Journal of Microbiology

Volume 49(3); June 2011

Review

REVIEW] Transcriptional Regulatory Elements in Fungal Secondary Metabolism: Wenbing Yin , Nancy P. Keller; J. Microbiol. 2011;49(3):329-339. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1009-1

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129 Citations

Abstract: Filamentous fungi produce a variety of secondary metabolites of diverse beneficial and detrimental activities to humankind. The genes required for a given secondary metabolite are typically arranged in a gene cluster. There is considerable evidence that secondary metabolite gene regulation is, in part, by transcriptional control through hierarchical levels of transcriptional regulatory elements involved in secondary metabolite cluster regulation. Identification of elements regulating secondary metabolism could potentially provide a means of increasing production of beneficial metabolites, decreasing production of detrimental metabolites, aid in the identification of ‘silent’ natural products and also contribute to a broader understanding of molecular mechanisms by which secondary metabolites are produced. This review summarizes regulation of secondary metabolism associated with transcriptional regulatory elements from a broad view as well as the tremendous advances in discovery of cryptic or novel secondary metabolites by genomic mining.

Research Support, Non-U.S. Gov't

Analyses of Bacterial Communities in Meju, a Korean Traditional Fermented Soybean Bricks, by Cultivation-Based and Pyrosequencing Methods: Yi-Seul Kim , Min-Cheol Kim , Soon-Wo Kwon , Soo-Jin Kim , In-Cheol Park , Jong-Ok Ka , Hang-Yeon Weon; J. Microbiol. 2011;49(3):340-348. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0302-3

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106 Citations

Abstract: Despite the importance of meju as a raw material used to make Korean soy sauce (ganjang) and soybean paste (doenjang), little is known about the bacterial diversity of Korean meju. In this study, the bacterial communities in meju were examined using both culture-dependent and independent methods in order to evaluate the diversity of the bacterial population. Analyses of the 16S rRNA gene sequences of the bacterial strains isolated from meju samples showed that the dominant species were related to members of the genera Bacillus, Enterococcus, and Pediococcus. The community DNAs extracted from nine different meju samples were analyzed by barcoded pyrosequencing method targeting of the V1 to V3 hypervariable regions of the 16S rRNA gene. In total, 132,374 sequences, with an average read length of 468 bp, were assigned to several phyla, with Firmicutes (93.6%) representing the predominant phylum, followed by Proteobacteria (4.5%) and Bacteroidetes (0.8%). Other phyla accounted for less than 1% of the total bacterial sequences. Most of the Firmicutes were Bacillus and lactic acid bacteria, mainly represented by members of the genera Enterococcus, Lactococcus, and Leuconostoc, whose ratio varied among different samples. In conclusion, this study indicated that the bacterial communities in meju were very diverse and a complex microbial consortium containing various microorganisms got involved in meju fermentation than we expected before.

Journal Article

Evaluation of Antibacterial Activity against Salmonella Enteritidis: Gaëlle Legendre , Fabienne Faÿ , Isabelle Linossier , Karine Vallée-Réhel; J. Microbiol. 2011;49(3):349-354. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0162-x

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Abstract: Salmonella enterica serovar Enteritidis is a well-known pathogenic bacterium responsible for human gastrointestinal enteritis mainly due to the consumption of eggs and egg-products. The first aim of this work was to study several virulence factors of a strain isolated from egg content: SEovo. First, bacterial growth was studied at several temperatures and cell morphology was observed by scanning electronic microscopy. These experiments showed Salmonella’s ability to grow at low temperatures and to produce exoproducts. Next, Salmonella motility was observed performing swimming, twitching, and swarming tests. Results indicated a positive flagellar activity and the cell ability to differentiate and become hyperflagellated under specific conditions. Moreover, SEovo adherence and biofilm formation was carried out. All of these tests enabled us to conclude that SEovo is a potential pathogen, thus it can be used as a model to perform antibacterial experiments. The second part of the study was dedicated to the evaluation of the antibacterial activity of different molecules using several methods. The antibacterial effect of silver and copper aluminosilicates was tested by two different kinds of methods. On the one hand, the effect of these two antibacterial agents was determined using microbiological methods: viable cell count and agar-well diffusion. And on the other hand, the antibacterial activity was evaluated using CLSM and SYTO Red/SYTOX Green dyeing. CLSM allowed for the evaluation of the biocide on sessile cells, whereas the first methods did not. Results showed that adhered bacteria were more resistant than planktonic counterparts and that CLSM was a good alternative to evaluate antibacterial activity on fixed bacteria without having to carry out a removing step.

