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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 10, Issue 6 - Dec 2000
Volume 10, Issue 5 - Oct 2000
Volume 10, Issue 4 - Aug 2000
Volume 10, Issue 3 - Jun 2000
Volume 10, Issue 2 - Apr 2000
Volume 10, Issue 1 - Feb 2000
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New Enzymes Acting on Peptides Containing D-Amino Acids: Their Properties and Application
Asano, Yasuhisa ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 573~579
Knowledge on the enzymes acting on p-amino-acid-containing peptides appears to be somewhat limited when compared with those acting on peptides composed on L-amino acids. Less than ten D-stereospecific enzymes are hitherto known. This review describes about several novel D-stereospecific peptidases and amidases of microbial origin, including D-aminopeptidase (E.C. 220.127.116.11), alkaline D-peptidase, and D-amino aicd amidase, which are applied to the synthesis of D-amino acid/or D-amino acid derivatives.
Purification and Characterization of NADH-Dependent Cr(VI) Reductase from Escherichia coli ATCD33456
Bae, Woo-Chul ; Kang, Tae-Gu ; Jung, Jae-Han ; Park, Chul-Jae ; Choi, Sung-Chan ; Jeong, Byeong-Chul ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 580~586
A soluble Cr(VI) reductase was purified from the Cr(VI) reducing strain Escherichia coli ATCC33456 by ammonium sulfate fractionation, and chromatographies on Q-Sepharose FF, Cibacron blue 3GA dye affinity, Mono-Q 5/5, and Superdex 200 HR 10/30 columns. The estimated molecular mass of the purified enzyme was 27 kDa on SDS-polyacrylamide gel electrophoresis and 54 kDa on gel filtration, thus indicating a dimeric structure. The isoelectric point of the enzyme was pH 4.85. The optimum reaction pH and storage pH were both 7.0, the optimum reaction temperature was
, and the storage temperature was
. NADH and NADPH both served as electron donors for the reductase, with
Cr(VI)/min/mg protein and Km of 7.6
M using HADH, and Vmax of 42.3
Cr(VI)/min/mg protein and Km of 14.6
using NADPH. When 1 mM EDTA was added, the Cr(VI) reducing activity increased 4-fold.
Production and Characterization of Monoclonal Antibodies to Glutamate Dehydrogenase from Thermophile Sulfolobus solfataricus
Cho, Sung-Woo ; Ahn, Jee-Yin ; Bahn, Jae-Hoon ; Jeon, Seong-Gyu ; Park, Jin-Seu ; Lee, Kil-Soo ; Choi, Soo-Young ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 587~594
Monoclonal antibodies against glutamate dehydrogenase (GDH) from Sulfolobus solfataricus were produced and characterized using epitope mapping and biosensor technology, Five monoclonal antibodies raised against S. solfataricus GDH were each identified as a single protein band that comigrated with purified S. solfataricus GDH on the SDS-polyacrylamide gel electrophoresis and immunoblot. Epitope mapping analysis showed that only one subgroup among the antibodies tested recognized the same peptide fragments of GDH. Using the anti-S. solfataricus GDH antibodies as probes, the cross-reactivities of GDHs from various sources were investigated and it was found that the mammalian GDH is not immunologically related to S. solfataricus GDH. The structural differences between the microbial and mammalian GDHs were further investigated using biosensor technology (Pharmacia BIAcore) and monoclonal antibodies against S. solfataricus and bovine brain. The binding affinity of S. solfataricus glutamate dehydrogenase anti-S. solfataricus for GDH (
=11 nM) was much tighter than that of anti-bovine for GDH (
=450 nM). These results, together with the epitope mapping analysis, suggest that there may be structural differences between the two GDH species, in addition to their different biochemical properties.
