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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal of Microbiology and Biotechnology
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Journal DOI :
The Korean Society for Applied Microbiology and Biotechnology
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Volume & Issues
Volume 19, Issue 12 - Dec 2009
Volume 19, Issue 11 - Nov 2009
Volume 19, Issue 10 - Oct 2009
Volume 19, Issue 9 - Sep 2009
Volume 19, Issue 8 - Aug 2009
Volume 19, Issue 7 - Jul 2009
Volume 19, Issue 6 - Jun 2009
Volume 19, Issue 5 - May 2009
Volume 19, Issue 4 - Apr 2009
Volume 19, Issue 3 - Mar 2009
Volume 19, Issue 2 - Feb 2009
Volume 19, Issue 1 - Jan 2009
Selecting the target year
Applications of Transposon-Based Gene Delivery System in Bacteria
Choi, Kyoung-Hee ; Kim, Kang-Ju ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 217~228
DOI : 10.4014/jmb.0811.669
Mobile genetic segments, or transposons, are also referred to as jumping genes as they can shift from one position in the genome to another, thus inducing a chromosomal mutation. According to the target site-specificity of the transposon during a transposition event, the result is either the insertion of a gene of interest at a specific chromosomal site, or the creation of knockout mutants. The former situation includes the integration of conjugative transposons via site-specific recombination, several transposons preferring a target site of a conserved AT-rich sequence, and Tn7 being site-specifically inserted at attTn7, the downstream of the essential glmS gene. The latter situation is exploited for random mutagenesis in many prokaryotes, including IS (insertion sequence) elements, mariner, Mu, Tn3 derivatives (Tn4430 and Tn917), Tn5, modified Tn7, Tn10, Tn552, and Ty1, enabling a variety of genetic manipulations. Randomly inserted transposons have been previously employed for a variety of applications such as genetic footprinting, gene transcriptional and translational fusion, signature-tagged mutagenesis (STM), DNA or cDNA sequencing, transposon site hybridization (TraSH), and scanning linker mutagenesis (SLM). Therefore, transposon-mediated genetic engineering is a valuable discipline for the study of bacterial physiology and pathogenesis in living hosts.
Molecular Phylogeny and Modular Structure of Hybrid NRPS/PKS Gene Fragment of Pseudoalteromonas sp. NJ6-3-2 Isolated From Marine Sponge Hymeniacidon perleve
Zhu, Peng ; Zheng, Yanling ; You, Yurong ; Yan, Xiaojun ; Shao, Jianzhong ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 229~237
DOI : 10.4014/jmb.0804.282
Among 12 marine bacterial strains from the China coast that exhibited interesting bioactivity (positive for both antimicrobial and cytotoxic activities), only four strains, namely, NJ6-3-1, NJ6-3-2, NB-6, and YTHM-17, had a KS domain or A domain when screened for PKS and NRPS genes using a PCR. Interestingly, two of these strains belonging to Pseudoalteromonas and associated with the marine sponge Hymeniacidon perleve were positive for both PKS and NRPS, whereas the other two strains of Pseudoalteromonas did not have a PKS or NRPS gene. A molecular phylogeny analysis and DGGE analysis of the Pseudoalteromonas sp. indicated that they had a specific affinity with the host marine sponge Hymeniacidon perleve. Furthermore, an analysis of a partial sequence of Pseudoalteromonas sp. NJ6-3-2 isolated from the marine sponge Hymeniacidon perleve obtained from genomic walking using a computational approach indicated a relatively complete PKS module including auxiliary domains (DH, KR, and Cy).
Listeria monocytogenes Serovar 4a is a Possible Evolutionary Intermediate Between L. monocytogenes Serovars 1/2a and 4b and L. innocua
Chen, Jianshun ; Jiang, Lingli ; Chen, Xueyan ; Luo, Xiaokai ; Chen, Yang ; Yu, Ying ; Tian, Guoming ; Liu, Dongyou ; Fang, Weihuan ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 238~249
DOI : 10.4014/jmb.0805.304
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.
