evaluate the genetic relatedness among isolates of the fungus Phoma macrostoma Montagne originating from Canada and Europe and to other species in the genus Phoma. Distinct differences were observed in genetic variation among nine species of the genus Phoma. Randomly amplified polymorphic DNA (RAPD) revealed the presence of intraspecific genetic variation among the isolates of P. macrostoma, with the isolates being used for biological weed control being distributed in a distinct phylogenetic cluster. Additional variation within the biocontrol isolate cluster in P. macrostoma was revealed by pulsed field gel electrophoresis (PFGE), which showed that biocontrol isolates generated two different chromosomal profiles, however the profiles did not relate to their Canadian ecozone origin. Mating studies showed that biocontrol isolates of P. macrostoma from Canada did not produce sexual reproductive structures and were incapable of crossing. These studies also confirmed that no obvious differentiation exists among the biocontrol isolates of P. macrostoma from Canadian Ecozones 3 and 4.     

Application of endophytic bacteria for controlling anthracnose disease (Colletotrichum lindemuthianum) on bean plants

Over 100 endophytic bacterial isolates were isolated from surface-sterilised roots of the Fabaceae family in East Azerbaijan farms. These isolates were screened for their in vitro biocontrol activity against Colletotrichum lindemuthianum by dual culture technique using potato dextrose agar (PDA) medium. Eight bacterial isolates (Bacillus subtilis subsp. subtilis, Bacillus atrophaeus, B. tequilensis, B. subtilis subsp. spizizenii, Streptomyces cyaneofuscatus, S. flavofuscus, S. parvus, S. acrimycini) showed promising inhibition on mycelial growth of C. lindemuthianum , and thus, these isolates were selected for greenhouse experiments. The disease control rate using these selected endophytic bacteria was varied from 40 to 76.80% in greenhouse without any negative effects on different growth performance, suggesting that these selected endophytic bacteria are potential to be developed as biocontrol agents.     

The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of extracellular senile plaques composed of amyloid beta-peptide (A beta). Whereas most cases of AD occur sporadically, about 10% of AD cases are inherited as a fully penetrant autosomal dominant trait. Mutations in the recently cloned Presenilin genes (PS-1 and PS-2) are by far the most common cause of early onset familial AD. MATERIALS AND METHODS: Cellular expression of endogenous and overexpressed PS proteins was analyzed by immunocytochemistry and metabolic labeling followed by immunoprecipitation. In vivo phosphorylation sites of PS proteins were analyzed by extensive mutagenesis. RESULTS: PS-1 as well as PS-2 proteins were localized predominantly within the endoplasmic reticulum (ER). However, small amounts of the PS proteins were detected within the Golgi compartment, where they colocalize with the beta-amyloid precursor protein (beta APP). The PS-2 protein was found to be highly phosphorylated, whereas very little phosphorylation was observed for PS-1. The selective phosphorylation of PS-2 occurs exclusively on serine residues. In vivo phosphorylation of PS-2 was mapped to serine residues 7, 9, and 19 within an acidic stretch at the N terminus, which is absent in PS-1. casein kinase (CK)-1 and CK-2 were shown to phosphorylate the N terminus of PS-2 in vitro. CONCLUSIONS: The majority of PS proteins were detected in the ER where little if any proteolytic processing of beta APP was reported. ER retention of PS proteins might occur by intramolecular aggregation. Small amounts of PS proteins were also detected in the Golgi where they colocalized with beta APP. This might suggest that potential interactions between PS proteins and beta APP could occur within the Golgi. Selective phosphorylation of PS-2 proteins within the acidic domain missing in PS-1 indicates differences in the biological functions and regulation of the two highly homologous proteins.     

Biocontrol Potential of Soybean Bacterial Endophytes Against Charcoal Rot Fungus, <Emphasis Type="Italic">Rhizoctonia bataticola</Emphasis>

A total of 137 bacterial isolates from surface sterilized root, stem, and nodule tissues of soybean were screened for their antifungal activity against major phytopathogens like Rhizoctonia bataticola, Macrophomina phaseolina, Fusarium udam, and Sclerotium rolfsii. Nine bacterial endophytes suppressed the pathogens under in vitro plate assay. These were characterized biochemically and identified at the genus level based on their partial sequence analysis of 16S rDNA. Eight of the isolates belonged to Bacillus and one to Paenibacillus. The phylogenetic relationship among the selected isolates was studied and phylogenetic trees were generated. The selected isolates were screened for biocontrol traits like production of hydrogen cyanide (HCN), siderophore, hydrolytic enzymes, antibiotics, and plant growth promoting traits like indole 3-acetic acid production, phosphate solubilization, and nitrogen fixation. A modified assessment scheme was used to select the most efficient biocontrol isolates Paenibacillus sp. HKA-15 (HKA-15) and Bacillus sp. HKA-121 (HKA-121) as potential candidates for charcoal rot biocontrol as well as soybean plant growth promotion.     

