Eu Jin Chung

Forest soil metagenome gene cluster involved in antifungal activity expression in Escherichia coli.

ABSTRACT Using two forest soils, we previously constructed two fosmid libraries containing 113,700 members in total. The libraries were screened to select active antifungal clones using Saccharomyces cerevisiae as a target fungus. One clone from the Yuseong pine tree rhizosphere soil library, pEAF66, showed S. cerevisiae growth inhibition. Despite an intensive effort, active chemicals were not isolated. DNA sequence analysis and transposon mutagenesis of pEAF66 revealed 39 open reading frames (ORFs) and indicated that eight ORFs, probably in one transcriptional unit, might be directly involved in the expression of antifungal activity in Escherichia coli. The deduced amino acid sequences of eight ORFs were similar to those of the core genes encoding type II family polyketide synthases, such as the acyl carrier protein (ACP), ACP synthases, aminotransferase, and ACP reductase. The gene cluster involved in antifungal activity was similar in organization to the putative antibiotic production locus of Pseudomonas putida KT2440, although we could not select a similar active clone from the KT2440 genomic DNA library in E. coli. ORFs encoding ATP binding cassette transporters and membrane proteins were located at both ends of the antifungal gene cluster. Upstream ORFs encoding an IclR family response regulator and a LysR family response regulator were involved in the positive regulation of antifungal gene expression. Our results suggested the metagenomic approach as an alternative to search for novel antifungal antibiotics from unculturable soil bacteria. This is the first report of an antifungal gene cluster obtained from a soil metagenome using S. cerevisiae as a target fungus.

Chitinophaga oryziterrae sp. nov., isolated from the rhizosphere soil of rice (Oryza sativa L.)

A novel bacterial strain, YC7001(T), was isolated from the rhizosphere soil of rice collected at Jinju, Korea, and was characterized using a polyphasic approach. Cells of strain YC7001(T) were Gram-negative-staining, yellow-pigmented, rod-shaped, aerobic, heterotrophic and non-motile. Strain YC7001(T) grew optimally at 25-30 °C, pH 5.0-8.0 and degraded gelatin, cellulose and chitin. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YC7001(T) belonged to the genus Chitinophaga in the family Chitinophagaceae. The most closely related species were Chitinophaga sancti NBRC 15057(T) (96.9%), Chitinophaga ginsengisoli Gsoil 052(T) (95.9%), Chitinophaga pinensis DSM 2588(T) (95.7%), Chitinophaga filiformis IFO 15056(T) (95.7%) and Chitinophaga niabensis JS13-10(T) (95.0%). The DNA-DNA relatedness value of strain YC7001(T) with C. sancti NBRC 15057(T) was 15.2 ± 0.6%. Strain YC7001(T) contained MK-7 as the major menaquinone and homospermidine as the major polyamine. The major fatty acids of strain YC7001(T) were C(15:0) iso, C(16:1)ω5c and C(17:0) iso 3-OH. The polar lipids were phosphatidylethanolamine, unknown aminolipids and unknown lipids. The total genomic DNA G+C content was 41.3 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, strain YC7001(T) represents a novel species of the genus Chitinophaga, in the family Chitinophagaceae, for which the name Chitinophaga oryziterrae sp. nov. is proposed. The type strain is YC7001(T) ( = KACC 14533(T) = JCM 16595(T)).

Bacillus oryzicola sp. nov., an Endophytic Bacterium Isolated from the Roots of Rice with Antimicrobial, Plant Growth Promoting, and Systemic Resistance Inducing Activities in Rice