Acinetobacter antiviralis sp. nov., from Tobacco Plant Roots
Lee, Jung-Sook ; Lee, Keun-Chul ; Kim, Kwang-Kyu ; Hwang, In-Cheon ; Jang, Cheol ; Kim, Nam-Gyu ; Yeo, Woon-Hyung ; Kim, Beom-Seok ; Yu, Yong-Man ; Ahn, Jong-Seog ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 250~256
DOI : 10.4014/jmb.0901.083
was isolated from tobacco plant roots during the screening of antiviral substances having inhibitory effects on Tobacco mosaic virus (TMV) and examined by phenotypic, chemotaxonomic, and genetic characterization. It was a nonmotile, Gram-negative bacterium. This strain contained Q-9 as the main respiratory quinone. The major cellular fatty acids of the isolate were 16:0, 18:1 w9c, and 16:1 w7c/15 iso 2OH. The DNA base composition was 44 mol%. Phylogenetic analysis based on the 16S rRNA sequence revealed that the isolate formed an evolutionary lineage distinct from other Acinetobacter species. Based on the evaluation of morphologic, physiologic, and chemotaxonomic characteristics, DNA-DNA hybridization values, and 16S rRNA sequence comparison, we propose the new species Acinetobacter antiviralis sp. nov., the type strain of which is
Gene Cloning, Expression, and Characterization of a
-Agarase, AgaB34, from Agarivorans albus YKW-34
Fu, Xiao Ting ; Pan, Cheol-Ho ; Lin, Hong ; Kim, Sang-Moo ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 257~264
DOI : 10.4014/jmb.0801.026
-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli
as a host. The purified rAgaB34 was a
-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at
and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to
. Its specific activity and
value for agarose were 242 U/mg and
, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT,
-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.
Oligonucleotide Array-based Detection and Genotyping of Mollicutes (Acholeplasma, Mycoplasma, and Ureaplasma)
Jang, Hyun-Jung ; Kim, Hyo-Myeung ; Kang, Byeong-Chul ; Kim, Cheol-Min ; Park, Hee-Kyung ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 265~270
DOI : 10.4014/jmb.0806.347
An oligonucleotide array was developed to detect and genotype mollicutes based on the internal transcribed spacer (ITS) sequence. The results of the assay were compared with those of a PCR-RFLP assay. The proposed oligonucleotide array containing 5 genus- and 23 species-specific probes was able to detect Mycoplasma species, including M. penetrans and M. spermatophilum, that were not detected by the PCR-RFLP assay. Therefore, the results demonstrated that the proposed oligonucleotide array was effective for the detection and discrimination of 23 species, including an acholeplasma, 21 mycoplasmas, and a ureaplasma, and showed promise as a countermeasure to ensure that biological products are safe and of good quality.
Quantifiable Downregulation of Endogenous Genes in Agaricus bisporus Mediated by Expression of RNA Hairpins
Costa, Ana S.M.B. ; Thomas, D. John I. ; Eastwood, Daniel ; Cutler, Simon B. ; Bailey, Andy M. ; Foster, Gary D. ; Mills, Peter R. ; Challen, Michael P. ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 271~276
DOI : 10.4014/jmb.0806.398
Functional gene studies in the cultivated white button mushroom Agaricus bisporus have been constrained by the absence of effective gene-silencing tools. Using two endogenous genes from A. bisporus, we have tested the utility of dsRNA hairpin constructs to mediate downregulation of specific genes. Hairpin constructs for genes encoding orotidine 5'-monophosphate decarboxylase (URA3) and carboxin resistance (CBX) were introduced into A. bisporus using Agrobacteriummediated transfection. Although predicted changes in phenotype were not observed in vitro, quantitative-PCR analyses indicated unambiguously that transcripts in several transformants were substantially reduced compared with the non-transformed controls. Interestingly, some hairpin transformants exhibited increased transcription of target genes. Our observations show that hairpin transgenic sequences can mediate downregulation of A. bisporus endogenous genes and that the technology has the potential to expedite functional genomics of the mushroom.
Cloning, Sequencing, and Expression of the Gene Encoding a Multidomain Endo-
-1,4-Xylanase from Paenibacillus curdlanolyticus B-6, and Characterization of the Recombinant Enzyme
Waeonukul, Rattiya ; Pason, Patthra ; Kyu, Khin Lay ; Sakka, Kazuo ; Kosug, Akihiko ; Mori, Yutaka ; Ratanakhanokchai, Khanok ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 277~285
DOI : 10.4014/jmb.0804.293
The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-
-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel,
-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.