Use of a breeding approach for improving biocontrol efficacy of Phlebiopsis gigantea strains against Heterobasidion infection of Norway spruce stumps

Sixty-four wild heterokaryotic isolates of Phlebiopsis gigantea were analysed for asexual spore production, growth rate and competitive ability against Heterobasidion in vitro , as well as growth rate in Norway spruce wood. These P. gigantea traits were considered important for controlling infection of Norway spruce stumps by spores of Heterobasidion spp. Ten most promising P. gigantea isolates were crossed with each other and 172 F₁ progeny heterokaryons were analysed for the above-mentioned traits. Thirteen most promising progeny heterokaryons were selected and their biocontrol ability against infection by Heterobasidion was compared with the parental isolates in stem pieces of Norway spruce. The results indicated that the progeny strains had generally better traits and control efficacy than the parental strains. The genetic effects accounted for a part of the variations between progeny and parental strains. This further suggests that there is a potential to improve the biocontrol properties of P. gigantea through breeding.     

Diversity of culturable root-associated/endophytic bacteria and their chitinolytic and aflatoxin inhibition activity of peanut plant in China

A total of 72 isolates of root-associated/endophytic (RAE) bacteria were isolated from peanut plants grown in the main producing areas of 6 provinces in China. The 16S rRNA gene sequences of these isolates were determined and phylogenetic analyses revealed that 72 isolates belonged to the classes Bacilli (49 isolates) and Gammaproteobacteria (23 isolates). The majority of RAE bacteria in Bacilli belonged to 2 genera, Bacillus and Lysinibacillus (48 and 1) while those in Gammaproteobacteria belonged to the genera Enterobacter, Serratia, Stenotrophomonas, and Pseudomonas (7, 11, 3 and 2 isolates, respectively). This is the first report of Lysinibacillus xylanilyticus isolate as biocontrol agent against AFs. All of the selected RAE bacteria showed inhibitory activities against Aspergillus parasiticus (A. parasiticus) growth and/or aflatoxins (AFs) production by visual agar plate assay and tip culture method. Most of the RAE bacteria strains (96?% strains) were determined to have decreased mycelia growth or AFs production levels by >50?% (p?<?0.05). Bacterial isolates were further characterized for chitinolytic activity and 22 strains (30?% strains) of identified RAE bacteria degraded colloidal chitin on the chitin medium plate. Ten selected chitinolytic RAE bacteria were tested for antifungal activity on peanuts and most of them significantly decreased mycelial growth and AFs production levels by >90?%. These results showed a wide distribution of biological control bacteria against AFs in Chinese peanut main producing areas and the selected RAE bacteria could potentially be utilized for the biocontrol of toxicogenic fungi.     

Biocontrol bacteria selected by a direct plant protection strategy against avocado white root rot show antagonism as a prevalent trait

Aim: This study was undertaken to study bacterial strains obtained directly for their efficient direct control of the avocado white root rot, thus avoiding prescreening by any other possible mechanism of biocontrol which could bias the selection. Methods and Results: A collection of 330 bacterial isolates was obtained from the roots and soil of healthy avocado trees. One hundred and forty‐three representative bacterial isolates were tested in an avocado/Rosellinia test system, resulting in 22 presumptive protective strains, all of them identified mainly as Pseudomonas and Bacillus species. These 22 candidate strains were screened in a more accurate biocontrol trial, confirming protection of some strains (4 out of the 22). Analyses of the potential bacterial traits involved in the biocontrol activity suggest that different traits could act jointly in the final biocontrol response, but any of these traits were neither sufficient nor generalized for all the active bacteria. All the protective strains selected were antagonistic against some fungal root pathogens. Conclusions: Diverse bacteria with biocontrol activity could be obtained by a direct plant protection strategy of selection. All the biocontrol strains finally selected in this work were antagonistic, showing that antagonism is a prevalent trait in the biocontrol bacteria selected by a direct plant protection strategy. Significance and Impact of the Study: This is the first report on the isolation of biocontrol bacterial strains using direct plant protection strategy in the system avocado/Rosellinia. Characterization of selected biocontrol bacterial strains obtained by a direct plant protection strategy showed that antagonism is a prevalent trait in the selected strains in this experimental system. This suggests that antagonism could be used as useful strategy to select biocontrol strains.     

