• Journal of Microbiology
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• v.43 no.3
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• pp.313-318
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• 2005

In this study, we demonstrate the use of a PCR-based method for the detection of the specific genes involved in natural-product biosynthesis. This method was applied, using specifically designed PCR primers, to the amplification of a gene segment encoding for sedo-heptulose 7-phosphate cyclase, which appears to be involved in the biosynthetic pathways of $C_7N$ aminoacyclitol or its keto analogue-containing metabolites, in a variety of actinomycetes species. The sequences of DNA fragments (about 540 bp) obtained from three out of 39 actinomycete strains exhibited a high degree of homology with the sedo-heptulose 7-phosphate cyclase gene, which has been implicated in acarbose biosynthesis. The selective cultivation conditions of this experiment induced the expression of these loci, indicating that the range of $C_7N$ aminoacyclitol or its keto analogue-group natural products might be far greater than was previously imagined. Considering that a total of approximately 20 $C_7N$ aminoacyclitol metabolites, or its keto analogue-containing metabolites, have been described to date, it appears likely that some of the unknown loci described herein might constitute new classes of $C_7N$ aminoacyclitol, or of its keto analogue-containing metabolites. As these metabolites, some of which contain valienamine, are among the most potent antidiabetic agents thus far discovered, the molecular detection of specific metabolite-producing actinomycetes may prove a crucial step in current attempts to expand the scope and diversity of natural-product discovery.

• Journal of Microbiology and Biotechnology
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• v.31 no.3
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• pp.483-491
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• 2021

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDS-PAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50℃ and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and insilico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

• The Korean Journal of Mycology
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• v.39 no.1
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• pp.22-30
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• 2011

Weonhyeong is one of important commercial strains. It has good characteristics of bundle formation, grey colored pilei and high productivity. We previously reported grouping of 70 strains of Pleurotus ostreatus in which one group contained 35 strains including Weonhyeong. Four strains in that group showed same profiles implicating no variety distinction for mushroom cultivation. Now we developed a specific marker for identification of Weonhyeong. Sequence Characterized Amplified Region (SCAR) marker was developed from the RAPD amplicon. SCAR marker 'S-OPO5' produced only one band specific to 2183, 2240, 2595 and 2725 strains showing similar banding patterns to Weonhyeong in RAPD-PCR results. The sequence of 'S-OPO5' marker was unknown when compared with the data in the Genbank using BLASTN. BLASTX results indicated that the marker showed significant alignment with the protein sequences in Tricholoma bakamatsutake reverse transcriptase. The results indicate that this new SCAR marker ('S-OPO5') will be valuable to distinguish the Weonhyeong similar strains from Pleurotus spp.

• The Plant Pathology Journal
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• v.36 no.1
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• pp.87-97
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• 2020

The development of reverse transcription-polymerase chain reaction using degenerate primers against conserved regions of most potyviral genomes enabled sampling of the potyvirome. However, these assays usually involve sampling potential host plants, but identifying infected plants when they are asymptomatic is challenging, and many plants, especially wild ones, contain inhibitors to DNA amplification. We used an alternative approach which utilized aphid vectors and indicator plants to identify potyviruses capable of infecting common bean (Phaseolus vulgaris). Aphids were collected from a range of asymptomatic leguminous weeds and trees in Iran, and transferred to bean seedlings under controlled conditions. Bean plants were tested serologically for potyvirus infections four-weeks postinoculation. The serological assay and symptomatology together indicated the presence of one potyvirus, and symptomology alone implied the presence of an unidentified virus. The partial genome of the potyvirus, encompassing the complete coat protein gene, was amplified using generic potyvirus primers. Sequence analysis of the amplicon confirmed the presence of an isolate of Wisteria vein mosaic virus (WVMV), a virus species not previously identified from Western Asia. Phylogenetic analyses of available WVMV sequences categorized them into five groups: East Asian-1 to 3, North American and World. The Iranian isolate clustered with those in the World group. Multiple sequence alignment indicated the presence of some genogroup-specific amino acid substitutions among the isolates studied. Chinese isolates were sister groups of other isolates and showed higher nucleotide distances as compared with the others, suggesting a possible Eastern-Asian origin of WVMV, the main region where Wisteria might have originated.

