Expression of Auxin Response Genes SlIAA1 and SlIAA9 in Solanum lycopersicum During Interaction with Acinetobacter guillouiae SW5.

Indole-3-acetic acid (IAA) production is a typical mechanism of plant growth promotion by some rhizobacteria. However, a functional genomic study is necessary to unravel the function and mechanism of IAA signaling during rhizobacteria-plant interactions. In this study, the expression of SlIAA1 and SlIAA9 among the auxin response genes in tomato was examined during the interaction between IAA-producing Acinetobacter guillouiae SW5 and tomato plants. When 3-day grown tomato seedlings were treated for 30 min with 10~100 µM of IAA produced by bacteria from tryptophan, the relative mRNA levels of SlIAA1 and SlIAA9 increased significantly compared with those of the control, demonstrating that IAA produced by this bacterium can induce the expressions of both genes. Inoculation of live A. guillouiae SW5 to tomato seedlings also increased the expressions of SlIAA1 and SlIAA9, with more mRNA produced at higher bacterial density. In contrast, treatment of tomato seedlings with dead A. guillouiae SW5 did not significantly affect the expression of SlIAA1and SlIAA9. When 3-day bacterial culture in tomato root exudates was administered to tomato seedlings, the relative mRNA level of SlIAA1 increased. This result indicated that the plant may take up IAA produced by bacteria in plant root exudates, which may increase the expression of the auxin response genes, with resulting promotion of plant growth.

Degradation of alkylphenols by white rot fungus Irpex lacteus and its manganese peroxidase.

Alkylphenols are common endocrine disrupters that are produced from the degradation of widely used surfactants. Since they cause various harmful effects on aquatic life and in humans, they should be removed from the environments being contaminated. White rot fungus Irpex lacteus can completely degrade 100 mg/L of octylphenol, nonylphenol, and phenylphenol during 1 day of incubation in the complex YMG medium, which was the highest degrading capability among nine strains of white rot fungi tested. In the N-limited Kirk's basal salts medium, I. lacteus could degrade almost 100 % of 100 mg/L octylphenol and nonylphenol in 1 h, and exhibited a high activity of manganese peroxidase (MnP; 1,790 U/L). MnP of I. lacteus was purified by ion exchange chromatography, and this degraded 99 % of 50 mg/L octylphenol and removed 80 % of estrogenic activity in 2 hours. In addition, the purified MnP (10 U/mL) degraded over 90 % of 50 mg/L nonylphenol in 1 h.

Genetic and phenotypic diversity of carbofuran-degrading bacteria isolated from agricultural soils.

Thirty-seven carbofuran-degrading bacteria were isolated from agricultural soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize carbofuran as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Rhodococcus, Sphingomonas, and Sphingobium, including new types of carbofuran-degrading bacteria, Bosea and Microbacterium. Among the 37 isolates, 15 different chromosomal DNA patterns were obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. Five of the 15 representative isolates were able to degrade carbofuran phenol, fenoxycarb, and carbaryl, in addition to carbofuran. Ten of the 15 representative isolates had 1 to 8 plasmids. Among the 10 plasmid-containing isolates, plasmid-cured strains were obtained from 5 strains. The cured strains could not degrade carbofuran and other pesticides anymore, suggesting that the carbofuran degradative genes were on the plasmid DNAs in these strains. When analyzed with PCR amplification and dot-blot hybridization using the primers targeting for the previously reported carbofuran hydrolase gene (mcd), all of the isolates did not show any positive signals, suggesting that their carbofuran hydrolase genes had no significant sequence homology with the mcd gene.

Growth promotion of Xanthium italicum by application of rhizobacterial isolates of Bacillus aryabhattai in microcosm soil.