Chitin-supplemented foliar application of chitinolytic Bacillus cereus reduces severity of Botrytis gray mold disease in chickpea under controlled conditions

AIM: To identify and evaluate chitinolytic bacteria for control of Botrytis gray mold (BGM), a devastating disease in chickpea. METHODS AND REsults: Two antifungal bacterial isolates, chitinolytic Bacillus cereus CRS 7 and nonchitinolytic Pseudomonas fluorescens CRS 31, from the rhizosphere of chickpea, were applied as a prophylactic foliar spray and evaluated for control of BGM. In a controlled environment, the two isolates reduced the severity of BGM on the susceptible cv. JG 62 to 6.0 and 5.6, respectively, compared with 9.0 in the control, measured on a 1-9 rating scale. Supplementation of the foliar application of CRS 7 with 0.5% and 1.0% colloidal chitin reduced BGM severity to 4.4 and 4.1 respectively, while chitin-supplemented application of CRS 31 was similar to CRS 31 applied alone. Partially purified 47-kDa chitinase from the cell-free culture filtrate of CRS 7 at 20 and 40 mug protein ml(-1) (enzyme activity 3.1 units ml(-1)) inhibited the germination and lysed the conidia of Botrytis cinerea, and as a prophylactic foliar spray reduced BGM severity to 5.4 and 4.8, respectively. CONCLUSION: Chitin supplementation improved the biocontrol of the foliar disease BGM by chitinolytic bacterium. Disease control with partially purified chitinase of CRS 7 supported the major role of chitinolysis in improved control of BGM. SIGNIFICANCE AND IMPACT OF THE STUDY: Enhanced control of BGM by chitin-supplemented application of CRS 7 is of significant in view of the frequent inconsistency in biocontrol of foliar diseases. This study supports further attempts on chitinolysis-based biocontrol methods for foliar disease biocontrol.     

Application of Soil‐borne Actinomycetes for Biological Control against Fusarium Wilt of Chickpea (Cicer arietinum) caused by Fusarium solani fsp pisi

Black root rot, caused by Fusarium solani f.sp. pisi, is a devastating soil‐borne disease in chickpea in Iran with no effective control measures. With the aim of finding applicable biocontrol agents to alleviate the malady, isolates of Actinomycetes isolated from soil and their antagonistic effect against F. solani f.sp. pisi were evaluated both in vitro and in vivo. More than 100 Actinomycetes isolates were screened for their antifungal activities against the pathogen. The most active isolates were evaluated in greenhouse for their biocontrol performance. Based on the results of dual cultures in screening evaluations, the size of inhibition zone of fungal growth, and the most effective antagonist isolates (S3, S12 and S40) were selected for further studies. Identity of active isolates was determined, in this regard, 16S rDNA of isolates were amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then performed using NCBI BLAST method. Comparison of the near full length 16S rRNA sequence of isolates to GenBank sequences demonstrated that isolates S3 and S12 were most similar to Streptomyces antibioticus, while isolate S40 was most similar to Streptomyces peruviensis. Biocontrol studies of these isolates in control of the disease in greenhouse significantly decreased the disease severity. Actinomycetes isolate S12 demonstrated the greatest effect in reducing disease than the other two. Results of this research are at preliminary stage for developing biocontrol agents. These data can be utilized as a platform for future studies with the aim of commercializing these biocontrol products and hoping to step towards sustainable agriculture.     