Rengyolone Inhibits Apoptosis via Etoposide-Induced Caspase Downregulation
Kim, Jin-Hee ; Lee, Choong-Hwan ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 286~290
DOI : 10.4014/jmb.0804.253
In the course of screening for substances inhibiting apoptosis of U937 human leukemia cells induced by etoposide (
), Forsythiae fructus, which showed a high level of inhibition, was selected. The regulating compounds were purified from the ethyl acetate extract by silica gel column chromatography and HPLC. The active substance was purified and identified as rengyolone by spectroscopic methods. This compound showed inhibitory activity on caspase-3 induction, a major protease of the apoptosis cascade, with an
after 8 h of etoposide treatment in U937 cells. The expression level of caspase-3 and poly(ADP-ribose) polymerase (PARP) were dose-dependently inhibited by the compound, suggesting that rengyolone inhibits etoposide-induced apoptosis via downregulation of caspases.
Effect of Initial Glucose Concentrations on Carbon and Energy Balances in Hydrogen-Producing Clostridium tyrobutyricum JM1
Jo, Ji-Hye ; Lee, Dae-Sung ; Kim, Jun-Hoon ; Park, Jong-Moon ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 291~298
DOI : 10.4014/jmb.0802.165
The carbon metabolism of newly isolated Clostridium tyrobutyricum JM1 was investigated at varying initial glucose concentrations (27.8-333.6mM). Because an understanding of metabolic regulations was required to provide guidance for further effective metabolic design or optimization, in this case, maximizing hydrogen production, carbon and energy balances by C. tyrobutyricum JM1 were determined and applied in anaerobic glucose metabolism. The overall carbon distribution suggested that initial glucose concentrations had strong influence on the stoichiometric coefficients of products and the molar production of ATP on the formation of biomass. C. tyrobutyricum JM1 had a high capacity for hydrogen production at the initial glucose concentration of 222.4 mM with high concentrations of acetate and butyrate.
Mechanism Analysis of Effect of Oxygen on Molecular Weight of Hyaluronic Acid Produced by Streptococcus zooepidemicus
Duan, Xu-Jie ; Niu, Hong-Xing ; Tan, Wen-Song ; Zhang, Xu ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 299~306
DOI : 10.4014/jmb.0801.073
Dissolved oxygen (DO) has a significant effect on the molecular weight of hyaluronic acid (HA) during the fermentation of Streptococcus zooepidemicus. Therefore, to further investigate the effect of DO on the yield and molecular weight of HA, this study compared the metabolic flux distribution of S. zooepidemicus under aerobic conditions at various DO levels. The metabolic flux analysis demonstrated that the HA synthesis pathway, considered a dependent network, was little affected by the DO level. In contrast, the fluxes of lactate and acetate were greatly influenced, and more ATP was generated concomitant with acetate at a high DO level. Furthermore, the has gene expression and HA synthase activity were both repressed under anaerobic conditions, yet not obviously affected under aerobic conditions at various DO levels. Therefore, it was concluded that the HA molecular weight would seem to depend on the concomitant effect of the generation of ATP and reactive oxygen species. It is expected that this work will contribute to a better understanding of the effect of the DO level on the mechanism of the elongation of HA chains.
Systemic Analysis of a Novel Coxsackievirus Gene Delivery System in a Mouse Model
Kim, Yeon-Jung ; Yun, Soo-Hyeon ; Lim, Byung-Kwan ; Park, Ki-Bum ; Na, Ha-Na ; Jeong, Soo-Young ; Kim, Dae-Sun ; Cho, Young-Joo ; Jeon, Eun-Seok ; Nam, Jae-Hwan ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 307~313
DOI : 10.4014/jmb.0803.237
In order to systemically investigate the possibility of using coxsackievirus B3 (CVB3) to deliver foreign genes in vivo, a recombinant strain of CVB3 encoding the renilla gene (CVB3-renilla) was constructed. The recombinant CVB3 resulted in extensive and transient expression of the renilla protein within mouse organs, especially the pancreas. The level of expression was generally dependent upon the viral titer present. Moreover, the CVB3-renilla strain was completely attenuated. Interestingly, the recombinant CVB3 vector was expressed much more strongly in mouse organs than was a comparable adenoviral vector. The CVB3-renilla strain did not express the renilla gene in mice with pre-existing coxsackievirus-specific neutralizing antibodies, but direct organ-specific administration of the virus during open-peritoneum surgery was able to circumvent this immunity. This coxsackievirus vector may represent a useful means for delivering and expressing foreign genes in mouse models in an acute and extensive fashion.