Research Support, Non-U.S. Gov'ts

Cultured Bacterial Diversity and Human Impact on Alpine Glacier Cryoconite: Yung Mi Lee , So-Yeon Kim , Jia Jung , Eun Hye Kim , Kyeung Hee Cho , Franz Schinner , Rosa Margesin , Soon Gyu Hong , Hong Kum Lee; J. Microbiol. 2011;49(3):355-362. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0232-0

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27 Citations

Abstract: The anthropogenic effect on the microbial communities in alpine glacier cryoconites was investigated by cultivation and physiological characterization of bacteria from six cryoconite samples taken at sites with different amounts of human impact. Two hundred and forty seven bacterial isolates were included in Actinobacteria (9%, particularly Arthrobacter), Bacteroidetes (14%, particularly Olleya), Firmicutes (0.8%), Alphaproteobacteria (2%), Betaproteobacteria (16%, particularly Janthinobacterium), and Gammaproteobacteria (59%, particularly Pseudomonas). Among them, isolates of Arthrobacter were detected only in samples from sites with no human impact, while isolates affiliated with Enterobacteriaceae were detected only in samples from sites with strong human impact. Bacterial isolates included in Actinobacteria and Bacteroidetes were frequently isolated from pristine sites and showed low maximum growth temperature and enzyme secretion. Bacterial isolates included in Gammaproteobacteria were more frequently isolated from sites with stronger human impact and showed high maximum growth temperature and enzyme secretion. Ecotypic differences were not evident among isolates of Janthinobacterium lividum, Pseudomonas fluorescens, and Pseudomonas veronii, which were frequently isolated from sites with different degrees of anthropogenic effect.

Molecular Analysis of a Prolonged Spread of Klebsiella pneumoniae Co-producing DHA-1 and SHV-12 β-Lactamases: Young Kyung Yoon , Hye Won Cheong , Hyunjoo Pai , Kyoung Ho Roh , Jeong Yeon Kim , Dae Won Park , Jang Wook Sohn , Seung Eun Lee , Byung Chul Chun , Hee Sun Sim , Min Ja Kim; J. Microbiol. 2011;49(3):363-368. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0491-9

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Abstract: The study investigated molecular mechanisms for prolonged nosocomial spread of multidrug-resistant Klebsiella pneumoniae co-producing plasmid-mediated AmpC β-lactamase DHA-1 and extended-spectrum β-lactamase SHV-12. Forty-eight clinical isolates of K. pneumonia, resistant to the extended-spectrum cephalosporins, were collected in a 750-bed university hospital over a year. The isolates were characterized for PCR-based β-lactamase genotypes, isoelectric focusing and pulsed-field gel electrophoresis (PFGE) profiles. Resistance transfer was performed by plasmid conjugation and confirmed by a duplex-PCR and Southern hybridization. On β-lactamase typing, the strains producing only the DHA-1 enzyme (n=17) or co-producing DHA-1 and SHV-12 enzymes (n=15) were predominant. Judging from a one year-distribution of PFGE profiles, the co-producer was spread primarily with single clonal expansion of the PFGE-type A with subtypes (n=14), whereas the strains producing only DHA-1 enzyme were spread simultaneously with the PFGE-type A (n=11) and other PFGE types (n=6). Transconjugants of the co-producers were confirmed to harbor either both blaDHA-1 and blaSHV-12 or only the blaDHA-1. In conclusion, this study indicated that the persistent nosocomial spread of multidrug-resistant K. pneumoniae strains was primarily associated with expansion of a clone harboring both the blaDHA-1 and blaSHV-12 or the blaDHA-1 only, and to a lesser extent with the horizontal transfer of the resistant plasmids. Our observations have clinical implication for the control and prevention of nosocomial dissemination of multidrug-resistant K. pneumoniae strains.