Synthesis of highly hydroxylated aromatics by evolved biphenyl dioxygenase and subsequent dihydrodiol dehydrogenase

The evolved bphA1 (2049) gene, in which nine amino acids from the Pseudomonas pseudoalcaligenes KF707 BphA1 were changed to those from the Burkholderia xenovorans LB400 BphA1 (M247I, H255Q, V258I, G268A, D303E, -313G, S324T, V325I, and T376N), was expressed in Escherichia coli along with the bphA2A3A4 and bphB genes derived from strain KF707. This recombinant E. coli cells converted biphenyl and several heterocyclic aromatic compounds into the highly hydroxylated products such as biphenyl-2,3,2′,3′-tetraol (from biphenyl), 2-(2,3-dihydroxyphenyl)benzoxazole-4,5-diol (from 2-phenylbenzoxazole), and 2-(2,5-dihydroxyphenyl)benzoxazole-4,5-diol [from 2-(2-hydroxyphenyl)benzoxazole]. The antioxidative activity of these generated compounds was markedly higher than that of the original substrate used.     

Genetic diversity of dioxygenase genes in polycyclic aromatic hydrocarbon-degrading bacteria isolated from mangrove sediments

To investigate the diversity of dioxygenase genes involved in polycyclic aromatic hydrocarbon (PAH)-degradation, a total of 32 bacterial strains were isolated from surface mangrove sediments, from the genera Mycobacterium, Sphingomonas, Terrabacter, Sphingopyxis, Sphingobium and Rhodococcus. Two sets of PCR primers were constructed to detect the nidA-like and nahAc-like sequences of the alpha subunit of the PAH ring-hydroxylating dioxygenase. PCR amplified the DNA fragments from all Gram-positive bacteria by using nidA-like primers and from all Gram-negative bacteria, except two, by using nahAc-like primers. The nidA-like primers showed three subtypes of nidA-like gene: (i) fadA1, clustering with nidA3 from M. vanbaalenii PYR-1, (ii) nidA, clustering with nidA from PYR-1, and (iii) fadA2 clustering with dioxygenase from Arthrobacter sp. FB24. The amplicons detected by nahAc-like primers had high sequence homologies to phnA1a from Sphingomonas sp. CHY-1 and were amplifiable from 8 of the 16 Gram-negative isolates. The primer also generated amplicons that had a 32-36% similarity to phnA1a and 53-93% identity to p-cumate dioxygenase. These results suggest that the nidA-like and nahAc-like genes are prevalent in the PAH-degrading bacteria and that they are useful for determining the presence of PAH-dioxygenase genes in environmental samples.     

Structural and functional analyses of human tryptophan 2,3‐dioxygenase

Tryptophan 2,3‐dioxygenase (TDO), one of the two key enzymes in the kynurenine pathway, catalyzes the indole ring cleavage at the C2‐C3 bond of l ‐tryptophan. This is a rate‐limiting step in the regulation of tryptophan concentration in vivo, and is thus important in drug discovery for cancer and immune diseases. Here, we report the crystal structure of human TDO (hTDO) without the heme cofactor to 2.90 Å resolution. The overall fold and the tertiary assembly of hTDO into a tetramer, as well as the active site architecture, are well conserved and similar to the structures of known orthologues. Kinetic and mutational studies confirmed that eight residues play critical roles in l ‐tryptophan oxidation. Proteins 2014; 82:3210–3216. © 2014 Wiley Periodicals, Inc.     

Isolation and characterization of a phorate degrading bacterium

Aims:  To study the degradation of phorate by a bacterium isolated from phorate-contaminated sites.
Methods and Results:  Ralstonia eutropha strain AAJ1 isolated from soil was found to degrade phorate (supplied as sole carbon source) upto 85% in 10 days in liquid medium. Half-life ( t _½) of phorate in the liquid medium in control (uninoculated) and in experimental (inoculated with R. eutropha , strain AAJ1) samples was recorded as 36·49 and 6·29 days, respectively. Kinetics revealed that phorate degradation depends on time and the reaction follows the first order kinetics. Diethyl dithiophosphate was one of the degradation products, which is markedly less toxic than the parent compound; other degradation products included phorate sulfoxide and phorate sulfone. Release of inorganic phosphates and sulfates indicated the potential of the isolate to further degrade the above-mentioned metabolites to simpler forms. The strain was also found to posses phosphomonoesterase and phosphodiesterase enzymatic activity, which are involved in biodegradation of organophosphorus compounds.
Conclusions:  Ralstonia eutropha AAJ1 could degrade and detoxify phorate upto 85% in 10 days in laboratory conditions.
Significance and Impact of the Study:  The isolate has the potential to be utilized for remediation of phorate-contaminated water and soil.