Biological control of major rice diseases has been attempted in several rice-growing countries in Asia during the last few decades and its application using antagonistic bacteria has proved to be somewhat successful for controlling various fungal diseases in field trials. Two novel endophytic Bacillus species, designated strains YC7007 and YC7010T, with anti-microbial, plant growth-promoting, and systemic resistance-inducing activities were isolated from the roots of rice in paddy fields at Jinju, Korea, and their multifunctional activities were analyzed. Strain YC7007 inhibited mycelial growth of major rice fungal pathogens strongly in vitro. Bacterial blight and panicle blight caused by Xanthomonas oryzae pv. oryzae (KACC 10208) and Burkholderia glumae (KACC 44022), respectively, were also suppressed effectively by drenching a bacterial suspension (107 cfu/ml) of strain YC7007 on the rhizosphere of rice. Additionally, strain YC7007 promoted the growth of rice seedlings with higher germination rates and more tillers than the untreated control. The taxonomic position of the strains was also investigated. Phylogenetic analyses based on 16S rRNA gene sequences indicated that both strains belong to the genus Bacillus, with high similarity to the closely related strains, Bacillus siamensis KACC 15859T (99.67%), Bacillus methylotrophicus KACC 13105T (99.65%), Bacillus amyloliquefaciens subsp. plantarum KACC 17177T (99.60%), and Bacillus tequilensis KACC 15944T (99.45%). The DNA-DNA relatedness value between strain YC7010T and the most closely related strain, B. siamensis KACC 15859T was 50.4±3.5%, but it was 91.5±11.0% between two strains YC7007 and YC7010T, indicating the same species. The major fatty acids of two strains were anteiso-C15:0 and iso C15:0. Both strains contained MK-7 as a major respiratory quinone system. The G+C contents of the genomic DNA of two strains were 50.5 mol% and 51.2 mol%, respectively. Based on these polyphasic studies, the two strains YC7007 and YC7010T represent novel species of the genus Bacillus, for which the name Bacillus oryzicola sp. nov. is proposed. The type strain is YC7010T (= KACC 18228T). Taken together, our findings suggest that novel endophytic Bacillus strains can be used for the biological control of rice diseases.

Nocardioides caricicola sp. nov., an endophytic bacterium isolated from a halophyte, Carex scabrifolia Steud.

A Gram-staining-positive, coccoid to rod-shaped bacterium, designated strain YC6903(T), was isolated from a halophytic plant (Carex scabrifolia Steud.) collected from sand dunes at Namhae Island, Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain YC6903(T) grew optimally at 30 °C and at pH 8.0. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YC6903(T) belongs to the genus Nocardioides in the family Nocardioidaceae. Strain YC6903(T) was related most closely to Nocardioides pyridinolyticus OS4(T) (97.0 % 16S rRNA gene sequence similarity), Nocardioides dokdonensis FR1436(T) (96.6 %), Nocardioides aquiterrae GW-9(T) (96.6 %) and Nocardioides hankookensis DS-30(T) (96.6 %). The cell-wall peptidoglycan contained LL-diaminopimelic acid and MK-8(H(4)) was the major respiratory quinone. The mean (±SD) level of DNA-DNA relatedness between strain YC6903(T) and N. pyridinolyticus OS4(T) was 53.5±5.5 %. The predominant cellular fatty acid of strain YC6903(T) was iso-C(16 : 0) (28.9 %). The DNA G+C content was 71.7 mol%. Phenotypic, phylogenetic and chemotaxonomic data indicated that strain YC6903(T) represents a novel species of the genus Nocardioides, for which the name Nocardioides caricicola sp. nov. is proposed. The type strain is YC6903(T) (=KACC 13778(T) =DSM 22177(T)).

Mucilaginibacter jinjuensis sp. nov., with xylan-degrading activity.

A gram-negative, rod-shaped, pale-orange-pigmented bacterial strain with xylan-degrading activity designated YC7004(T) was isolated from a rotten-wood sample collected at Jinju, Korea, and its taxonomic position was investigated by using a polyphasic approach. The strain grew optimally on R2A medium at 30 °C and at pH 6. The major isoprenoid quinone was MK7 and major fatty acids were summed feature 3, iso-C15 : 0, C16 : 0, iso-C17 : 0 3-OH, iso-C17 : 1ω9c and C16 : 1ω5c. The G+C content of the genomic DNA was 40.0 mol%. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the strain belongs to the genus Mucilaginibacter in the family Sphingobacteriaceae. The most closely related species were Mucilaginibacter daejeonensis (95.5 %), Mucilaginibacter frigoritolerans (94.6 %) and Mucilaginibacter mallensis (94.0 %). Based on the phylogenetic and chemotaxonomic data analyses, strain YC7004(T) represents a novel species of the genus Mucilaginibacter with the proposed name of Mucilaginibacter jinjuensis sp. nov. The type strain is YC7004(T) ( = KACC 16571(T) = NBRC 108856(T)).

