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Volume 49(3); June 2011
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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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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.

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  • Soil extracellular enzymes as drivers of soil carbon storage under nitrogen addition
    Xiao Chen, Junji Cao, Robert L. Sinsabaugh, Daryl L. Moorhead, Richard D. Bardgett, Nicolas Fanin, Andrew T. Nottingham, Xunhua Zheng, Ji Chen
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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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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).

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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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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).

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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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AbstractAbstract PDF
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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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.

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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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AbstractAbstract PDF
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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Research Support, N.I.H., Extramural
Sulfolipid Accumulation in Mycobacterium tuberculosis Disrupted in the mce2 Operon
Olivera Marjanovic , Anthony T. Iavarone , Lee W. Riley
J. Microbiol. 2011;49(3):441-447.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0435-4
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AbstractAbstract PDF
Mycobacterium tuberculosis, the causative agent of tuberculosis, has a lipid-rich cell wall that serves as an effective barrier against drugs and toxic host cell products, which may contribute to the organism’s persistence in a host. M. tuberculosis contains four homologous operons called mce (mce1-4) that encode putative ABC transporters involved in lipid importation across the cell wall. Here, we analyzed the lipid composition of M. tuberculosis disrupted in the mce2 operon. High resolution mass spectrometric and thin layer chromatographic analyses of the mutant’s cell wall lipid extracts showed accumulation of SL-1 and SL1278 molecules. Radiographic quantitative analysis and densitometry revealed 2.9, 3.9 and 9.8-fold greater amount of [35S] SL-1 in the mce2 operon mutant compared to the wild type M. tuberculosis during the early/mid logarithmic, late logarithmic and stationary phase of growth in liquid broth, respectively. The amount of [35S] SL1278 in the mutant also increased progressively over the same growth phases. The expression of the mce2 operon genes in the wild type strain progressively increased from the logarithmic to the stationary phase of bacterial growth in vitro, which inversely correlated with the proportion of radiolabel incorporation into SL-1 and SL1278 at these phases. Since the mce2 operon is regulated in wild type M. tuberculosis, its cell wall may undergo changes in SL-1 and SL1278 contents during a natural course of infection and this may serve as an important adaptive strategy for M. tuberculosis to maintain persistence in a host.

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Journal Article
Epidemiological Features and Resistance Pattern in Uropathogens Isolated from Chronic Bacterial Prostatitis
Tommaso Cai , Sandra Mazzoli , Francesca Meacci , Vieri Boddi , Nicola Mondaini , Gianni Malossini , Riccardo Bartoletti
J. Microbiol. 2011;49(3):448-454.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0391-z
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AbstractAbstract PDF
Chronic bacterial prostatitis (CBP) is, usually, caused by uropathogens, especially gram-negative bacilli, although infection is sometimes due to Gram-positive and atypical microorganisms. A recent increasing in prevalence of Gram-positive strains has been reported. The aim of this study was to explore the epidemiological features and resistance rates in uropathogens isolated from CBP outpatients in last 10 years. All consecutive outpatients with demonstrated CBP attending a single Sexually Transmitted Disease centre from January 1997 and December 2008, were enrolled and underwent microbiological cultures in first void early morning urine, midstream urine, expressed prostatic secretion, and post prostate massage urine. Prevalence of different bacterial strains was stratified in four different periods: 1997-1999, 2000-2002, 2003-2005, 2006-2008. Any changes observed in epidemiological features and resistance rates in uropathogens over the whole study period have been analyzed. The present study has been planned, thus, as in vitro study. From 6,221 patients, 4,601 Gram-positive and 1,620 Gram-negative bacterial strains have been isolated. Enterococcus faecalis and Escherichia coli strains are the first and second frequent pathogens found, respectively. Significant differences between E. faecalis prevalence in the 1997-1999 and 2006-2008 periods were found. E. coli showed a significant difference between prevalence in 1997-1999 and 2006-2008 periods. Gram-positive organisms showed a decreasing of susceptibility to ciprofloxacin as well as Gram-negative strains, while a good susceptibility to the levofloxacin was evidenced. E. faecalis prevalence seemed to be raised in 2006-2008 periods. Moreover, a decreasing of activity of ciprofloxacin and a good activity profile of levofloxacin have been reported.