A Detection Kit for Aeromonas hydrophila Using Antibody Sensitized Latex
Shin, En-Joo ; Lee, Soon-Deuk ; Lee, Kyung-Won ; Lee, Yeon-Hee ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 595~598
Aeromonas hydrophila is a pathogen to fish as well as human. It is a food-borne disease, and causes severe mortality in fish, and sometimes severe septicemia in human. In this study, a rapid detection method using latex agglutination has been developed for A. hydrophila. Polyclonal antibodies were raised against membrane and whole cells of three isolates from rainbow trout. Among these, latex particles coated with antibodies raised against whole cells of isolate No. 2 showed the best sensitivity. With latex particles coated with this antibody, we could detect
CFU of A. hydrophila in 5 min. The cross-reactivity with bacteria constituting the normal intestinal microflora and other pathogens for rainbow trout was insignificant. This latex agglutination assay method produced positive reaction with all clinical isolates of A. hydrophila which were identified by species-specific PCR for 16S rRNA in A. hydrophila.
Lipopolysaccharide Synergizes with Interferon-
to Induce Expression of Mig mRNA in Mouse Peritoneal Macrophages
Kim, Young-Ho ; Kim, Hee-Sun ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 599~605
Lipopolysaccharide (LPS) is responsible for the tissue injury that occurs following the invasion of multicelluar organisms by Gram-negative microbes. The effect of LPS on IFN-
-induced chemokine Mig gene expression in mouse peritoneal macrophages was investigated. Very little Mig mRNA was detectable upon exposure to LPS without IFN-
. Although LPS alone is only minimally effective, LPS plus IFN-
synergized to produce a high level of Mig mRNA in the peritoneal macrophages. This synergy was not dependent on a new protein synthesis, and was not controlled at the level of the gene transcription. Futhermore, LPS did not increase IFN-
-induced Mig mRNA stability. Accordingly, it is suggested the LPS may synergize the expression of IFN-
-induced Mig mRNA through a process that depends on a pretranscriptional level or concurrent Mig mRNA translation.
Effects of the Myosin ATPase Inhibitor, 2,3-Butanedione-2-Monoxime, on Growth and Dimorphic Switches of Candida albicans
Woo, Mi-Young ; Jwa, Mi-Ri ; Kim, Jin-Mi ; Song, Ki-Won ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 606~611
Dimorphic yeast Candida albicans reversibly switches between the form of yeast and hyphae depending on external conditions. We investigated possible roles of the myosin family in the growth and dimorphic switches of C. albicans with a general myosin ATPase inhibitor, 2,3-butanedione-2-monoxime (BDM). Transition to hyphae as well as proliferation by budding was completely inhibited by BDM at 16 mM. Presence of 16 mM BDM did not affect hyphae-to-bud transition but it blocked budding. The effects of BDM on yeast growth and dimorphic switches were reversible. More than 70% of the BDM-treated cells demonstrated defects in the amount and the polarized localization of F-actin as well as in the shape and migration of the nucleus, suggesting that myosin activities are needed in these cellular processes of C. albicans.
Overexpression and Characterization of Vibrio mimicus Metalloprotease
Shin, Seung-Yeol ; Lee, Jong-Hee ; Huh, Sung-Hoi ; Park, Young-Seo ; Kim, Jin-Man ; Kong, In-Soo ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 612~619
To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with
for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.
Analysis of Genetic Polymorphisms of Epstein-Barr Virus Isolates from Cancer Patients and Healthy Carriers
Cho, Sung-Gyu ; Lee, Won-Keun ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 620~627
To determine the prevalence of genetic polymorphisms in Epstein-Barr virus (EBV) strains in the Korean population, the restriction site polymorphisms for BamHI and XhoI enzymes were analyzed with 16 EBV isolates from cancer patients and 7 EBV isolates from healthy carriers, using polymerase chain reaction techniques. None of the 23 isolates were found to carry an extra BamHI site in the BamHI F-fragment (f-variant). Of the 12 type-1 isolates from the cancer patients, 10 lost both the LMP1 XhoI site and the BamHI site between the BamHi W1* and I1* fragments (a W1*I1* fusion variant or type C). The latter W1*I1* fusion variant was due to a mutation of thymidine to adenine, as evidenced by a sequence analysis. The remaining two type-1 isolates showed either no variation at both sites or the loss of only the XhoI site. In contrast, two type-2 isolates and two intertypic recombinants with a type-1 allele at the EBNA2 locus and type-2 alleles at all or some of the EBNA3 loci retained both enzyme sites. In similar analyses of the 7 isolates from the healthy carriers, five of six type-1 isolates lost these two sites, however, one type-2 isolate did not. These results clearly indicate a strong association of both the LMP1 XhoI site loss and the W1*I1* fusion variant with the type-1 rather than the type-2 EBV strains circulating in the immunocompetent Korean carriers.