Chitinophaga eiseniae sp. nov., isolated from vermicompost.

A Gram-negative, rod-shaped bacterial strain, YC6729(T), was isolated from vermicompost collected at Masan, Korea, and its taxonomic position was investigated by a polyphasic taxonomic approach. Strain YC6729(T) grew optimally at 30 °C and at pH 6.5-8.5. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YC6729(T) belongs to the genus Chitinophaga in the family Chitinophagaceae. It was related most closely to Chitinophaga terrae KP01(T) (96.4 % 16S rRNA gene sequence similarity), Chitinophaga ginsengisegetis Gsoil 040(T) (96.1 %), Chitinophaga arvensicola IAM 12650(T) (96.1 %) and Chitinophaga pinensis DSM 2588(T) (93.3 %). Strain YC6729(T) contained MK-7 as the major menaquinone and homospermidine as the major polyamine. The fatty acids of strain YC6729(T) were iso-C(15 : 0), C(16 : 1)ω5c, iso-C(17 : 0) 3-OH, C(16 : 0), anteiso-C(18 : 0) and/or C(18 : 2)ω6,9c, iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c, C(14 : 0), iso-C(15 : 0) 3-OH, iso-C(15 : 1) G, C(18 : 1)ω5c, iso-C(15 : 1) I and/or C(13 : 0) 3-OH, C(13 : 0) 2-OH, C(16 : 0) 3-OH and unknown fatty acid ECL 13.565. The polar lipid profile contained phosphatidylethanolamine, unknown aminolipids and unknown lipids. The total DNA G+C content of strain YC6729(T) was 48.9 mol%. The phenotypic, chemotaxonomic and phylogenetic data showed that strain YC6729(T) represents a novel species of the genus Chitinophaga, for which the name Chitinophaga eiseniae sp. nov. is proposed. The type strain is YC6729(T) ( = KACC 13774(T)  = DSM 22224(T)).

MicroTom - A Model Plant System to Study Bacterial Wilt by Ralstonia solanacearum

MicroTom is a miniature tomato plants with various properties that make it as a model system for experiments in plant molecular biology. To extend its utility as a model plant to study a plant - bacterial wilt system, we investigated the potential of the MicroTom as a host plant of bacterial wilt caused by Ralstonia solanacearum. We compared the disease progress on standard tomato and MicroTom by two inoculation methods, root dipping and soil drenching, using a race 1 strain GMI1000. Both methods caused the severe wilting on MicroTom comparable to commercial tomato plant, although initial disease development was faster in root dipping. From the diseased MicroTom plants, the same bacteria were successfully reisolated using semiselective media to fulfill Koch`s postulates. Race specific and isolate specific virulence were investigated by root dipping with 10 isolates of R. solanacearum isolated from tomato and potato plants. All of the tested isolates caused the typical wilt symptom on MicroTom. Disease severities by isolates of race 3 was below 50% until 15 days after inoculation, while those by isolates of race 1 reached over 50% to death until 15 days. This result suggested that MicroTom can be a model host plant to study R. solanacearum - plant interaction.

Rhizobium halophytocola sp. nov., isolated from the root of a coastal dune plant.

