Hee-Wan Kang

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.

Phylogenetic Analysis of Erwinia Species Based on 16s rRNA Gene Sequences

The phylogenetic relationships of the type strains of 16 Erwinia species were investigated by performing a comparative analysis of the sequences of the 16S rRNA genes of these organisms. The sequence data were analyzed by the neighbor-joining method, and each branch was supported by moderate bootstrap values. The phylogenetic tree and sequence analyses confirmed that the genus Erwinia is composed of species that exhibit considerable heterogeneity and form four clades that are intermixed with members of other genera, such as Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens. Cluster I includes the type strains of Erwinia herbicola, Erwinia milletiae, Erwinia ananas, Erwinia uredovora, and Erwinia stewartii and corresponds to Dyes herbicola group. Cluster II consists of Erwinia persicinus, Erwinia rhapontici, Erwinia amylovora, and Erwinia cypripedii. Cluster III consists of Erwinia carotovora subspecies and Erwinia chrysanthemi and is characterized by the production of pectate lyases and cellulases. Erwinia salicis, Erwinia rubrifaciens, and Erwinia nigrifluens form the cluster that is most distantly related to other Erwinia species. The data from the sequence analyses are discussed in the context of biochemical and DNA-DNA hybridization data.

Whole genome and global gene expression analyses of the model mushroom Flammulina velutipes reveal a high capacity for lignocellulose degradation.

Flammulina velutipes is a fungus with health and medicinal benefits that has been used for consumption and cultivation in East Asia. F. velutipes is also known to degrade lignocellulose and produce ethanol. The overlapping interests of mushroom production and wood bioconversion make F. velutipes an attractive new model for fungal wood related studies. Here, we present the complete sequence of the F. velutipes genome. This is the first sequenced genome for a commercially produced edible mushroom that also degrades wood. The 35.6-Mb genome contained 12,218 predicted protein-encoding genes and 287 tRNA genes assembled into 11 scaffolds corresponding with the 11 chromosomes of strain KACC42780. The 88.4-kb mitochondrial genome contained 35 genes. Well-developed wood degrading machinery with strong potential for lignin degradation (69 auxiliary activities, formerly FOLymes) and carbohydrate degradation (392 CAZymes), along with 58 alcohol dehydrogenase genes were highly expressed in the mycelium, demonstrating the potential application of this organism to bioethanol production. Thus, the newly uncovered wood degrading capacity and sequential nature of this process in F. velutipes, offer interesting possibilities for more detailed studies on either lignin or (hemi-) cellulose degradation in complex wood substrates. The mutual interest in wood degradation by the mushroom industry and (ligno-)cellulose biomass related industries further increase the significance of F. velutipes as a new model.

Functional analysis of pilQ gene in Xanthomanas oryzae pv. oryzae, bacterial blight pathogen of rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease in rice. A virulence-attenuated mutant strain HNU89K9 of X. oryzae pv. oryzae (KACC10331), with a transposon insertion in the pilQ gene was used for this study. The pilQ was involved in the gene cluster pilMNOPQ of the Xoo genome. Growth rate of the pilQ mutant was similar to that of wild-type. At level of amino acids, PilQ of Xoo showed that a high sequence identities more than 94% and 70% to Xanthomonas species and to Xyllela fastidiosa, respectively but a low sequence homology less than 30% to other bacterial species. The twitching motility forming a morginal fringe on PSA media was observed on colony of the wild-type strain KACC10331, but not in mutant HNU89K9. Wild-type Xoo cells formed a biofilm on the surface of the PVC plastic test tube, while the mutant strain HNU89K9 did not form a biofilm. The results suggest that the pilQ gene of X. oryzae pv. oryzae plays a critical role in pathogenicity, twitching motility, and biofilm formation.

Genomic Differentiation Among Oyster Mushroom Cultivars Released in Korea by URP-PCR Fingerprinting

URP primers of 20 mer derived from repetitive sequence of rice were used to assess genetic variation of oyster mushroom consisting of 10 cultivars of Pleurotus ostreatus, two cultivars of P. florida and two cultivars of P. sajor-caju which were registered in Korea. URP2 F and URP38 F primers produced cultivar-specific PCR polymorphic bands in the Pleurotus species. UPGMA cluster analysis using the URP-PCR data showed that 14 Pleurotus cultivars are genetically clustered into large three groups. The URP-PCR data provided important information for more efficient breeding strategies of Pleurotus cultivars.

Phellinins A1 and A2, new styrylpyrones from the culture broth of Phellinus sp. KACC93057P: I. Fermentation, taxonomy, isolation and biological properties.