Increased Poly(3-Hydroxybutyrate) Accumulation in Recombinant Escherichia coli from Whey by Agitation Speed Control
Kim, Beom-Soo ; Brian K. O'Neill ; Lee, Sang-Yup ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 628~631
The timing of poly(3-Hydroxybutyrate) (PHB) biosynthesis was controlled by varying the agitation speed of a stirred tank fermentor during the pH-stat fed-batch culture of recombinant Escherichia coli strain GCSC 6576 harboring pSYL107. Using a concentrated whey solution containing ca. 200 g/l lactose as the nutrient feed, the PHB content was only 57% after 35h due to volumetric limitation of the fermentor. However, by limiting the oxygen by maintaining the agitation speed at 300 rpm, the final PHB content increased to 70% after 70h with a cell concentration of 15 g/l. When the agitation speed was increased up to 500 rpm, a cell concentration of 31 g/l with 80% PHB was obtained after 52h. A further increase in the maximum agitation speed increased the cell concentration, PHB concentration, and PHB productivity, however, the PHB content decreased to 56-58%.
Refolding of Bacillus macerans Cyclodextrin Glucanotransferase Expressed as Inclusion Bodies in Recombinant Escherichia coli
Kim, Chung-Im ; Kim, Myoung-Dong ; Park, Yong-Cheol ; Han, Nam-Soo ; Seo, Jin-Ho ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 632~637
This research was undertaken to restore the biological activity of cyclodextrin glucanotransferase (CGTase) of Bacillus macerans origin expressed as inclusion bodies in recombinant Escherichia coli. The optimum concentration of urea used as a denaturant was 8 M. The supplementation of 0.5 M urea into a dialysis buffer increased the refolding efficiency by preventing any protein aggregation. The influence of the protein concentration, temperature, and pH were also investigated. The protein concentration was found to be the most important factor in the refolding efficiency. The optimum temperature was 15-
and the optimum pH was 6.0. The maximum specific activity of the CGTase refolded under the optimum conditions was 92.2 U/mg, corresponding to 72% of the native CGTase. A comparison of the secondary structure between the native and the refolded CGTase showed that the relative ratio of the
-helix content in the native to the refolded CGTase was 1:0.82.
Purification and Characterization of a Thermostable
from Thermus caldophilus GK24
Yoo, Jin-Sang ; Han, Ki-Woong ; Kim, Hyun-Kyu ; Kim, Min-Hong ; Kwon, Suk-Tae ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 638~642
-D-glucosidase activities has been purified from Thermus caldophilus GK24. The enzyme was monomeric with a molecular mass of 49 kDa, as evidenced by SDS-PAGE. The
values for p-nitrophenyl
(p-NPGal), and p-nitrophenyl
(p-NPGlu) were 0.23 mM, 6.25 mM, and 0.28 mM, respectively. The enzyme showed optimal pH ranging between 5.5-6.5 and maximum temperature in the range of
for all the above mentioned activities. The half-life of the enzyme in sodium phosphate buffer (pH 6.0) at
was approximately 7 h. The p-NPGal hydrolyzing activity of Tca
was strongly activated by L-histidine, while the p-NPFuc and p-NPGlu hydrolyzing activities of Tca
were not affected at all by the amino acid. These results suggest differences in the conformation or in the reactive residues at the active site of Tca
Physiological and Phylogenetic Analysis of Burkholderia sp. HY1 Capable of Aniline Degradation
Kahng, Hyung-Yeel ; Jerome J. Kukor ; Oh, Kye-Heon ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 643~650
A new aniline-utilizing microorganism, strain HY1 obtained from an orchard soil, was characterized by using the BIOLOG system, an analysis of the total cellular fatty acids, and a 16S rDNA sequence. Strain HY1 was identified as a Burkholderia species, and was designated Burkholderia sp. HY1. GC and HPLC analyses revealed that Burkholderia sp. HY1 was able to degrade aniline to produce catechol, which was subsequently converted to cis,cis-muconic acid through an ortho-ring fission pathway under aerobic conditions. Strain HY1 exhibited a drastic reduction in the rate of aniline degradation when glucose was added to the aniline media. However, the addition of peptone or nitrate to the aniline media dramatically accelerated the rate of aniline degradation. A fatty acid analysis showed that strain HY1 was able to produce lipids 16:0 2OH, and 11 methyl 18:1