Identification of an Entomopathogenic Bacterium, Serratia sp. ANU101, and Its Hemolytic Activity
Kim, Yong-Gyun ; Kim, Keun-Seob ; Seo, Ji-Ae ; Shrestha, Sony ; Kim, Hosanna-H. ; Nalini, Madanagopal ; Yi, Young-Keun ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 314~322
DOI : 10.4014/jmb.0806.360
Four different bacterial colonies were isolated from an old stock of an entomopathogenic nematode, Steinernema monticolum. They all showed entomopathogenicity to final instar larvae of beet armyworm, Spodoptera exigua, by hemocoelic injection. However, they varied in colony form, susceptibility to antibiotics, and postmortem change of the infected host insects. Biolog microbial identification and 16S rDNA sequence analyses indicate that these are four different species classified into different bacterial genera. Owing to high entomopathogenicity and a cadaver color of infected insect host, Serratia sp. was selected as a main symbiotic bacterial species and analyzed for its pathogenicity. Although no virulence of Serratia sp. was detected at oral administration, the bacteria gave significant synergistic pathogenicity to fifth instar S. exigua when it was treated along with a spore-forming entomopathogenic bacterium, Bacillus thuringiensis. The synergistic effect was explained by an immunosuppressive effect of Serratia sp. by its high cytotoxic effect on hemocytes of S. exigua, because Serratia sp. caused septicemia of S. exigua when the bacterial cells were injected into S. exigua hemocoel. The cytotoxic factor(s) was present in the culture medium because the sterilized culture broth possessed high potency in the cytotoxicity, which was specific to granular cells and plasmatocytes, two main immune-associated hemocytes in insects.
Novel Diagnostic Algorithm Using tuf Gene Amplification and Restriction Fragment Length Polymorphism is Promising Tool for Identification of Nontuberculous Mycobacteria
Shin, Ji-Hyun ; Cho, Eun-Jin ; Lee, Jung-Yeon ; Yu, Jae-Yon ; Kang, Yeon-Ho ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 323~330
DOI : 10.4014/jmb.0804.267
Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infections in immunocompromised patients, making the reliable and rapid identification of NTM to the species level very important for the treatment of such patients. Therefore, this study evaluated the usefulness of the novel target genes tuf and tmRNA for the identification of NTM to the species level, using a PCRrestriction fragment length polymorphism analysis (PRA). A total of 44 reference strains and 17 clinical isolates of the genus Mycobacterium were used. The 741 bp or 744 bp tuf genes were amplified, restricted with two restriction enzymes (HaeIII/MboI), and sequenced. The tuf gene-PRA patterns were compared with those for the tmRNA (AvaII), hsp65 (HaeIII/HphI), rpoB (MspI/HaeIII), and 16S rRNA (HaeIII) genes. For the reference strains, the tuf gene-PRA yielded 43 HaeIII patterns, of which 35 (81.4%) showed unique patterns on the species level, whereas the tmRNA, hsp65, rpoB, and 16S rRNA-PRAs only showed 10 (23.3%), 32 (74.4%), 19 (44.2%), and 3 (7%) unique patterns after single digestion, respectively. The tuf gene-PRA produced a clear distinction between closely related NTM species, such as M. abscessus (557-84-58) and M. chelonae (477-84-80-58), and M. kansasii (141-136-80-63-58-54-51) and M. gastri (141-136-117-80-58-51). No difference was observed between the tuf-PRA patterns for the reference strains and clinical isolates. Thus, a diagnostic algorithm using a tuf gene-targeting PRA is a promising tool with more advantages than the previously used hsp65, rpoB, and 16S rRNA genes for the identification of NTM to the species level.
Development of an In Vitro Test System Measuring Transcriptional Downregulatory Activities on IL-13
Choi, Jeong-June ; Park, Bo-Kyung ; Park, Sun-Young ; Yun, Chi-Young ; Kim, Dong-Hee ; Kim, Jin-Sook ; Hwang, Eun-Sook ; Jin, Mi-Rim ;
Journal of Microbiology and Biotechnology, volume 19, issue 3, 2009, Pages 331~337
DOI : 10.4014/jmb.0806.358
Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a well-known antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.