Dong Wan Lee

Antimicrobial Cyclic Peptides for Plant Disease Control

Antimicrobial cyclic peptides derived from microbes bind stably with target sites, have a tolerance to hydrolysis by proteases, and a favorable degradability under field conditions, which make them an attractive proposition for use as agricultural fungicides. Antimicrobial cyclic peptides are classified according to the types of bonds within the ring structure; homodetic, heterodetic, and complex cyclic peptides, which in turn reflect diverse physicochemical features. Most antimicrobial cyclic peptides affect the integrity of the cell envelope. This is achieved through direct interaction with the cell membrane or disturbance of the cell wall and membrane component biosynthesis such as chitin, glucan, and sphingolipid. These are specific and selective targets providing reliable activity and safety for non-target organisms. Synthetic cyclic peptides produced through combinatorial chemistry offer an alternative approach to develop antimicrobials for agricultural uses. Those synthesized so far have been studied for antibacterial activity, however, the recent advancements in powerful technologies now promise to provide novel antimicrobial cyclic peptides that are yet to be discovered from natural resources.

Comparative evaluation of microbial diversity and metabolite profiles in doenjang, a fermented soybean paste, during the two different industrial manufacturing processes

Two different doenjang manufacturing processes, the industrial process (IP) and the modified industrial process (mIP) with specific microbial assortments, were subjected to metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). The multivariate analyses indicated that both primary and secondary metabolites exhibited distinct patterns according to the fermentation processes (IP and mIP). Microbial community analysis for doenjang using denaturing gradient gel electrophoresis (DGGE), exhibited that both bacteria and fungi contributed proportionally for each step in the process viz., soybean, steaming, drying, meju fermentation, cooling, brining, and aging. Further, correlation analysis indicated that Aspergillus population was linked to sugar metabolism, Bacillus spp. with that of fatty acids, whereas Tetragenococcus and Zygosaccharomyces were found associated with amino acids. These results suggest that the components and quality of doenjang are critically influenced by the microbial assortments in each process.

Microbial community composition and PAHs removal potential of indigenous bacteria in oil contaminated sediment of Taean coast, Korea

The tidal flats near Sinduri beach in Taean, Korea, have been severely contaminated by heavy crude oils due to the Koreas worst oil spill accident, say the Hebei Spirit Oil Spill, in 2007. Crude oil compounds, including polycyclic aromatic hydrocarbons (PAHs), pose significant environmental damages due to their wide distribution, persistence, high toxicity, mutagenicity, and carcinogenicity. Microbial community of Sinduri beach sediments samples was analyzed by metagenomic data with 16S rRNA gene amplicons. Three phyla (Proteobacteria, Firmicutes, and Bacteroidetes) accounted for approximately ≥93.0% of the total phyla based on metagenomic analysis. Proteobacteria was the dominant phylum in Sinduri beach sediments. Cultivable bacteria were isolated from PAH-enriched cultures, and bacterial diversity was investigated through performing culture characterization followed by molecular biology methods. Sixty-seven isolates were obtained, comprising representatives of Actinobacteria, Firmicutes, α- and γ-Proteobacteria, and Bacteroidetes. PAH catabolism genes, such as naphthalene dioxygenase (NDO) and aromatic ring hydroxylating dioxygenase (ARHDO), were used as genetic markers to assess biodegradation of PAHs in the cultivable bacteria. The ability to degrade PAHs was demonstrated by monitoring the removal of PAHs using a gas chromatography mass spectrometer. Overall, various PAH-degrading bacteria were widely present in Sinduri beach sediments and generally reflected the restored microbial community. Among them, Cobetia marina, Rhodococcus soli, and Pseudoalteromonas agarivorans were found to be significant in degradation of PAHs. This large collection of PAH-degrading strains represents a valuable resource for studies investigating mechanisms of PAH degradation and bioremediation in oil contaminated coastal environment, elsewhere.

Chryseobacterium cucumeris sp. nov., an endophyte isolated from cucumber (Cucumis sativus L.) root and emended description of Chryseobacterium arthrosphaerae.