This study was conducted using rhizobacteria, which are able to exert beneficial effects upon plant growth in the infertile soil collected from barren lakeside areas. Four strains of plant growth promoting bacteria were isolated from the rhizosphere of a common wild plant, Erigeron canadensis. Isolated strains LS9, LS11, LS12, and LS15 were identified as Bacillus aryabhattai by 16S rDNA sequence analysis. B. aryabhattai LS9, LS11, LS12, and LS15 could solubilize 577.9, 676.8, 623.6, and 581.3 mg/L of 0.5% insoluble calcium phosphate within 2 days of incubation. Production of indole acetic acid, a typical growth promoting phytohormone auxin, by strain LS15 was 471.3 mg/L in 2 days with the addition of auxin precursor L-tryptophan. All the strains also produced other phytohormones such as indole butyric acid, gibberellins, and abscisic acid, and strain LS15 showed the highest production rate of gibberellin (GA(3)), 119.0 µg/mg protein. Isolated bacteria were used in a microcosm test for growth of wild plant Xanthium italicum, which can be utilized as a pioneer plant in barren lands. Seed germination was facilitated, and the lengths of roots, and shoots and the dry weights of germinated seedlings after 16 days were higher than those of the uninoculated control plants. Root lengths of seedlings of X. italicum increased by 121.1% in LS11-treated samples after 16 days. This plant growth-promoting capability of B. aryabhattai strains may be utilized as an environmentally friendly means of revegetating barren lands, especially sensitive areas such as lakeside lands.

Changes in the activities of enzymes involved in the degradation of butylbenzyl phthalate by Pleurotus ostreatus.

Degradation of butylbenzyl phthalate (BBP) by the white rot fungus Pleurotus ostreatus and the activities of some degrading enzymes were examined in two different media containing 100 mg/l of the compound. P. ostreatus pregrown for 7 days in complex YMG medium was able to completely degrade BBP within an additional 24 h but degraded only 35 mg/l of BBP in 5 days of incubation in minimal medium. Fungal cell mass in the culture in YMG medium was higher in the presence than in the absence of BBP. The esterase activity of the fungal culture in YMG medium was higher than that in minimal medium and increased with the addition of BBP. On the contrary, laccase activity was higher in minimal medium and it did not increase upon the addition of BBP. General peroxidase activity increased for a few days after the addition of BBP to both media. The degradation of BBP and its metabolites by P. ostreatus thus may be attributed mostly to esterase rather than lignin-degrading laccase. In addition, the activities of the enzymes involved in BBP degradation and their changes varied significantly in the different media and culture conditions.

Nitroreductase II involved in 2,4,6-trinitrotoluene degradation: purification and characterization from Klebsiella sp. Cl.

Three 2,4,6-trinitrotoluene (TNT) nitroreductases from Klebsiella sp. CI have different reduction capabilities that can degrade TNT by simultaneous utilization of two initial reduction pathways. Of these, nitroreductase II was purified to homogeneity by sequential chromatographies. Nitroreductase II is an oxygen-insensitive enzyme and reduces both TNT and nitroblue tetrazolium. The N-terminal amino acid sequence of the enzyme did not show any sequence similarity with those of other nitroreductases reported. However, it transformed TNT by the reduction of nitro groups like nitroreductase I. It had a higher substrate affinity and specific activity for TNT reduction than other nitroreductases, and it showed a higher oxidation rate of NADPH with the ortho-substituted isomers of TNT metabolites (2-hydroxylaminodinitrotoluene and 2-aminodinitrotoluene) than with para-substituted compounds (4-hydroxylaminodinitrotoluene and 4-amino-dinitrotoluene).

Effect of fungal pellet morphology on enzyme activities involved in phthalate degradation.

Pellet size of white rot fungus, Pleurotus ostreatus may affect the secretion of its degradative enzymes and accompanying biodegrading capability, but could be controlled by several physical culture conditions in liquid culture. The pellet size of P. ostreatus was affected by the volume of inoculum, flask, and medium, but the agitation speed was the most important control factor. At the lower agitation speed of 100 rpm, the large pellets were formed and the laccase activity was higher than that of small pelleted culture at 150 rpm, which might be due to loose intrapellet structure. However, the biodegradation rates of benzylbutylphthalate and dimethylphthalate were higher in the small pelleted culture, which indicated the involvement of other degradative enzyme rather than laccase. The activity of esterase which catalyzes the nonphenolic compounds before the reaction of ligninolytic enzymes was higher in the small pelleted culture, and coincided with the degradation pattern of phthalates. This study suggests the optimization of pellet morphology and subsequent secretion of degradative enzymes is necessary for the efficient removal of recalcitrants by white rot fungi.