• Microbiology and Biotechnology Letters
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• v.47 no.1
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• pp.25-33
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• 2019

Lactobacillus rhamnosus BFE5264, isolated from a Maasai fermented milk product ("kule naoto"), was previously shown to exhibit bile acid resistance, cholesterol assimilation, and adhesion to HT29-MTX cells in vitro. In this study, we re-annotated and analyzed the previously reported complete genome sequence of strain BFE5264. The genome consists of a circular chromosome of 3,086,152 bp and a putative plasmid, which is the largest one identified among L. rhamnosus strains. Among the 2,883 predicted protein-coding genes, those with carbohydrate-related functions were the most abundant. Genome analysis of strain BFE5264 revealed two consecutive CRISPR regions and no known virulence factors or antimicrobial resistance genes. In addition, previously known highly variable regions in the genomes of L. rhamnosus strains were also evident in strain BFE5264. Pairwise comparison with the most studied probiotic strain L. rhamnosus GG revealed strain BFE5264-specific deletions, probably due to insertion sequence-mediated recombination. The latter was associated with loss of the spaCBA pilin gene cluster and exopolysaccharide biosynthetic genes. Comparative genomic analysis of the sequences from all available L. rhamnosus strains revealed that they were clustered into two groups, being within the same species boundary based on the average nucleotide identities. Strain BFE5264 had a sister group relationship with the group that contained strain GG, but neither ANI-based hierarchical clustering nor core-gene-based phylogenetic tree construction showed a clear distinctive pattern associated with the isolation source, implying that the genotype alone cannot account for their ecological niches. These results provide insights into the probiotic mechanisms of strain BFE5264 at the genomic level.

• Journal of Life Science
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• v.25 no.2
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• pp.189-196
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• 2015

Bacterial diversity was studied in the rhizosphere of Suaeda japonica Makino, which is native to Suncheon Bay in South Korea. Soil samples from several sites were diluted serially, and pure isolation was performed by subculture using marine agar and tryptic soy agar media. Genomic DNA was extracted from 29 pure, isolated bacterial strains, after which their 16S rDNA sequences were amplified and analyzed. Phylogenetic analysis was performed to confirm their genetic relationship. The 29 bacterial strains were classified into five groups: phylum Firmicutes (44.8%), Gamma proteobacteria group (27.6%), Alpha proteobacteria group (10.3%), phylum Bacteriodetes (10.3%), and phylum Actinobacteria (6.8%). The most widely distributed genera were Bacillus (phylum Firmicutes), and Marinobacterium, Halomonas, and Vibrio (Gamma proteobacteria group). To confirm the bacterial diversity in rhizospheres of S. japonica, the diversity index was used at the genus level. The results show that bacterial diversity differed at each of the sampling sites. These 29 bacterial strains are thought to play a major role in material cycling at Suncheon Bay, in overcoming the sea/mud flat-specific environmental stress. Furthermore, some strains are assumed to be involved in a positive interaction with the halophyte S. japonica, as rhizospheric flora, with induction of growth promotion and plant defense mechanism.

• Research in Plant Disease
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• v.17 no.3
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• pp.369-375
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• 2011

Fusarium verticillioides (teleomorph: Gibberella moniliformis), a member of the Gibberellea fujikuroi species complex, causes rots of corn stalks and ears, and produces a group of mycotoxins known as fumonisins that are harmful to animals and humans. Here, we focus on the development of a species-specific PCR primer set for differentiating F. verticillioides from other fumonisin-producing Fusarium species belonging to the species complex, such as F. proliferatum, F. fujikuroi, and F. subglutinans that are frequently associated with corn. The specific primers (RVERT1 and RVERT2) derived from the nucleotide sequences of RNA polymerase II beta subunit (RPB2) gene amplified a 208 bp-DNA fragment from only F. verticillioides isolates among the potential fumonisin-producing species examined; all of these isolates were shown to carry FUM1 required for fumonisin biosynthesis. The PCR detection limit using this specific primer set was approximately 0.125 pg/${\mu}l$ genomic DNA of F. verticillioides. In addition, the F. verticillioides-specfic fragment was successfully amplified from genomic DNAs of corn samples contaminated with Fusarium spp. This primer set would provide a useful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains in cereal samples.