approximately 3.7-, 2.2-, and 6-fold more, respectively, when grown on aniline media than when grown on TSA. An analysison the alignment of a 1,435 bp fragment. A phylogenetic analysis of the 16S rDNA sequence based on a 1,420 bp multi-alignment sowed of the 16s rDNA sequence revealed that strain HY1 was very closely related to Burkholderia graminis with 95% similarity based that strain HY1 was placed among three major clonal types of
-Proteobacteria, including Burkholderia graminis, Burkholderia phenazinium, and Burkholderia glathei. The sequence GAT(C or G)
, which is highly conserved in several locations in the 16S rDNA gene among the major clonal type strains of
-Proteobacteria, was frequently replaced with GAT(C or G)
in the 16S rDNA sequence from strain HY1.
Rapid and Accurate Species-Specific Detection of Phytophthora infestans Through Analysis of ITS Regions in Its rDNA
Kim, Kyoung-Su ; Lee, Youn-Su ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 651~655
Polymerase chain reaction (PCR) was used to specifically detect Phytophthora infestans by analyzing the sequences of the ribosomal internal transcribed spacer regions (ITS) in the rDNA of the Phytophthora species. Based on the sequence data, PISP-1 together with the ITS3 primer were used to detect p. infestans. A single ca. 450 bp segment was observed in P. infestans, but not in the other fungal or bacterial isolates. Two factors, the annealing temperature and template DNA quantity, were investigated to determine the optimal conditions. Using these species-specific primers, a unique band was obtained within annealing temperatures of
and template DNA levels of 10 pg-100 ng.
Staphylococcus haemolyticus Lipase; High-Level Expression in Escherichia coli and Activation of Nonionic Detergent
Oh, Byung-Chul ; Kim, Hyung-Kwoun ; Kim, Myung-Hee ; Lee, Jung-Kee ; Oh, Tae-Kwang ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 656~662
A high level of Staphylococcus haemolyticus L62 lipase was expressed in an Escherichia coli transformant. The expressed lipase activity in the cell-free extract was 70,800 U/l, which corresponded to 30% of the total cellular protein. Pre-mixing of the l62 lipase with some nonionic detergents enhanced its hydrolytic activity towards olive oil: Tween detergents activated the L62 lipase by 3 fold. Gel filtration chromatography of the Tween-80-L62 lipase mixture demonstrated a polymerized complex (∼180 kDa) formed exclusively between Tween-80 and the L62 lipase. The lipase enzyme in the complex showed a higher specific activity towards most triacylglycerols than the intact L62 lipase. The activity enhancement towards each substrate was quite different depending on the acyl chain length; the activity towards tributyrin, trilinolein, and trilinolenin was much more enhanced than the towards the medium and the long-chain saturated triglycerides.
Construction of a New Gene-Fusion Expression Vector, pMONSTER
Baek, Chang-Ho ; Wee, Sec-Han ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 663~669
The fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion expression vector, pMONSTER, was then used to make fusion proteins with
-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur
-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-
-D-galactoside plate and the resulting 133 kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29 kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified Fur-HIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.
Purification and Characterization of Two Alkaline Proteases Produced by Pseudomonas sp. BK7
이은구 ; 박은희 ; 현형환 ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 667~667
Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enganced by the increase of agitation speed. Two formas of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The
value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0 and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both protease were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were 50℃ and 45℃, respectively. About 56% of the original protease BK7-2 activity remained after being treated at 50℃ for 30 min but protease BK7-1 was rapidly inactivated at above 25℃. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.