Quantitative improvement of 16S rDNA DGGE analysis for soil bacterial community using real-time PCR.

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments has been frequently used to profile a structure of the bacterial community in a given soil. However, this procedure has various types of intrinsic error and bias, thus often misleads the relative abundance of bacterial populations. In order to establish a reliability for the current 16S rDNA DGGE method, we investigated various parameters and potential sources of errors in the DGGE procedures, such as primer mismatch, dNTP concentration, DNA polymerase, PCR cycles, uneven amplification of templates, secondary structure of PCR product, melting domain profiles, and acrylamide/bis concentration. Our result showed that the relative band intensities of the corresponding 16S rDNA templates were closely correlated with the differences of the melting temperature between the higher and lower melting domains of the PCR products. In addition, application of i) real-time PCR, ii) combination of PCR primers and iii) optimization of both dNTP and acrylamide/bis concentrations significantly improved the quantitative representation of bacterial 16S rDNA levels in the mixed samples. Especially, identification of the inflection points of DNA samples through the real-time PCR was crucial for the accurate representation of soil bacterial populations. Beyond these points DNA templates can be over-amplified to a saturated level independently of their initial amounts. Therefore for the accurate analysis of soil bacterial community, a quantitative 16S rDNA DGGE analysis needs to be performed in combination with a real-time PCR.

Genetic and phenotypic diversity of fenitrothion-degrading bacteria isolated from soils.

Twenty-seven fenitrothion-degrading bacteria were isolated from different soils, and their genetic and phenotypic characteristics were investigated. Analysis of the 16S rDNA sequence showed that the isolates were related to members of the genera Burkholderia, Pseudomonas, Sphingomonas, Cupriavidus, Corynebacterium, and Arthrobacter. Among the 27 isolates, 12 different chromosomal DNA fingerprinting patterns were obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. The isolates were able to utilize fenitrothion as a sole source of carbon and energy, producing 3-methyl-4- nitrophenol as the intermediate metabolite during the complete degradation of fenitrothion. Twenty-two of 27 isolates were able to degrade parathion, methyl-parathion, and p-nitrophenol, but only strain BS2 could degrade EPN (O-ethyl-O-p-nitrophenyl phenylphosphorothioate) as a sole source of carbon and energy for growth. Eighteen of the 27 isolates had plasmids. When analyzed with PCR amplification and dot-blotting hybridization using various specific primers targeted to the organophosphorus pesticide hydrolase genes of the previously reported isolates, none of the isolates showed positive signals, suggesting that the corresponding genes of our isolates had no significant sequence homology with those of the previously isolated organophosphate pesticide-degrading bacteria.

Biodegradation and saccharification of wood chips of Pinus strobus and Liriodendron tulipifera by white rot fungi.

Degradation and glucose production from wood chips of white pine (Pinus strobus) and tulip tree (Liriodendron tulipifera) by several white rot fungi were investigated. The highest weight losses from 4 g of wood chips of P. strobus and L. tulipifera by the fungal degradation on yeast extractmalt extract-glucose agar medium were 38% of Irpex lacteus and 93.7% of Trametes versicolor MrP 1 after 90 days, respectively. When 4 g of wood chips of P. strobus and L. tulipifera biodegraded for 30 days were treated with cellulase, glucose was recovered ot the highest values of 106 mg/g degraded wood by I. lacteus and 450 mg/g degraded wood by T. versicolor. The weight loss of 10 g of wood chip of L. tulipifera by T. versicolor on the nutrient non-added agar under the nonsterile conditions was 35% during 7 weeks of incubation, and the cumulative amount of glucose produced during this period was 239 mg without cellulase treatment. The activities of ligninolytic enzymes (lignin peroxidase, manganese peroxidase, and laccase) of fungi tested did not show a high correlation with degradation of the wood chips and subsequent glucose formation. These results suggest that the selection of proper wood species and fungal strain and optimization of glucose recovery are all necessary for the fungal pretreatment of woody biomass as a carbon substrate.