The Gram-stain-negative, yellow-pigmented, rod-shaped bacterial strain GSE06T, isolated from the surface-sterilized root of a cucumber plant grown in a field in Gunsan, Korea, was characterized by not only cultural and morphological features but also physiological, biochemical and molecular analyses. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain GSE06T was most closely related to species of the genus Chryseobacterium. Furthermore, strain GSE06T exhibited the highest sequence similarities with the type strains Chryseobacterium indologenes ATCC 29897T (98.9 %), Chryseobacterium gleum ATCC 35910T (98.8 %), Chryseobacterium arthrosphaerae CC-VM-7T (98.7 %), Chryseobacterium contaminans C26T (98.5 %), Chryseobacterium artocarpi UTM-3T (98.3 %), and Chryseobacterium gallinarum 100T (97.9 %). Average nucleotide identity values between genome sequences of strain GSE06T and the above-mentioned reference strains ranged from 81.2 to 86.9 %, which were lower than the threshold of 95 % (corresponding to a DNA-DNA reassociation value of 70 %). The DNA G+C content of strain GSE06T was 36.1 mol%; the predominant respiratory quinone of the strain was MK-6. The major fatty acids were iso-C15 : 0, summed feature 9 (iso-C17 : 1ω9c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, three aminolipids, one aminophospholipid, four glycolipids and one unidentified lipid. These results of phenotypic and genotypic characteristics could differentiate strain GSE06T from closely related type strains belonging to the genus Chryseobacterium. Thus, strain GSE06T is proposed as a representative of a novel species in the genus Chryseobacterium, Chryseobacterium cucumeris sp. nov. The type strain is GSE06T (=KACC 18798T=JCM 31422T).

Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin biosynthesis and branched-chain α-keto acid dehydrogenase complex expression

Streptomyces sp. M10 is a valinomycin-producing bacterial strain that shows potent bioactivity against Botrytis blight of cucumber plants. During studies to increase the yield of valinomycin (a cyclododecadepsipeptide) in strain M10, additional antifungal metabolites, including bafilomycin derivatives (macrolide antibiotics), were identified. To examine the effect of bafilomycin biosynthesis on valinomycin production, the bafilomycin biosynthetic gene cluster was cloned from the genome of strain M10, as were two branched-chain α-keto acid dehydrogenase (BCDH) gene clusters related to precursor supply for bafilomycin biosynthesis. A null mutant (M10bafm) of one bafilomycin biosynthetic gene (bafV) failed to produce bafilomycin, but resulted in a 1.2- to 1.5-fold increase in the amount of valinomycin produced. In another null mutant (M10bkdFm) of a gene encoding a subunit of the BCDH complex (bkdF), bafilomycin production was completely abolished and valinomycin production increased fourfold relative to that in the wild-type M10 strain. The higher valinomycin yield was likely the result of redistribution of the metabolic flux from bafilomycin to valinomycin biosynthesis, because the two antibiotics share a common precursor, 2-ketoisovaleric acid, a deamination product of valine. The results show that directing precursor flux toward active ingredient biosynthesis could be used as a prospective tool to increase the competence of biofungicides.

Five New Records of Soil-Derived Trichoderma in Korea: T. albolutescens, T. asperelloides, T. orientale, T. spirale, and T. tomentosum

Abstract Despite the huge worldwide diversity of Trichoderma (Hypocreaceae, Ascomycota), only about 22 species have been reported in Korea. Thus, between 2013 and 2015, soil-derived Trichoderma spp. were isolated to reveal the diversity of Korean Trichoderma. Phylogenetic analysis of translation elongation factor 1 alpha gene was used for identification. Among the soil-derived Trichoderma, Trichoderma albolutescens, T. asperelloides, T. orientale, T. spirale, and T. tomentosum have not been previously reported in Korea. Thus, we report the five Trichoderma species as new in Korea with morphological descriptions and images.

Biosurfactant-assisted bioremediation of crude oil by indigenous bacteria isolated from Taean beach sediment

Crude oil and its derivatives are considered as one group of the most pervasive environmental pollutants in marine environments. Bioremediation using oil-degrading bacteria has emerged as a promising green cleanup alternative in more recent years. The employment of biosurfactant-producing and hydrocarbon-utilizing indigenous bacteria enhances the effectiveness of bioremediation by making hydrocarbons bioavailable for degradation. In this study, the best candidates of biosurfactant-producing indigenous bacteria were selected by screening of biochemical tests. The selected bacteria include Bacillus algicola (003-Phe1), Rhodococcus soli (102-Na5), Isoptericola chiayiensis (103-Na4), and Pseudoalteromonas agarivorans (SDRB-Py1). In general, these isolated species caused low surface tension values (33.9-41.3 mN m-1), high oil spreading (1.2-2.4 cm), and hydrocarbon emulsification (up to 65%) warranting active degradation of hydrocarbons. FT-IR and LC-MS analyses indicated that the monorhamnolipid (Rha-C16:1) and dirhamnolipid (Rha-Rha-C6-C6:1) were commonly produced by the bacteria as potent biosurfactants. The residual crude oil after the biodegradation test was quantitated using GC-MS analysis. The bacteria utilized crude oil as their sole carbon source while the amount of residual crude oil significantly decreased. In addition the cell-free broth containing biosurfactants produced by bacterial strains significantly desorbed crude oil in oil-polluted marine sediment. The selected bacteria might hold additional capacity in crude oil degradation. Biosurfactant-producing indigenous bacteria therefore degrade crude oil hydrocarbon compounds, produce biosurfactants that can increase the emulsification of crude oil and are thus more conducive to the degradation of crude oil.