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Research Support, Non-U.S. Gov'ts
Purification and Partial Characterization of a Detergent and Oxidizing Agent Stable Alkaline Protease from a Newly Isolated Bacillus subtilis VSG-4 of Tropical Soil
Sib Sankar Giri , V. Sukumaran , Shib Sankar Sen , M. Oviya , B. Nazeema Banu , Prasant Kumar Jena
J. Microbiol. 2011;49(3):455-461.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0427-4
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AbstractAbstract PDF
An extracellular detergent tolerant protease producing strain VSG-4 was isolated from tropical soil sample and identified as Bacillus subtilis based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The VSG-4 protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and sephadex G-200 gel permeation chromatography with a 17.4 purification fold. The purified enzyme was active and stable over a broad range of pH (8.0-11.0, optimum at 9.0) and temperature (40°C to 60°C, optimum at 50°C). The thermostability of the enzyme was significantly increased by the addition CaCl2. This enzyme was strongly inhibited by PMSF and DFP, suggesting that it belongs to the serine protease superfamily. The purified VSG-4 alkaline protease showed remarkable stability in anionic (5 mM SDS) and ionic (1% Trion X-100 and 1% Tween 80) detergents. It retained 97±2% and 83.6±1.1% of its initial activity after 1 h preincubation in the presence of 1% H2O2 and 1% sodium perborate, respectively. Furthermore, the purified enzyme showed excellent stability and compatibility with some commercial laundry detergents besides its stain removal capacity. Considering these promising properties, VSG-4 protease may find tremendous application in laundry detergent formulations.

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Predicting Genetic Traits and Epitope Analysis of apxIVA in Actinobacillus pleuropneumoniae
Min-Kyoung Shin , Seung-Bin Cha , Won-Jung Lee , Han Sang Yoo
J. Microbiol. 2011;49(3):462-468.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0449-y
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AbstractAbstract PDF
Actinobacillus pleuropneumoniae causes a severe hemorrhagic pneumonia in pigs. Fifteen serotypes of A. pleuropneumoniae express four different Apx toxins that belong to the pore-forming repeats-in-toxin (RTX) group of toxins. ApxIV, which is conserved and up-regulated in vivo, could be an excellent candidate for the development of a protective cross-serotype immunity vaccine, and could aid in the differential diagnosis of diseases caused by A. pleuropneumoniae. We identified and sequenced apxIVA from A. pleuropneumoniae serotype 2 isolated in Korea (Kor-ApxIVA). The Kor-ApxIVA was closely related to Switzerland (AF021919), China (CP000687), and China (GQ332268), showing 98.6%, 98.4%, and 97.2% amino acid homology, respectively. The level of amino acid homology, however, was higher than the nucleotide homology. The structural characteristics of ApxIVA showed RTX proteins, including N-terminal hydrophobic domains, signature sequences for potential acylation sites, and repeated glycine-rich nonapeptides in the C-terminal region of the protein. Thirty glycine-rich nonapeptides with the consensus sequence, L/V-X-G-G-X-G-N/D-D-X, were found in the C-terminus of the Kor-ApxIVA. In addition, the Kor-ApxIVA was predicted for the linear B-cell epitopes and conserved domains with determined peptide sequences. This genetic analysis of the Kor-ApxIVA might be an important foundation for future biological and functional research on ApxIVA.