Enhancement of growth and yield of tomato by Rhodopseudomonas sp. under greenhouse conditions.

A greenhouse test was carried out to examine the effects on tomato growth of application of purple non-sulfur bacterium Rhodopseudomonas sp. which had enhanced germination and growth of tomato seed under axenic conditions. The shoot length of tomato plant inoculated by Rhodopseudomonas sp. KL9 increased by 34.6% compared to that of control in 8 weeks of cultivation. During the same period, this strain increased 120.6 and 78.6% of dry weight of shoot and root of tomato plants, respectively. The formation ratio of tomato fruit from flower was also raised by inoculation of KL9. In addition, Rhodopseudomonas sp. KL9 treatment enhanced the fresh weight and lycopene content in the harvested tomato fruits by 98.3 and 48.3%, respectively compared to those of the uninoculated control. When the effect on the indigenous bacterial community and fate of the inoculated Rhodopseudomonas sp. KL9 were monitored by denaturing gradient gel electrophoresis analysis, its application did not affect the native bacterial community in tomato rhizosphere soil, but should be repeated to maintain its population size. This bacterial capability may be applied as an environment-friendly biofertilizer to cultivation of high quality tomato and other crops including lycopene-containing vegetables and fruits.

Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor.

A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57-97% homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.

Biodegradation of endocrine-disrupting phthalates by Pleurotus ostreatus.

Biodegradation of endocrine-disrupting phthalates [diethyl phthalate (DEP), dimethyl phthalate (DMP), butylbenzyl phthalate (BBP)] was investigated with 10 white rot fungi isolated in Korea. When the fungal mycelia were added together with 100 mg/l of phthalate into yeast extract-malt extract-glucose (YMG) medium, Pleurotus ostreatus, Irpex lacteus, Polyporus brumalis, Merulius tremellosus, Trametes versicolor, and T. versicolor MrP1 and MrP13 (transformant of the Mn-repressed peroxidase gene of T. versicolor) could remove almost all of the 3 kinds of phthalates within 12 days of incubation. When the phthalates were added to 5-day pregrown fungal cultures, most fungi except I. lacteus showed the increased removal of the phthalates compared with those of the nonpregrown cultures. In both culture conditions, P. ostreatus showed the highest degradation rates for the 3 phthalates tested. BBP was degraded with the highest rates among the 3 phthalates by all fungal strains. Only 14.9% of 100 mg/l BBP was degraded by the supernatant of P. ostreatus culture in YMG medium in 4 days of incubation, but the washed or homogenized mycelium of P. ostreatus could remove 100% of BBP within 2 days even in distilled water, indicating that the initial BBP biodegradation by P. ostreatus may be attributed to mycelium-associated enzymes rather than extracellular enzymes. The biodegradation rate of BBP by the immobilized cells of P. ostreatus was almost the same as that in the suspended culture. The estrogenic activity of 100 mg/l DMP decreased during biodegradation by P. ostreatus.

Molecular analysis of bacterial community structures in paddy soils for environmental risk assessment with two varieties of genetically modified rice, Iksan 483 and Milyang 204.