The Impacts of Excessive Nitrogen Additions on Enzyme Activities and Nutrient Leaching in Two Contrasting Forest Soils: Haryun Kim , Hojeong Kang; J. Microbiol. 2011;49(3):369-375. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0421-x

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28 Citations

Abstract: Nitrogen (N) deposition has increased dramatically worldwide, which may affect forest soils in various ways. In this study, we conducted a short-term manipulation experiment of N addition on two types of forest soils (urban and rural soils) found in Korea. N addition significantly decreased phenol oxidase activities in urban soil samples; however, it did not affect those in rural soils. Furthermore, N addition did not change β-glucosidase and N-acetylglucosaminidase activities, except for β-glucosidase activities in the O layer of rural soils. Changes in microbial biomass and general activity (dehydrogenase activity) were not induced by N addition, except for dehydrogenase in the A layer of urban soils. Although N addition did not change the extractable soil nutrients, organic matter, and water contents significantly, it enhanced nutrient leaching and resulted in lower pH leachate. These results suggest that excessive N addition to forest soils may induce nutrient leaching in the long-term. Overall results of our study also suggest that N addition may induce retardation of organic matter decomposition in soils; however, such a response may depend on the intensity of previous exposure to N deposition.

Biochemical Analysis of a Fibrinolytic Enzyme Purified from Bacillus subtilis Strain A1: Won Sik Yeo , Min Jeong Seo , Min Jeong Kim , Hye Hyeon Lee , Byoung Won Kang , Jeong Uck Park , Yung Hyun Choi , Yong Kee Jeong; J. Microbiol. 2011;49(3):376-380. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1165-3

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Abstract: A fibrinolytic enzyme from Bacillus subtilis strain A1 was purified by chromatographic methods, including DEAE Sephadex A-50 column chromatography and Sephadex G-50 column gel filtration. The purified enzyme consisted of a monomeric subunit and was estimated to be approximately 28 kDa in size by SDS-PAGE. The specific activity of the fibrinolytic enzyme was 1632-fold higher than that of the crude enzyme extract. The fibrinolytic activity of the purified enzyme was approximately 0.62 and 1.33 U/ml in plasminogen-free and plasminogen-rich fibrin plates, respectively. Protease inhibitors PMSF, DIFP, chymostatin, and TPCK reduced the fibrinolytic activity of the enzyme to 13.7, 35.7, 15.7, and 23.3%, respectively. This result suggests that the enzyme purified from B. subtilis strain A1 was a chymotrypsin-like serine protease. In addition, the optimum temperature and pH range of the fibrinolytic enzyme were 50°C and 6.0-10.0, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as Q-T-G-G-S-I-I-D-P-I-N-G-Y-N, which was highly distinguished from other known fibrinolytic enzymes. Thus, these results suggest a fibrinolytic enzyme as a novel thrombolytic agent from B. subtilis strain A1.

Shewanella upenei sp. nov., a Lipolytic Bacterium Isolated from Bensasi Goatfish Upeneus bensasi: Kyung-Kil Kim , Young-Ok Kim , Sooyeon Park , So-Jung Kang , Bo-Hye Nam , Doo Nam Kim , Tae-Kwang Oh , Jung-Hoon Yoon; J. Microbiol. 2011;49(3):381-386. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0175-5

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15 Citations

Abstract: A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23RT, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23RT belonged to the genus Shewanella. Strain 20-23RT exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192T, Shewanella haliotis DW01T, and Shewanella chilikensis JC5T, respectively. Strain 20-23RT exhibited 93.1-96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3-98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23RT contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20-23RT, S. algae KCTC 22552T and S. haliotis KCTC 12896T were similar; major components were iso-C15:0, C16:0, C16:1 ω7c and/or iso-C15:0 2-OH and C17:1 ω8c. The DNA G+C content of strain 20-23RT was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20-23RT, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23RT represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20-23RT (=KCTC 22806T =CCUG 58400T).

Flavobacterium koreense sp. nov., Flavobacterium chungnamense sp. nov., and Flavobacterium cheonanense sp. nov., Isolated from a Freshwater Reservoir: Siwon Lee , Hang-Yeon Weon , Soo-Jin Kim , Tae-Young Ahn; J. Microbiol. 2011;49(3):387-392. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0382-0

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14 Citations

Abstract: Taxonomic studies were performed on three strains isolated from Cheonho reservoir in Cheonan, Korea. The isolates were Gram-negative, aerobic, rod-shaped, non-motile, catalase-positive, and oxidase-positive. Colonies on solid media were cream-yellow, smooth, shiny, and circular. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belong to the genus Flavobacterium. The strains shared 98.6-99.4% sequence similarity with each other and showed less than 97% similarity with members of the genus Flavobacterium with validly published names. The DNA-DNA hybridization results confirmed the separate genomic status of strains ARSA-42T, ARSA-103T, and ARSA-108T. The isolates contained menaquinone-6 as the predominant menaquinone and iso-C15:0, iso-C15:0 3-OH, iso-C15:1 G, and iso-C16:0 3-OH as the major fatty acids. The genomic DNA G+C content of the isolates were 31.4-33.2 mol%. According to the phenotypic and genotypic data, these organisms are classified as representative of three novel species in the genus Flavobacterium, and the name Flavobacterium koreense sp. nov. (strain ARSA-42T =KCTC 23182T =JCM 17066T =KACC 14969T), Flavobacterium chungnamense sp. nov. (strain ARSA-103T =KCTC 23183T =JCM 17068T =KACC 14971T), and Flavobacterium cheonanense sp. nov. (strain ARSA-108T =KCTC 23184T =JCM 17069T =KACC 14972T) are proposed.