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Journal Article
High Efficiency Transformation by Electroporation of Yarrowia lipolytica
Jia-Hung Wang , Wenpin Hung , Shu-Hsien Tsai
J. Microbiol. 2011;49(3):469-472.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0433-6
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AbstractAbstract PDF
Yarrowia lipolytica was usually transformed by heat shock, but linearized integrative vectors always resulted in a low transformation efficiency when electroporation was used. To develop a high efficiency integrative transformation method by electroporation of Y. lipolytica, we report here that pretreatment of Y. lipolytica with 150 mM LiAc for 1 h before electroporation will approximately 30-fold of increase transformation efficiency. A cell concentration of 1010/ml and instrument settings of 1.5 kV will generate the highest transformation efficiencies. We have developed a procedure to transform Y. lipolytica that will be able to yield an efficiency of 2.1×104 transformants/μg for integrative linear DNA. With our modifications, the electroporation procedures became a very efficient and reliable tool for Y. lipolytica transformation.

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Research Support, Non-U.S. Gov'ts
Isolation and Characterization of a Reducing Polyketide Synthase Gene from the Lichen-Forming Fungus Usnea longissima
Yi Wang , Jung A Kim , Yong Hwa Cheong , Yogesh Joshi , Young Jin Koh , Jae-Seoun Hur
J. Microbiol. 2011;49(3):473-480.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0362-4
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  • 15 Scopus
AbstractAbstract PDF
The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (UlPKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding UlPKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A UlPKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRads1, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located UlPKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UlPKS3 had six intervening introns and that UlPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.
Complementation System for Helicobacter pylori
Jinmoon Kim , Sung-Whan Kim , Sungil Jang , D. Scott Merrell , Jeong-Heon Cha
J. Microbiol. 2011;49(3):481-486.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-1196-9
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AbstractAbstract PDF
Previously Langford et al. (2006) developed the pIR203C04 complementation system for Helicobacter pylori, which can be used to complement and restore phenotypic effects in H. pylori mutant, and furthermore they used the complementation system in vivo experiments to animals without altering the ability of strain SS1 to colonize mice. In their previous study, the pIR203C04 was able to transform 26695, SS1, J99, and 43504 H. pylori strains by an electroporation method. However, in the present study using a natural transformation the pIR203C04 transformed only 26695 H. pylori but not SS1, J99, 7.13, and G27 H. pylori strains. Since the useful complementation system has a limitation of narrow selection among H. pylori strains, we redesigned the complementation system for the improvement. The same intergenic chromosomal site between hp0203 and hp0204 was utilized for the new complementation system because the insertion at the intergenic site didn’t show any polar effects and disruption of other H. pylori genes. The genome sequence analysis showed that the intergenic regions among H. pylori strains may have too low homology to each others to do a homologous recombination. Thus, in addition to the short intergenic region, the fragments of the new complementation system included 3′ conserved parts of hp0203 and hp0204 coding regions. Between the fragments there are a chloramphenicol acetyltransferase cassette and multicloning sites, resulting in pKJMSH. DNA fragment of the interest can be cloned into the multicloning sites of pKJMSH and the fragment can be integrated at the intergenic region of H. pylori chromosome by the homologous recombination. Indeed, by the natural transformation, pKJMSH was able to transform all five H. pylori strains of 26695, SS1, J99, 7.13, and G27, which are common for the investigation of molecular pathogenesis. Thus, the new pKJMSH complementation system is applicable to most H. pylori wild-type stains.

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  • AI-2 Induces Urease Expression Through Downregulation of Orphan Response Regulator HP1021 in Helicobacter pylori
    Huang Yang, Xiaoxing Huang, Xiaochuan Zhang, Xiaoyan Zhang, Xiaohong Xu, Feifei She, Yancheng Wen
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Ruminococcus faecis sp. nov., Isolated from Human Faeces
Min-Soo Kim , Seong Woon Roh , Jin-Woo Bae
J. Microbiol. 2011;49(3):487-491.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0505-7
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AbstractAbstract PDF
Bacterial strain Eg2T, an anaerobic, Gram-positive, non-motile, and non-spore-forming coccus, was isolated from human faeces. The optimal temperature for its growth was 37°C. Oxidase activity was negative, but catalase activity was positive. The strain was able to hydrolyze esculin and to produce acids from the fermentation of several substrates, including glucose. Lactic and acetic acids were the main products of glucose fermentation. The major fatty acids present in this strain were C16:0, C14:0, and C18:1 cis11 DMA. The G+C content was 43.4 mol%. Based on the 16S rRNA gene sequence, strain Eg2T was closely related to species of the genus Ruminococcus (96.3% similarity to R. torques and 96.2% similarity to R. lactaris), and its taxonomic position was placed within the Clostridium cluster XIVa. Based on phenotypic, chemotaxonomic, genotypic, and phylogenetic evidence, we propose that this novel strain be assigned to the genus Ruminococcus and be named Ruminococcus faecis sp. nov. The type strain is Eg2T (=KCTC 5757T =JCM 15917T).

