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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal of Microbiology and Biotechnology
Journal Basic Information
Journal DOI :
The Korean Society for Applied Microbiology and Biotechnology
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Volume & Issues
Volume 21, Issue 12 - Dec 2011
Volume 21, Issue 11 - Nov 2011
Volume 21, Issue 10 - Oct 2011
Volume 21, Issue 9 - Sep 2011
Volume 21, Issue 8 - Aug 2011
Volume 21, Issue 7 - Jul 2011
Volume 21, Issue 6 - Jun 2011
Volume 21, Issue 5 - May 2011
Volume 21, Issue 4 - Apr 2011
Volume 21, Issue 3 - Mar 2011
Volume 21, Issue 2 - Feb 2011
Volume 21, Issue 1 - Jan 2011
Selecting the target year
Characterization of Two Metagenome-Derived Esterases That Reactivate Chloramphenicol by Counteracting Chloramphenicol Acetyltransferase
Tao, Weixin ; Lee, Myung-Hwan ; Yoon, Mi-Young ; Kim, Jin-Cheol ; Malhotra, Shweta ; Wu, Jing ; Hwang, Eul-Chul ; Lee, Seon-Woo ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1203~1210
DOI : 10.4014/jmb.1107.07034
Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (
) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3-acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at
, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.
Time-Dependent Hepatic Proteome Analysis in Lean and Diet-Induced Obese Mice
Oh, Tae-Seok ; Kwon, Eun-Young ; Choi, Jung-Won ; Choi, Myung-Sook ; Yun, Jong-Won ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1211~1227
DOI : 10.4014/jmb.1107.07056
C57BL/6J mice have been widely used as a diet-induced obesity model because they trigger common features of the human metabolic syndrome. In the present study, C57BL/6J male mice were fed either a high-fat diet (HFD) or normal diet (ND) during a 24-week period, and then the age-dependent liver proteome of mice in two groups was analyzed using 2-DE combined with MALDI-TOF-MS. Among identified proteins, up-regulated proteins were subdivided to early (during the first 4 weeks) and late (20~24 weeks) markers that played a role in diet-induced obesity development. Important early markers included ketohexokinase and prohibitin, and late markers included the 75 kDa glucose-regulated protein, citrate synthase, and selenium-binding liver protein. Of these, the 75 kDa glucosere-gulated protein has already been linked to obesity; however, prohibitin protein involved in obesity was identified for the first time in this study. In order to validate the proteomic results and gain insight into metabolic changes between the two groups, we further confirmed the expression pattern of some proteins of interest by Western blot analysis. Combined results of proteomic analysis with Western blot analysis revealed that antioxidant enzymes were progressively decreased, whereas cytoskeletal proteins were time-dependently increased in HFD mice.
Identification of Virulence Factors in Vibrio vulnificus by Comparative Transcriptomic Analyses between Clinical and Environmental Isolates Using cDNA Microarray
Kim, In-Hwang ; Kim, Byung-Soo ; Lee, Kyung-Shin ; Kim, Ik-Joong ; Son, Jee-Soo ; Kim, Kun-Soo ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1228~1235
DOI : 10.4014/jmb.1111.11016
We compared the gene expression among four clinical and five environmental V. vulnificus isolates, using a cDNA microarray containing 131 genes possibly associated with pathogenicity, transport, signal transduction, and gene regulations in the pathogen. cDNAs from total RNAs of these isolates were hybridized into the cDNA microarray using the cDNA of the wild-type strain MO6-24/O as a reference. We focused on selecting differentially expressed (DE) genes between clinical and environmental isolates using a modified t-statistic. We could detect two statistically significant DE genes between virulent isolates and less-virulent isolates with a marginal statistical significance (p-value of 0.008). These were genes putatively encoding pilin and adenlyate cylase. Real time-PCR confirmed that these two selected genes transcribed in significantly higher levels in virulent isolates than in less-virulent isolates. Mutants with lesions in the gene encoding pilin showed significantly higher
values than that of wild type.