Mucilaginibacter composti sp. nov., with Ginsenoside Converting Activity, Isolated from Compost: Chang-Hao Cui , Tae-Eun Choi , Hongshan Yu , Fengxie Jin , Sung-Taik Lee , Sun-Chang Kim , Wan-Taek Im; J. Microbiol. 2011;49(3):393-398. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1176-0

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Abstract: The Gram-negative, strictly aerobic, non-motile, non-spore-forming, rod shaped bacterial strain designated TR6-03T was isolated from compost, and its taxonomic position was investigated by using a polyphasic approach. Strain TR6-03T grew at 4-42°C and at pH 6.0-8.0 on R2A and nutrient agar without NaCl supplement. Strain TR6-03T had β-glucosidase activity, which was responsible for its ability to transform ginsenoside Re (one of the dominant active components of ginseng) to Rg2. On the basis of 16S rRNA gene sequence similarity, strain TR6-03T was shown to belong to the family Sphingobacteriaceae and to be related to Mucilaginibacter lappiensis ANJLI2T (96.3% sequence similarity), M. dorajii FR-f4T (96.1%), and M. rigui WPCB133T (94.1%). The G+C content of the genomic DNA was 45.6%. The predominant respiratory quinone was MK-7 and the major fatty acids were summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2OH), iso-C15:0 and iso-C17:0 3OH. DNA and chemotaxonomic data supported the affiliation of strain TR6-03T to the genus Mucilaginibacter. Strain TR6-03T could be differentiated genotypically and phenotypically from the recognized species of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter composti sp. nov. is proposed, with the type strain TR6-03T (=KACC 14956T =KCTC 12642T =LMG 23497T).

Molecular Cloning, Purification, and Characterization of a Superoxide Dismutase from a Fast-Growing Mycobacterium sp. Strain JC1 DSM 3803: Ji-Sun Nam , Jee-Hyun Yoon , Hyun-Il Lee , Si Wouk Kim , Young-Tae Ro; J. Microbiol. 2011;49(3):399-406. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1046-9

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Abstract: A cytosolic superoxide dismutase (SOD) was purified and characterized from a fast-growing Mycobacterium sp. strain JC1 DSM 3803 grown on methanol. The native molecular weight of the purified SOD was estimated to be 48 kDa. SDS-PAGE revealed a subunit of 23 kDa, indicating that the enzyme is a homodimer. The enzyme activity was inhibited by H2O2 and azide. The purified SOD contained 1.12 and 0.56 g-atom of Mn and Fe per mol of enzyme, respectively, suggesting that it may be a Fe/Mn cambialistic SOD. The apo-SOD reconstitution study revealed that Mn salts were more specific than Fe salts in the SOD activity. The gene encoding the SOD was identified from the JC1 cosmid genomic library by PCR screening protocol. The cloned gene, sodA, had an open reading frame (ORF) of 624 nt, encoding a protein with a calculated molecular weight of 22,930 Da and pI of 5.33. The deduced SodA sequence exhibited 97.6% identity with that of Mycobacterium fortuitum Mn-SOD and clustered with other mycobacterial Mn-SODs. A webtool analysis on the basis of SOD sequence and structure homologies predicted the SOD as a tetrameric Mn-SOD, suggesting that the protein is a dimeric Mn-SOD having tetramer-specific sequence and structure characteristics.