Pseudokineococcus basanitobsidens sp. nov., isolated from volcanic rock

A novel Gram-strain-positive, non-spore-forming bacterial strain, designated SKC1-2T, was isolated from volcanic rock of the scoria cone of Seobjikoji, Jeju, Republic of Korea. Cells were aerobic, catalase-positive, oxidase-negative, motile and cocci. Colonies of cells were dark orange-coloured, circular, smooth and convex. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the isolate was related to members of the genus Pseudokineococcus. Phylogenetic neighbours were P. marinus KCCM 42250T (98.2 %, 16S rRNA gene sequence similarity) and P. lusitanus DSM 23768T (98.0 %). The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant respiratory quinone was MK-9(H2). The predominant respiratory quinone was MK-9(H2) and the major fatty acid was anteiso-C15 : 0. The polar lipid profile included major amounts of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two unidentified phospholipids and two unidentified phosphoglycolipids. The DNA G+C content was 74.9 mol%. DNA-DNA relatedness values between strain SKC1-2T and P. lusitanus DSM 23768T or P. marinus KCCM 42250T were 37.5-38.1 % or 45.4-46.4 %, respectively. On the basis of the phenotypic differences and DNA-DNA relatedness data, the isolate represents a new species of the genus Pseudokineococcus, for which the name Pseudokineococcus basanitobsidens sp. nov. is proposed. The type strain is SKC1-2T (=DSM 103726T=KCCM 43221T).

Nocardioides litoris sp. nov., isolated from the Taean seashore

A novel Gram-stain-positive, irregularly rod-shaped actinomycete, designated strain 002-2T, was isolated from sand beach sediment collected from the Taean seashore, Republic of Korea. Cells were aerobic, oxidase-negative and catalase-positive. Colonies of cells were bright yellow, circular, smooth and convex. The diagnostic diamino acid in the cell wall was ll-diaminopimelic acid. The predominant menaquinone was MK-8(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unknown phospholipid and two unknown amino phospholipids. The major fatty acids were iso-C16 : 0 and C17 : 1ω8c. The DNA G+C content was 68.8 mol%. 16S rRNA gene sequence analysis revealed that strain 002-2T belongs to the family Nocardioidaceae and formed a cluster with Nocardioides rubroscoriae Sco-A25T (98.1 % sequence similarity) and Nocardioides plantarum NCIMB 12834T (97.6 %). On the basis of the phenotypic and phylogenetic data, strain 002-2T (=KCTC 39838T=DSM 103718T) is considered to represent a novel species of the genus Nocardioides, for which the name Nocardioides litoris sp. nov. is proposed.

The chejuenolide biosynthetic gene cluster harboring an iterative trans -AT PKS system in Hahella chejuensis strain MB-1084

Hahella chejuensis MB-1084 is a Gram-negative marine bacterial strain that produces unusual 17-membered carbocyclic tetraenes, chejuenolide A and B. Two fosmid clones responsible for chejuenolide production were identified from the genomic DNA library of the MB-1084 strain. Systematic inactivation of the open reading frames (ORFs) in the sequenced region defines the boundaries of the chejuenolide (che) biosynthetic gene cluster (24.9 kbp) that encodes one non-ribosomal peptide synthase (NRPS)-polyketide synthase (PKS) hybrid protein, three modular PKSs, two PKS domains, and an amine oxidase homolog. Based on the results, we found that the che PKSs have non-canonical features such as trans-AT system and insufficient number of KS domains (five KS domains) for chejuenolide production (requires eight rounds of Claisen condensation reaction). Heterologous expression of the che PKSs in the E. coli BAP1 strain provides strong evidence of the iterative characteristic of the modular PKSs. Additionally, the phylogenetic relatedness of the KS domains of che PKSs and other trans-AT PKSs was analyzed to propose a possible pathway for chejuenolide biosynthesis.