Novel styrylpyrones, phellinins A1 and A2, were isolated together with known styrylpyrone compounds, hispidin and 1,1-distyrylpyrylethan, from the cultured broth of Phellinus sp. KACC93057P. These compounds were purified by solvent partition, Sephadex LH-20 column chromatography, C18-solid phase extraction and finally by reversed-phase (ODS) TLC. To identify the phellinin producer Phellinus sp. KACC93057P, the ribosomal DNA (rDNA) internal transcribed space regions containing 5.8 rDNA were sequenced and compared with those of the known Phellinus isolates. Phellinus sp. KACC93057P was 94.8% identical to P. baumii and P. linteus, all of which did not produce phellinins A1 and A2. These compounds significantly scavenged free radicals such as 1,1-diphenyl-2-picrylhydrazyl, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and superoxide.

Efficient Recovery of Lignocellulolytic Enzymes of Spent Mushroom Compost from Oyster Mushrooms, Pleurotus spp., and Potential Use in Dye Decolorization

Abstract This study was conducted in order to perform efficient extraction of lignocellulolytic enzymes amylase (EC 3.2.1.1), cellulase (EC 3.2.1.4), laccase (EC 1.10.3.2), and xylanase (EC 3.2.1.8) from spent mushroom compost (SMC) of Pleurotus ostreatus, P. eryngii, and P. cornucopiae. Optimal enzyme recovery was achieved when SMCs were extracted with 50 mM sodium citrate (pH 4.5) buffer at 4°C for 2 hr. Amylase, cellulase, and xylanase activities showed high values in extracts from P. ostreatus SMC, with 2.97 U/g, 1.67 U/g, and 91.56 U/g, respectively, whereas laccase activity and filter paper degradation ability were highest in extracts from P. eryngii SMC, with values of 9.01 U/g and 0.21 U/g, respectively. Enzymatic activities varied according to the SMCs released from different mushroom farms. The synthetic dyes remazol brilliant blue R and Congo red were decolorized completely by the SMC extract of P. eryngii within 120 min, and the decolorization ability of the extract was comparable to that of 0.3 U of commercial laccase. In addition, laccase activity of the SMC extract from P. eryngii was compared to that of commercial enzymes or its industrial application in decolorization.

PCR Based Detection of Phellinus linteus using Specific Primers Generated from Universal Rice Primer (URP) Derived PCR Polymorphic Band

Abstract This study was carried out to develop specific primers for PCR detection of Phellinus linteus. Diverse genomes of 15 Phellinus spp. including five Phellinus linteus isolates were fingerprinted by Primer Universal rice primer (URP)1F. The URP-PCR pattern differentiated P. linteus isolates from other phellinus spp. A polymorphic band (2.8 kb), which is unique for P. linteus isolates, was isolated and sequenced. Twenty four-oligonucleotide primer pairs were designed based on information of DNA sequence. The primer set (PLSPF2/PLSPR1) amplified single band (2.2 kb) of expected size with genomic DNA from seven Phellinus linteus, but not with that of other Phellinus species tested. The primers could be used identically in both DNA samples from mycelium and fruit bodies. This specific primers could offer a useful tool for detecting and identifying P. linteus rapidly.

Virulence analysis and gene expression profiling of the pigment-deficient mutant of Xanthomonas oryzae pathovar oryzae

Xanthomonas oryzae pathovar oryzae (Xoo) causes bacterial blight disease in rice (Oryza sativa L.). For a study of function, we constructed a random insertion mutant library of Xoo using a Tn5 transposon and isolated the mutant strain (M11; aroK::Tn5) that had extremely low pigment production. In addition, M11 had decreased virulence against the susceptible rice cultivar IR24. Thermal asymmetric interlaced-PCR and sequence analysis of M11 revealed that the transposon was inserted into the aroK gene (which encodes a shikimate kinase). To investigate the expression patterns of the pigment- and virulence-deficient mutant, DNA microarray analysis was performed. In addition, reverse transcriptase-PCR was performed to confirm the expression levels of several genes, including the aro genes of the aroK mutant. Our findings reveal that several crucial genes for virulence, including cellulase and hypersensitive response and pathogenicity (hrp) genes, were regulated by mutations in the aroK gene.

Production of Lignocellulytic Enzymes from Spent Mushroom Compost of Pleurotus eryngii

The lignocellulytic enzymes including a-amylase (EC 3.2.1.1), lignin peroxidase (EC 1.11.1.14), laccase (EC 1.10.3.2), xylanase (EC 3.2.1.8), -xylosidase (EC 3.2.1.37), -glucosidase (EC 3.2.1.21) and cellulase (EC 3.2.1.4) were extracted from spent mushroom compost (SMC) of Pleurotus eryngii. Different extraction buffers and conditions were tested for optimal recovery of the enzymes. The optimum extraction was shaking incubation (200 rpm) for 2 h at . -Amylase was extracted with the productivity range from 1.20 to 1.6 Unit/SMC g. Cellulase was recovered with the productivity range from 2.10 to 2.80 U/gf. -glucosidase and -xylosidase productivities showed lowest recovery producing 0.1 U/g and 0.02 U/g, respectively. The P. eryngii SMCs collected from three different mushroom farms showed different recovery on laccase and xylanse, cellulase. Furthermore, the water extracted SMC was compared to commercial enzymes for its industrial application in decolorization and cellulase activity.