• Korean Journal of Breeding Science
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• v.42 no.5
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• pp.547-554
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• 2010

Low-molecular-weight glutenin subunit (LMW-GS) in common wheat (Triticum aestivum L.) is important for quality processing of bread and noodles. The objectives of this study were to clarify the composition of LMW-GSs and to identify their corresponding proteins. Using LMW-GS specific primers we cloned and characterized 43 LMW-GS genes in the wheat cultivar 'Jokyoung'. Some of these genes contain polypeptides different in size due to the presence of various deletions or insertions within repetitive and glutamine-rich domains. The comparison of deduced amino acid sequence of the LMW-GS genes in Jokyoung with that of 12 groups LMW-GSs of wheat cultivar Norin 61 showed that the deduced amino acid sequences were nearly the same to LMW-GS groups of 1, 2, 3/4, 5, 7, 10 and 11. All LMW-GS genes contain eight cysteine residues, which are conserved among all of the typical LMW-GS sequences. The relative positions of cysteine residues are also conserved, except those of the first and seventh. Based on phylogenetic analysis, the 43 sequences with the same N-terminal and C-terminal amino acid sequences were clustered in the same group. To identify the proteins containing the corresponding amino acid sequences, we determined the N-terminal amino acid sequence of 7 spots of LMW-GSs of Jokyoung separated by two-dimensional gel electrophoresis (2DE). Of them, Glu-B3 (LMW-m and LMW-s) and Glu-D3 (LMW-m) were detected in two and three spots, respectively and the others were not clear. Collectively, we classified diverse LMW-GSs and identified their corresponding protein products. These results will be helpful in breeding programs for improvement of wheat flour quality.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.238-249
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• 2009

The genus Listeria consists of six closely related species and forms three phylogenetic groups: L. monocytogenes-L. innocua, L. ivanovii-L. seeligeri-L. welshimeri, and L. grayi. In this report, we attempted to examine the evolutionary relationship in the L. monocytogenes-L. innocua group by probing the nucleotide sequences of 23S rRNA and 16S rRNA, and the gene clusters lmo0029-lmo0042, ascB-dapE, rplS-infC, and prs-ldh in L. monocytogenes serovars 1/2a, 4a, and 4b, and L. innocua. Additionally, we assessed the status of L. monocytogenes-specific inlA and inlB genes and 10 L. innocua-specific genes in these species/serovars, together with phenotypic characterization by using in vivo and in vitro procedures. The results indicate that L. monocytogenes serovar 4a strains are genetically similar to L. innocua in the lmo0035-lmo0042, ascB-dapE, and rplS-infC regions and also possess L. innocua-specific genes lin0372 and lin1073. Furthermore, both L. monocytogenes serovar 4a and L. innocua exhibit impaired intercellular spread ability and negligible pathogenicity in mouse model. On the other hand, despite resembling L. monocytogenes serovars 1/2a and 4b in having a nearly identical virulence gene cluster, and inlA and inlB genes, these serovar 4a strains differ from serovars 1/2a and 4b by harboring notably altered actA and plcB genes, displaying strong phospholipase activity and subdued in vivo and in vitro virulence. Thus, by possessing many genes common to L. monocytogenes serovars 1/2a and 4b, and sharing many similar gene deletions with L. innocua, L. monocytogenes serovar 4a represents a possible evolutionary intermediate between L. monocytogenes serovars 1/2a and 4b and L. innocua.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1290-1297
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• 2008

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting $G_{1PAO},\;G_{2PAO},\;and\;G_{3PAO}$ groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non-Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (GINPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the $G_{4PAO}$ group of Accumulibacter phosphatis, which suggests that GINPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.