Molecular Characterization of crp, the Cyclic AMP Receptor Protein Gene of Serratia marcescens KTCC 1272
Yoo, Ju-Soon ; Kim, Hae-Sun ; Chung, Soo-Yeol ; Choi, Yong-Lark ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 670~676
Several clones obtained from Serratia marcescens stimulated E. coli TP2139 (
) cells to use maltose as a carbon source. The crp gene clone, pCKB12, was confirmed to stimulate the
-galactosidase activity, by Southern hybridization . The nucleotide sequence of the crp region consisting of 1,979 bp was determined. The sequencing of the fragment led to the identification of two open reading frames: One of these, the crp gene, encoded 210 amino acid and the other encoded a truncated protein. The S. marcescens and E. coli crp genes showed a higher degree of divergence in their nucleotide sequence with 120 changes, however, the corresponding amino acid sequences showed only two amino acid differences. Yet, an analysis of the amino acid divergence revealed that the catabolite gene activator protein, the crp gene product, was the most conserved protein observed so far. Using a crp-lac protein fusion, it was demonstrated that S. marcescens CRP could repress its own expression, probably via a mechanism similar to that previously described for the E. coli crp gene.
Purification and Characterization of Two Alkaline Protease Produced by Pseudomonas sp. BK7
Lee, Eun-Goo ; Park, Eun-Hee ; Hyun, Hyung-Hwan ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 677~684
Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The
value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were
, respectively. About 56% of the original protease BK7-2 activity remained after being treated at
for 30 min but protease BK7-1 was rapidly inactivated at above
. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.
A New and Rapid Testing Method for Drug Susceptibility of Mycobacterium leprae Using RT-PCR
Kim, Min-Joo ; Lee, Ju-Hang ; You, Ji-Chang ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 685~689
Due to the uncultivable nature of Mycobacterium leprae in vitro, the fast, easy, and accurate measurement of the antimicrobial drug susceptibility of this microbe has been difficult. Conventional methods for such testing are subjective, cumbersome, and expensive in some cases. Here, the utility of a reverse transcriptase-PCR (RT-PCR)-based assay for testing was examined and compared with a Buddmeyer-type radiorespirometric assay. The susceptibility of M. leprae to rifampin was determined by probing the presence of M.leprae-specific 18 kDa gene mRNA in M. leprae-infected IC-21 macrophage cells after drug treatment. The results showed that, as the refampin concentration was increased, the 360-bp cDNA products generated by the RT-PCR-based assay decreased in a dose-dependent manner as in the drug susceptibility observed in the Buddmeyer-type assay. The drug susceptibility testing of M. leprae by the RT-PCR based assay was found to be not only faster but also nearly
-fold more sensitive than the Buddmeyer-type assay. Moreover, it was also found that, unlike the RT-PCR based assay, the same testing by a DNA-PCR resulted in no differences in the 360-bp signal, regardless of the rifampin concentrations used. Accordingly, these results demonstrated that the drug susceptibility of M. leprae can be determined effectively by an RT-PCR-based assay, thereby providing a new, fast, and sensitive testing method.
Recombinant Human Proinsulin: A New Approach in Gene Assembly and Protein Expression
Mergulaho, Filipe J.M. ; Monteiro, Gabriel A. ; Kelly, Andrew G. ; Taipa, Maria A. ; Joaquim, M.S. Cabral ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 690~693
Efficient intron deletion with the correct splicing of the two exons of the human proinsulin gene was accomplished by a novel stepwise method using genomic DNA . The two exons were separately amplified in two steps, using the second step primers that incorporated additional bases complementary to the other exon. The fragments were combined in a third PCR reaction. Cloning and sequencing of the PCR product demonstrated the correct splicing of the two exons. Expression studies, using the pET9a vector, revealed a protein band with the correct size with respect to human proinsulin as confirmed by SDS-PAGe and Western blot. Proinsulin concentration was estimated to be around 200 mg per liter culture, expressed as inclusion bodies. Protein secretion to the culture medium and periplasmic space was achieved by cloning in the pEZZ18 vector.