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    Ji-Sun Kim, Keun Chul Lee, Min Kuk Suh, Kook-Il Han, Mi Kyung Eom, Ju Huck Lee, Seung-Hwan Park, Se Won Kang, Jam-Eon Park, Byeong Seob Oh, Seung Yeob Yu, Seung-Hyeon Choi, Dong Ho Lee, Hyuk Yoon, Byung-Yong Kim, Seung-Jo Yang, Jung-Sook Lee
    Journal of Microbiology.2019; 57(1): 38.     CrossRef
  • Schaedlerella arabinosiphila gen. nov., sp. nov., a D-arabinose-utilizing bacterium isolated from faeces of C57BL/6J mice that is a close relative of Clostridium species ASF 502
    Melissa Soh, Sou Miyake, Austin Lim, Yichen Ding, Henning Seedorf
    International Journal of Systematic and Evolutionary Microbiology.2019; 69(11): 3616.     CrossRef
  • List of new names and new combinations previously effectively, but not validly, published

    International Journal of Systematic and Evolutionary Microbiology .2011; 61(11): 2563.     CrossRef
Diversity of Bovine Rumen Methanogens In Vitro in the Presence of Condensed Tannins, as Determined by Sequence Analysis of 16S rRNA Gene Library
Hui Yin Tan , Chin Chin Sieo , Chin Mei Lee , Norhani Abdullah , Juan Boo Liang , Yin Wan Ho
J. Microbiol. 2011;49(3):492-498.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0319-7
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AbstractAbstract PDF
Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.
A Modified Immunoblot Method to Identify Substrates of Protein Kinases
Choong-Min Kang , Wan Jin Jahng , Robert N. Husson , Sang Hee Lee
J. Microbiol. 2011;49(3):499-501.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0465-y
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AbstractAbstract PDF
While protein kinases are key components in multiple cellular processes, efficient identification of cognate in vivo substrates remains challenging. Here we describe a powerful method to screen potential substrates of protein kinases by partial transfer of proteins from a 2D-PAGE gel to a Western blot membrane. This approach allowed precise pinpointing of candidate substrate spots in the 2D gel, and identifying physiological substrates of protein kinases in Mycobacterium tuberculosis.
Paralcaligenes ureilyticus gen. nov., sp. nov. Isolated from Soil of a Korean Ginseng Field
Soo-Jin Kim , Seung-Hee Yoo , Hang-Yeon Weon , Yi-Seul Kim , Rangasamy Anandham , Jang-Sun Suh , Soon-Wo Kwon
J. Microbiol. 2011;49(3):502-507.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-0076-7
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AbstractAbstract PDF
A bacterial strain, designated GR24-5T, was isolated from soil cultivated with Korean ginseng. Cells were Gram-negative, strictly aerobic, catalase- and oxidase-positive, non-spore-forming motile rods. Based on the 16S rRNA gene sequence, strain GR24-5T could be assigned to the family Alcaligenaceae. Strain GR24-5T showed the highest sequence similarities with Parapusillimonas granuli Ch07T (97.1%), Pusillimonas noertemannii BN9T (96.9%), Pigmentiphaga kullae DSM 13608T (96.5%), and Castellaniella defragrans 54PinT (96.3%). Strain GR24-5T demonstrated a low DNA-DNA relatedness (23%) with P. granuli Ch07T. The major respiratory quinone is ubiquinone 8 (Q-8) and the major fatty acids are C16:0, C17:0 cyclo, and summed feature 1 (C14:03-OH/iso-C16:1 I/C12:0 alde). Putrescine, spermidine, and 2-hydroxyputrescine are the major polyamines. The major polar lipids are phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unknown aminophospholipid. Polar lipid patterns of strain GR24-5T were unique in having a large amount of phosphatidylmethylethanolamine. Based on phylogenetic analysis and physiological and biochemical characteristics, strain GR245T represents a novel genus and species, for which the name Paralcaligenes ureilyticus gen. nov., sp. nov. is proposed. The type strain of P. aralcaligenes ureilyticus is GR24-5T (=KACC 13888T =DSM 24591T).