Biochemical Characteristization of Propionyl-Coenzyme A Carboxylase Complex of Streptomyces toxytricini: Atanas V. Demirev , Anamika Khanal , Nguyen Phan Kieu Hanh , Kyung Tae Nam , Doo Hyun Nam; J. Microbiol. 2011;49(3):407-412. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1122-1

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Abstract: Acyl-CoA carboxylases (ACC) are involved in important primary or secondary metabolic pathways such as fatty acid and/or polyketides synthesis. In the 6.2 kb fragment of pccB gene locus of Streptomyces toxytricini producing a pancreatic inhibitor lipstatin, 3 distinct subunit genes of presumable propionyl-CoA carboxylase (PCCase) complex, assumed to be one of ACC responsible for the secondary metabolism, were identified along with gene for a biotin protein ligase (Bpl). The subunits of PCCase complex were α subunit (AccA3), β subunit (PccB), and auxiliary ε subunit (PccE). In order to disclose the involvement of the PCCase complex in secondary metabolism, some biochemical characteristics of each subunit as well as their complex were examined. In the test of substrate specificity of the PCCase complex, it was confirmed that this complex showed much higher conversion of propionyl-CoA rather than acetyl-CoA. It implies the enzyme complex could play a main role in the production of methylmalonyl-CoA from propionyl-CoA, which is a precursor of secondary polyketide biosynthesis.

Journal Article

Genotypic and Phenotypic Characteristics of Tunisian Isoniazid-Resistant Mycobacterium tuberculosis Strains: Alya Soudani , Meriem Zribi , Feriel Messaadi , Taieb Messaoud , Afef Masmoudi , Mohamed Zribi , Chedlia Fendri; J. Microbiol. 2011;49(3):413-417. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0268-1

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Abstract: Forty three isoniazid (INH)-resistant Mycobacterium tuberculosis isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA -15C→T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity). Typing for resistance mutations was performed by Multiplex Allele-Specific PCR and sequencing reaction. Mutations at either codon were detected in 67.5% of isolates: katG315 in 37.2, mabA -15C→T in 27.9 and both of them in 2.4%, respectively. katG sequencing showed a G insertion at codon 325 detected in 2 strains and leading to amino acid change T326D which has not been previously reported. Distribution of each mutation, among the investigated strains, showed that katG S315T was associated with multiple-drug profile, high-level INH resistance and loss or decreased catalase activity; whereas the mabA -15C→T was more prevalent in mono-INH resistant isolates, but it was not only associated with a low-level INH resistance. It seems that determination of catalase activity aids in the detection of isolates for which MICs are high and could, in conjunction with molecular methods, provide rapid detection of most clinical INH-resistant strains.

Research Support, Non-U.S. Gov'ts

The MpkB MAP Kinase Plays a Role in Post-karyogamy Processes as well as in Hyphal Anastomosis During Sexual Development in Aspergillus nidulans: Sang-Cheol Jun , Sei-Jin Lee , Hyun-Joo Park , Ji-Young Kang , Young-Eun Leem , Tae-Ho Yang , Mi-Hee Chang , Jung-Mi Kim , Seung-Hwan Jang , Hwan-Gyu Kim , Dong-Min Han , Keon-Sang Chae , Kwang-Yeop Jahng; J. Microbiol. 2011;49(3):418-430. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0193-3

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24 Citations

Abstract: Two genes encoding MAP kinase homologs, designated as mpkB and mpkC, were isolated from Aspergillus nidulans by PCR with degenerate primers. Deletion and over-expression mutants of mpkC showed no detectable phenotypes under any external stress tested. Deletion of mpkB caused pleiotropic phenotypes including a failure in forming cleistothecia under any induction conditions for sexual development, increased Hülle cell production, slow hyphal growth and aberrant conidiophore morphology. Over-expression of mpkB led to increased cleistothecium production. While the transcripts of mpkB and mpkC were constitutively synthesized through the entire life cycle, their size and amount differed with developmental stages. An outcross test using fluorescent protein reporters showed that the mpkB deletion mutant could not form heterokaryons with wild type. Protoplast fusion experiments showed that the fusant of the mpkB mutant with wild type could undergo normal sexual development. However, heterokaryotic mycelia that were produced from a fusant between two mpkB deletion mutants could not form cleistothecia, although they did appear to form diploid nuclei. These results suggest that the MpkB MAP kinase is required for some post-karyogamy process as well as at the hyphal anastomosis stage to accomplish sexual development successfully.

Identification of the Genes Involved in 1-Deoxynojirimycin Synthesis in Bacillus subtilis MORI 3K-85: Kyung-Don Kang , Yong Seok Cho , Ji Hye Song , Young Shik Park , Jae Yeon Lee , Kyo Yeol Hwang , Sang Ki Rhee , Ji Hyung Chung , Ohsuk Kwon , Su-Il Seong; J. Microbiol. 2011;49(3):431-440. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1238-3

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34 Citations

Abstract: 1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation. The genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.

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