Nutrient Amendments Influence Endophytic Colonization of Rice by Serratia marcescens IRBG500 and Herbaspirillum seropedicae Z67
Gyaneshwar, P. ; Reddy, Pallavolu M. ; Ladha, Jagdish K. ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 694~699
Serratia marcescens IRBG500 and Herbaspirillum seropedicae Z67 grow endophytically in rice. The ability of these bacteria to colonize rice grown under increased nutrient availability was assessed in variety IR72 using strains marked with transposon-based gusA. The endophytic colonization was monitored via bacterial enumeration and histochemical visualization of GUS expression of bacteria in plant tissues. Rhizoplane and endophytic colonization by both bacteria was significantly inhibited in the rice plants grown in the presence of 10 mM
. In contrast, the addition of 10 mM
showed no adverse effect on colonization. Increasing the concentration of
to 5 mM significantly reduced endophytic colonization by both bacterial strains, whereas the addition of 0.5 mM
substantially lowered the colonization of roots by S. marcescens IRBG500 but showed no effect on colonization by H. seropedicae Z67. Taken together, these finding suggest that, like in legume-rhizobial symbiosis as well as plant-pathogen interactions, nutrient status, particularly
concentrations in the surrounding medium, plays an important role in the regulation of endophytic infection and colonization processes in rice.
Cloning, DNA Sequence Determination, and Analysis of Growth-Associated Expression of the sodF Gene Coding for Fe- and Zn-Containing Superoxide Dismutase of Streptomyces griseus
Kim, Ju-Sim ; Lee, Jeong-Kug ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 700~706
Iron- and zinc-containing superoxide dismutase (FeZnSOD) and nickel-containing superoxide dismutase (NiSOD) are cytoplamic enzymes in Streptomyces griseus. The sodF gene coding for FeZnSOD was cloned from genomic Southern hybridization analysis with a 0.5-kb DNA probe, which was PCR-amplified with facing primers corresponding to the N-terminal amino acid of the purified FeZnSOD of S. griseus and a C-terminal region which is conserved among bacterial FeSODs and MnSODs. The sodF open reading frame (ORF) was comprised of 213 amino acid (22,430 Da), and the deduced sequence of the protein was highly homologous (86% identity) to that of FeZnSOD of Streptomyces coelicolor. The FeZnSOD expression of exponentially growing S. griseus cell was approximately doubled as the cell growth reached the early stationary phase. The growth-associated expression of FeZnSOD was mainly controlled at the transcriptional level, and the regulation was exerted through the 110 bp regulatory DNA upstream from the ATG initiation codon of the sodF gene.
In vitro Selection of the 2'-Fluoro-2'-Deoxyribonucleotide Decoy RNA Inhibitor of Myasthenic Autoantibodies
Seo, Hwa-Seon ; Lee, Seong-Wook ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 707~713
Myasthenia gravis (MG) is caused mainly by autoantibodies directed against acetylcholine receptors located in the postsynaptic muscle cell membrane. Using in vitro selection techniques, we isolated an RNA containing 2'-fluoro pyrimidines that can specifically and avidly (
~25 nM) bind rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the acetylcholine receptors. This RNA can act as a very effective decoy and block mAb198 binding to the receptors in vitro. Furthermore, this RNA decoy can prevent the antigenic modulation of the acetylcholine receptor caused by mAb198 in human muscle cell cultures with and
. These results indicate that the RNA selected in this study is a more potent decly inhibitor of myashthenic antibodies than the previously identified RNA with 2'-amino pyrimidines . Moreover, this RNA cross-reacts with autoantibodies from patients with MG and can protect human cells from the effects of these antibodies. These observations have important implications for developing an antigen-specific treatment of autoimmune diseases including MG, which is based on decoy RNAs selected in vitro.