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  • Neopusillimonas aromaticivorans sp. nov. isolated from poultry manure
    Shih-Yao Lin, Tzu-Yu Lin, Asif Hameed, Yu-Shan Tang, Chiu-Chung Young
    International Journal of Systematic and Evolutionary Microbiology .2023;[Epub]     CrossRef
  • Paracandidimonas caeni sp. nov., isolated from sludge
    Li Yao, Yuhan Lai, Fei Xue, Lina Sun, Jialian Wang
    International Journal of Systematic and Evolutionary Microbiology .2019; 69(11): 3332.     CrossRef
  • Achromobacter panacis sp. nov., isolated from rhizosphere of Panax ginseng
    Priyanka Singh, Yeon Ju Kim, Hina Singh, Mohamed El-Agamy Farh, Deok-Chun Yang
    Journal of Microbiology.2017; 55(6): 428.     CrossRef
  • Paralcaligenes ginsengisoli sp. nov., isolated from ginseng cultivated soil
    Jong-Pyo Kang, Ngoc-Lan Nguyen, Yeon-Ju Kim, Van-An Hoang, Kwi-Sik Bae, Deok-Chun Yang
    Antonie van Leeuwenhoek.2015; 108(3): 619.     CrossRef
  • Paenalcaligenes suwonensis sp. nov., isolated from spent mushroom compost
    Ji-Young Moon, Jun-Muk Lim, Jae-Hyung Ahn, Hang-Yeon Weon, Soon-Wo Kwon, Soo-Jin Kim
    International Journal of Systematic and Evolutionary Microbiology .2014; 64(Pt_3): 882.     CrossRef
  • Eoetvoesia caeni gen. nov., sp. nov., isolated from an activated sludge system treating coke plant effluent
    Tamás Felföldi, Anita Vengring, Zsuzsa Kéki, Károly Márialigeti, Peter Schumann, Erika M. Tóth
    International Journal of Systematic and Evolutionary Microbiology .2014; 64(Pt_6): 1920.     CrossRef
  • List of new names and new combinations previously effectively, but not validly, published – Validation List No. 141

    International Journal of Systematic and Evolutionary Microbiology .2011; 61(9): 2025.     CrossRef
RNase G Participates in Processing of the 5′-end of 23S Ribosomal RNA
Woo-Seok Song , Minho Lee , Kangseok Lee
J. Microbiol. 2011;49(3):508-511.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-1198-7
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AbstractAbstract PDF
In Escherichia coli, primary rRNA transcripts must be processed by a complex process in which several ribonucleases are involved in order to generate mature 16S, 23S, and 5S rRNA molecules. While it is known that RNase G, a single-stranded RNA-specific endoribonuclease encoded by the rng gene, plays an active role in the maturation of the 5′-end of 16S rRNA, its involvement in the maturation of the 5′-end of 23S rRNA remains unclear. Here we show that E. coli cells deleted for the rng gene accumulate the 23S rRNA precursor containing an extra 77 nucleotides at its mature 5′-end. In vitro cleavage assays show that RNase G cleaves synthetic RNA containing a sequence encompassing the 5′-end to 77 nucleotides upstream of mature 23S rRNA at two sites present in single-stranded regions. Our results suggest the involvement of RNase G in the processing of the 5′-region of 23S rRNA precursors.