The impacts of planted transgenic rice varieties on bacterial communities in paddy soils were monitored using both cultivation and molecular methods. The rice field plot consisted of eighteen subplots planted with two genetically modified (GM) rice and four non-GM rice plants in three replicates. Analysis with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes revealed that the bacterial community structures were quite similar to each other in a given month, suggesting that there were no significant differences in bacterial communities between GM and non- GM rice soils. The bacterial community structures appeared to be generally stable with the seasons, as shown by a slight variation of microbial population levels and DGGE banding patterns over the year. Comparison analysis of 16S rDNA clone libraries constructed from soil bacterial DNA showed that there were no significant differences between GM and non-GM soil libraries but revealed seasonal differences of phyla distribution between August and December. The composition profile of phospholipid fatty acids (PLFA) between GM and non-GM soils also was not significantly different to each other. When soil DNAs were analyzed with PCR by using primers for the bar gene, which was introduced into GM rice, positive DNA bands were found in October and December soils. However, no bar gene sequence was detected in PCR analysis with DNAs extracted from both cultured and uncultured soil bacterial fractions. The result of this study suggested that, in spite of seasonal variations of bacterial communities and persistence of the bar gene, the bacterial communities of the experimental rice field were not significantly affected by cultivation of GM rice varieties.

Effects of application of Rhodopseudomonas sp. on seed germination and growth of tomato under axenic conditions.

Purple nonsulfur bacteria were isolated from river sediments and their growth promoting capabilities on tomato were examined. Isolated strains KL9 and BL6 were identified as Rhodopseudomonas spp. by 16S rDNA sequence analysis. Rhodopseudomonas strain KL9 maximally produced 5.56 mM/ min/mg protein and 67.2 microM/min/mg protein of indole-3-acetic acid (IAA) and 5-aminolevulinic acid (ALA), respectively, which may be one of the mechanisms of plant growth enhancement. The germination percentage of tomato seed, total length, and dry mass of germinated tomato seedling increased by 30.2%, 71.1%, and 270.8%, respectively, compared with those of the uninoculated control 7 days after inoculation of strain KL9. The lengths of the root and shoot of germinated seedling treated with 3 mM tryptophan, a precursor of IAA, increased by 104.4% and 156.5%, respectively, 7 days after inoculation of strain KL9. Rhodopseudomonas KL9 increased 123.5% and 54% of the root and shoot lengths of germinated seedling, respectively, treated with 15 mM glycine and succinate, precursors of ALA. This plant growth promoting capability of purple nonsulfur bacteria may be a candidate for a biofertilizer in agriculture.

Cloning and expression of a parathion hydrolase gene from a soil bacterium, Burkholderia sp. JBA3.

A bacterium, Burkholderia sp. JBA3, which can mineralize the pesticide parathion, was isolated from an agricultural soil. The strain JBA3 hydrolyzed parathion to p-nitrophenol, which was further utilized as the carbon and energy sources. The parathion hydrolase was encoded by a gene on a plasmid that strain JBA3 harbored, and it was cloned into pUC19 as a 3.7-kbp Sau3AI fragment. The ORF2 (ophB) in the cloned fragment encoded the parathion hydrolase composed of 526 amino acids, which was expressed in E. coli DH10B. The ophB gene showed no significant sequence similarity to most of other reported parathion hydrolase genes.

Biodegradation of endocrine-disrupting bisphenol A by white rot fungus Irpex lacteus.

Biodegradation of endocrine-disrupting bisphenol A was investigated with several white rot fungi (Irpex lacteus, Trametes versicolor, Ganoderma lucidum, Polyporellus brumalis, Pleurotus eryngii, Schizophyllum commune) isolated in Korea and two transformants of T versicolor (strains MrP 1 and MrP 13). I. lacteus degraded 99.4% of 50 mg/l bisphenol A in 3 h incubation and 100% in 12 h incubation. which was the highest degradation rate among the fungal strains tested. T. versicolor degraded 98.2% of 50 mg/l bisphenol A in 12 h incubation. Unexpectedly, the transformant of the Mn-repressed peroxidase gene of T. versicolor, strain MrP 1, degraded 76.5% of 50 mg/l bisphenol A in 12 h incubation, which was a lower degradation rate than wild-type T. versicolor. The removal of bisphenol A by I. lacteus occurred mainly by biodegradation rather than adsorption. Optimum carbon sources for biodegradation of bisphenol A by I. lacteus were glucose and starch, and optimum nitrogen sources were yeast extract and tryptone in a minimal salts medium; however, bisphenol A degradation was higher in nutrient-rich YMG medium than that in a minimal salts medium. The initial degradation of endocrine disruptors was accompanied by the activities of manganese peroxidase and laccase in the culture

Microcosm study for revegetation of barren land with wild plants by some plant growth-promoting rhizobacteria.