Immunoelectron Microscopic Localization and Analysis of Herpes simplex Virus Type 1 Antigens
Chung, Charles C. ; Lee, Hyung-Hoan ; Cho, Myung-Hwan ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 714~720
Antigens of Herpes simplex virus type 1 (HSV-1) strain F were immunoblotted to identify the most immunodominant one, and the localization of this antigen was then studied using immunoelectron microscopy. The 67.8 kDa antigen appeared to be the most immunodominant one in a mouse model, and it showed randomly scattered and partially clustered distribution on the surface of the virion. The localization study was performed using immunogold with polyclonal anti-HSV-1 sera produced from BALB/c mice, and immunofluorescence demonstrated that the viral products in the HSV-2 infected Vero cells were distributed throughout the infected host cell, however, mainly on the surface of the host membrane.
-Catalyzed Transesterification of Phosphatidylcholine with Nervonic Acid in Organic Solvent
Park, Chang-Won ; Park, Ki-Won ; Han, Jeong-Jun ; Chung, Guk-Hoon ; Rhee, Joon-Shick ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 721~723
-catalyzed transesterification of phosphatidylcholine (PC, 95%) with nervonic acid (NA, 95%) was successfully carried out in an organic solvent. The maximum yield after 48 h was 10.3% (w/w) at
with an initial water activity (
) of 0.16, and a molar ratio of NA to PC of 20 in 5 ml ethyl acetate.
Degradation of Phenanthrene by Sphingomonas sp. 1-21 Isolated from Oil-Contaminated Soil
Ryeom, Tai-Kyung ; Lee, Il-Gyu ; Son, Seung-Yeol ; Ahn, Tae-Young ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 724~727
A Phenanthrene-degrading bacterium, Strain 1-21 was isolated from oil-contaminated soil. This strain was a Gram-negative, aerobic, and rod-shaped bacterium, and exhibited a 99% sequence similarity of 16S rDNA to that of Sphingomonas subarctica. The major cellular fatty acid was a summed feature 7(18:1 w7c, 18:1 w9t, 18:1 s12t), which is a characteristic of the Sphingomonas species. When 200 and 1,000 ppm of phenanthrene was added as the sole carbon source, Strain 1-21 degraded 98% and 67% after 10 days of incubation, respectively. Futhermore, this strain was also able to utilized naphthalene and fluorene as sole carbon and energy sources.
Optimization of Propagation of Anagrapha falcifera Nuclear Polyhedrosis Virus in Spodoptera Frugiperda 21 Cells
Lee, Jong-Min ; Chang, Kyung-Hwa ; Park, Jin-O ; Park, Jong-Hwa ; Hwang, In-Sook ; Lee, Youn-Hyung ; Yang, Jai-Myung ; Chung, In-Sik ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 728~732
Propagation of Anagrapha falcifera nuclear polyhedrosis virus(AfNPV) was investigated using well-plates and split-flow air-lift bioreactors. In well-plate experiments, the effects of pH, cell density at a point of infection, serum concentration, DEAE-dextran, and lipid on virus propagation were all closely examined. The AfNPV titer in well-plates was optimal at pH 6.8 and
. The virus titer was not dramatically affected when the fetal bovine serum concentration was reduced from 10% to 5%. The addition of cholesterol at AfNPV infection of Sf21 cells enhanced the virus titer, whereas the addition of DEAE-dextran did not improve the titer. The AfNPV titer (
) at optimized conditions for well-plate experiments was 2.5-fold higher than for the control. In bioreactor experiments, the AfNPV titer showed its maximum level at air flow rates of 20-40 ml/min. In a split-flow air-lift bioreactor, AfNPV titer (
) was 1.5-fold higher than the control when the culture was at pH 6.8 and supplemented with 0.34 mM cholesterol.
Comparison of the Solution Structure of Vancomycin with Its X-ray Crystallographic Structure
Lee, Chul-Hoon ; Kyung, Han-Soo ; Lim, Yoong-Ho ;
Journal of Microbiology and Biotechnology, volume 10, issue 5, 2000, Pages 733~736
Since pathogens resistant against vancomycin occur rapidly, the development of a new drug is needed. To make a new drug based on a rational drug design, the structural study of vancomycin is necessary. Accordingly, this study reports on a comparison of the solution structure of vancomycin determined by NMR spectroscopy, which was performed in the present work, with the X-ray crystallographic structure previously deposited in the Protein Data Bank (PDB).