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  • Bacterial ribonucleases and their roles in RNA metabolism
    David H. Bechhofer, Murray P. Deutscher
    Critical Reviews in Biochemistry and Molecular Biology.2019; 54(3): 242.     CrossRef
  • Intracellular ribonucleases involved in transcript processing and decay: Precision tools for RNA
    Cecília Maria Arraiano, Fabienne Mauxion, Sandra Cristina Viegas, Rute Gonçalves Matos, Bertrand Séraphin
    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms.2013; 1829(6-7): 491.     CrossRef
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    Ashley D. Frazier, W. Scott Champney
    Current Microbiology.2013; 67(2): 226.     CrossRef
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    Ashley D. Frazier, W. S. Champney
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Providencia Isolates Carrying blaPER-1 and blaVIM-2 Genes: Biofilm-Forming Capacity and Biofilm Inhibitory Concentrations for Carbapenem Antibiotics
Jungmin Kim , Shukho Kim , Hee Woo Lee , Sung Min Kim , Sung Yong Seol
J. Microbiol. 2011;49(3):512-515.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-1221-z
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AbstractAbstract PDF
Multidrug-resistant clinical isolates of Providencia carrying blaPER-1 and blaVIM-2 were evaluated for the abilities to form biofilm and high biofilm forming capacity was demonstrated in them. Minimum biofilm inhibitory concentrations (MBICs), minimum biofilm eradication concentrations (MBECs), and minimum inhibitory concentrations (MICs) for imipenem and meropenem were also determined. In all tested strains, the MBICs were higher than the MICs for both drugs. Interestingly, the MBICs and the MBEC50 for meropenem were lower than those for imipenem in the isolates producing high amounts of biofilm, suggesting that meropenem is superior to imipenem in the growth inhibition and eradication of biofilm forming Providencia strains.
Packaging of Porcine Reproductive and Respiratory Syndrome Virus Replicon RNA by a Stable Cell Line Expressing Its Nucleocapsid Protein
Byung-Hak Song , Jeong-Min Kim , Jin-Kyoung Kim , Han-Saem Jang , Gil-Nam Yun , Eun-Jin Choi , Jae-Young Song , Sang-Im Yun , Young-Min Lee
J. Microbiol. 2011;49(3):516-523.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-1280-1
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AbstractAbstract PDF
Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, is one of the most common and economically important swine pathogens. Although both live-attenuated and killed-inactivated vaccines against the virus have been available for a decade, PRRSV is still a major problem in the swine industry worldwide. To explore the possibility of producing single-round infectious PRRSV replicon particles as a potential vaccine strategy, we have now generated two necessary components: 1) a stable cell line (BHK/Sinrep19/PRRSV-N) that constitutively expresses the viral nucleocapsid (N) protein localized to the cytoplasm and the nucleolus and 2) a PRRSV replicon vector (pBAC/PRRSV/Replicon-ΔN) with a 177-nucleotide deletion, removing the 3′-half portion of ORF7 in the viral genome, from which the self-replicating propagation-defective replicon RNAs were synthesized in vitro by SP6 polymerase run-off transcription. Transfection of this replicon RNA into N protein-expressing BHK-21 cells led to the secretion of infectious particles that packaged the replicon RNA, albeit with a low production efficiency of 0.4×102 to 1.1×102 infectious units/ml; the produced particles had only single-round infectivity with no cell-to-cell spread. This trans-complementation system for PRRSV provides a useful platform for studies to define the packaging signals and motifs present within the viral genome and N protein, respectively, and to develop viral replicon-based antiviral vaccines that will stop the infection and spread of this pathogen.

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  • Research progress on the N protein of porcine reproductive and respiratory syndrome virus
    Yajie Zheng, Gan Li, Qin Luo, Huiyang Sha, Hang Zhang, Ruining Wang, Weili Kong, Jiedan Liao, Mengmeng Zhao
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