During a study of endophytic bacteria from coastal dune plants, a bacterial strain, designated YC6881(T), was isolated from the root of Rosa rugosa collected from the coastal dune areas of Namhae Island, Korea. The bacterium was found to be Gram-staining-negative, motile, halophilic and heterotrophic with a single polar flagellum. Strain YC6881(T) grew at temperatures of 4-37 °C (optimum, 28-32 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0), and at NaCl concentrations in the range of 0-7.5% (w/v) (optimum, 4-5% NaCl). Strain YC6881(T) was catalase- and oxidase-positive and negative for nitrate reduction. According to phylogenetic analysis using 16S rRNA gene sequences, strain YC6881(T) belonged to the genus Rhizobium and showed the highest 16S rRNA gene sequence similarity of 96.9% to Rhizobium rosettiformans, followed by Rhizobium borbori (96.3%), Rhizobium radiobacter (96.1%), Rhizobium daejeonense (95.9%), Rhizobium larrymoorei (95.6%) and Rhizobium giardinii (95.4%). Phylogenetic analysis of strain YC6881(T) by recA, atpD, glnII and 16S-23S intergenic spacer (IGS) sequences all confirmed the phylogenetic arrangements obtained by using 16S rRNA gene sequences. Cross-nodulation tests showed that strain YC6881(T) was a symbiotic bacterium that nodulated Vigna unguiculata and Pisum sativum. The major components of the cellular fatty acids were C(18:1)ω7c (53.7%), C(19:0) cyclo ω8c (12.6%) and C(12:0) (8.1%). The DNA G+C content was 52.8 mol%. Phenotypic and physiological tests with respect to carbon source utilization, antibiotic resistance, growth conditions, phylogenetic analyses of housekeeping genes recA, atpD and glnII, and fatty acid composition could be used to discriminate strain YC6881(T) from other species of the genus Rhizobium in the same sublineage. Based on the results obtained in this study, strain YC6881(T) is considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium halophytocola sp. nov. is proposed. The type strain is YC6881(T) ( = KACC 13775(T) = DSM 21600(T)).

Mucilaginibacter gynuensis sp. nov., isolated from rotten wood.

A Gram-staining-negative, rod-shaped, aerobic bacterial strain designated YC7003(T), was isolated from a piece of rotten wood collected at Jinju, Korea. The taxonomic position of the strain was investigated using a polyphasic approach. The strain was catalase- and oxidase-positive, grew at 4-35 °C (optimum, 30 °C) and at pH 5.0-10.0 (optimum, pH 6.5-7.0). The major cellular fatty acids were C(16:1)ω7c and/or iso-C(15:0) 2-OH (summed feature 3), iso-C(15:0) and C(16 : 1)ω5c and the major respiratory quinone was MK-7. The total genomic DNA G+C content was 49.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YC7003(T) belonged to the genus Mucilaginibacter in the family Sphingobacteriaceae with 94.4-97.2% sequence similarities with type strains of species of the genus Mucilaginibacter. The most closely related species was Mucilaginibacter mallensis MP1X4(T) (97.2%). The DNA-DNA relatedness value between strain YC7003(T) and M. mallensis MP1X4(T) was 21.7 ± 3.3%. Based on these data, strain YC7003(T) represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter gynuensis sp. nov. is proposed. The type strain is YC7003(T) ( =KACC 15532(T) =JCM 17705(T)).

Sphingomonas oryziterrae sp. nov. and Sphingomonas jinjuensis sp. nov. isolated from rhizosphere soil of rice (Oryza sativa L.).

Two Gram-reaction-negative, yellow-orange-pigmented, rod-shaped bacterial strains, designated YC6722(T) and YC6723(T), were isolated from rhizosphere soil samples collected from rice fields in Jinju, Korea. Strains YC6722(T) and YC6723(T) grew optimally at 25-30 °C and at pH 7.0-8.5. Phylogenetic analyses of 16S rRNA gene sequences showed that strain YC6722(T) was most closely related to Sphingomonas jaspsi TDMA-16(T) (96.6 % sequence similarity) and strain YC6723(T) was related most closely to Sphingomonas aquatilis JSS7(T) (96.9 %). The two strains contained ubiquinone-10 (Q-10) as the major respiratory quinone system and sym-homospermidine as the major polyamine. The G+C contents of the genomic DNA of strains YC6722(T) and YC6723(T) were 63.3 and 61.0 mol%, respectively. The major fatty acid was C(18 : 1)ω7c. The polar lipids detected in the two strains were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, sphingoglycolipid, phosphatidyldimethylethanolamine and other unknown lipids. On the basis of their phylogenetic positions, and their biochemical and phenotypic characteristics, strains YC6722(T) and YC6723(T) represent two novel species of the genus Sphingomonas, for which the names Sphingomonas oryziterrae sp. nov. ( = KCTC 22476(T)  = DSM 21455(T)) and Sphingomonas jinjuensis sp. nov. (KCTC 22477(T)  = DSM 21457(T)) are proposed.