Growth promotion of wild plants by some plant growth-promoting rhizobacteria (PGPR) was examined in the microcosms composed of soils collected separately from a grass-covered site and a nongrass-covered site in a lakeside barren area at Lake Paro, Korea. After sowing the seeds of eight kinds of wild plants and inoculation of several strains of PGPR, the total bacterial number and microbial activity were measured during 5 months of study period, and the plant biomasses grown were compared at the end of the study. Acridine orange direct counts in the inoculated microcosms, 1.3-9.8 x 10(9) cells x g soil(-1) in the soil from the grass-covered area and 0.9-7.2 x 10(9) cells x g soil(-1) in the soil from the nongrass-covered site, were almost twice higher than those in the uninoculated microcosms. The number of Pseudomonas sp., well-known bacteria as PGPR, and the soil dehydrogenase activity were also higher in the inoculated soils than the uninoculated soils. The first germination of sowed seeds in the inoculated microcosm was 5 days earlier than the uninoculated microcosm. Average lengths of all plants grown during the study period were 26% and 29% longer in the inoculated microcosms starting with the grass-covered soil and the nongrass-covered soil, respectively, compared with those in the uninoculated microcosms. Dry weights of whole plants grown were 67-82% higher in the inoculated microcosms than the uninoculated microcosms. Microbial population and activity and growth promoting effect by PGPR were all higher in the soils collected from the grass-covered area than in the nongrass-covered area. The growth enhancement of wild plants seemed to occur by the activities of inoculated microorganisms, and this capability of PGPR may be utilized for rapid revegetation of some barren lands.

Purification and characterization of an intracellular NADH: quinone reductase from Trametes versicolor.

Intracellular NADH:quinone reductase involved in degradation of aromatic compounds including lignin was purified and characterized from white rot fungus Trametes versicolor. The activity of quinone reductase was maximal after 3 days of incubation in fungal culture, and the enzyme was purified to homogeneity using ion-exchange, hydrophobic interaction, and gel filtration chromatographies. The purified enzyme has a molecular mass of 41 kDa as determined by SDS-PAGE, and exhibits a broad temperature optimum between 20-40 degrees C , with a pH optimum of 6.0. The enzyme preferred FAD as a cofactor and NADH rather than NADPH as an electron donor. Among quinone compounds tested as substrate, menadione showed the highest enzyme activity followed by 1,4-benzoquinone. The enzyme activity was inhibited by CuSO(4), HgCl(2), MgSO(4), MnSO(4), AgNO(3), dicumarol, KCN, NaN(3), and EDTA. Its Km and Vmax with NADH as an electron donor were 23 microM and 101 mM/mg per min, respectively, and showed a high substrate affinity. Purified quinone reductase could reduce 1,4-benzoquinone to hydroquinone, and induction of this enzyme was higher by 1,4-benzoquinone than those of other quinone compounds.

Generation of a transformant showing higher manganese peroxidase (Mnp) activity by overexpression of Mnp gene in Trametes versicolor.

Trametes versicolor has a lignin degrading enzyme system, which is also involved in the degradation of diverse recalcitrant compounds. Manganese-dependent peroxidase (MnP) is one of the lignin degrading enzymes in T. versicolor. In this study, a cDNA clone of a putative MnP-coding gene was cloned and transferred into an expression vector (pBARGPE1) carrying a phosphinothricin resistance gene (bar) as a selectable marker to yield the expression vector, pBARTvMnP2. Transformants were generated through genetic transformation using pBARTvMnP2. The genomic integration of the MnP clone was confirmed by PCR with bar-specific primers. One transformant showed higher enzyme activity than the recipient strain did, and was genetically stable even after 10 consecutive transfers on non-selective medium.