Screening and Identification of Antimicrobial Compounds from Streptomyces bottropensis Suppressing Rice Bacterial Blight
Park, Sait-Byul ; Lee, In-Ae ; Suh, Joo-Won ; Kim, Jeong-Gu ; Lee, Choong-Hwan ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1236~1242
DOI : 10.4014/jmb.1106.06047
Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating pathogen to Oryza sativa and has been shown to cause bacterial blight. Two bioactive compounds showing antimicrobial activities against Xoo strain KACC 10331 were isolated from a Streptomyces bottropensis strain. The ethyl acetate extract was fractionated on a Sephadex LH-20 column, and then purified by preparative HPLC. The purified compounds were identified as bottromycin A2 and dunaimycin D3S by HR/MS and
NMR analyses. The MIC value against Xoo and the lowest concentration still capable of suppressing rice bacterial blight were 2
/ml and 16
/ml for bottromycin A2, and 64
/ml and 0.06
/ml for dunaimycin D3S, respectively. These two compounds were shown to exert different bioactivities in vitro and in rice leaf explants.
Antimicrobial Treatment of Grapes Using Sodium Hypochlorite in Winemaking and Its Effects on the Chemical and Sensory Characteristics of Wines
Yoo, Ki-Seon ; Ahn, Ji-Eun ; Han, Jin-Soo ; Seo, Eun-Young ; Otgonbayar, Gan-Erdene ; Han, Nam-Soo ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1243~1249
DOI : 10.4014/jmb.1105.05009
This study was performed to examine the use of NaOCl as an alternative antimicrobial compound in winemaking because of the potential health problems that may arise as a result of the use of
. For this, the blank (non-treated), control (
-added), and sample (NaOCl-treated) wines were made, and microbial and chemical changes including sensory characteristics were analyzed during the fermentation periods. Treatment of grapes with NaOCl decreased the initial contaminating microbial population in grape must, resulting in higher growth of yeast and lactic acid bacteria. After 200 days of fermentation, the chemical analysis of sample wine revealed that it had higher ethanol content, redness (
), and concentrations of fruity ester compounds and lower total acidity than the control. In the sensory analyses, the sample wine obtained a higher overall acceptability score (5.70) than the control (4.26). This result reveals that NaOCl can be used as an alternative to
in winemaking for inhibiting the growth of contaminating microorganisms.
Production of Alkaline Protease by Entrapped Bacillus licheniformis Cells in Repeated Batch Process
Mashhadi-Karim, Mohammad ; Azin, Mehrdad ; Gargari, Seyyed Latif Mousavi ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1250~1256
DOI : 10.4014/jmb.1105.05037
In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.
External and Internal Glucose Mass Transfers in Succinic Acid Fermentation with Stirred Bed of Immobilized Actinobacillus succinogenes under Substrate and Product Inhibitions
Galaction, Anca-Irina ; Rotaru, Roxana ; Kloetzer, Lenuta ; Vlysidis, Anestis ; Webb, Colin ; Turnea, Marius ; Cascaval, Dan ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1257~1263
DOI : 10.4014/jmb.1107.07024
This paper is dedicated to the study on the external and internal mass transfers of glucose for succinic acid fermentation under substrate and product inhibitions using a bioreactor with stirred bed of immobilized Actinobacillus succinogenes cells. By means of the substrate mass balance for a single particle of biocatalysts, considering the kinetic model adapted for both inhibitory effects, specific mathematical models were developed for describing the profiles of the substrate concentration in the outer and inner regions of biocatalysts and for estimating the substrate mass flows in the liquid boundary layer surrounding the particle and inside the particle. The values of the mass flows were significantly influenced by the internal diffusion velocity and rate of the biochemical reaction of substrate consumption. These cumulated influences led to the appearance of a biological inactive region near the particle center, its magnitude varying from 0 to 5.3% of the overall volume of particles.
Secretory Expression and Purification of the Recombinant Duck Interleukin-2 in Pichia pastoris
Du, Cuihong ; Han, Long ; Xiao, Anfeng ; Cao, Minjie ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1264~1269
DOI : 10.4014/jmb.1106.06057
Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular functions and immunity of animals. In this study, the recombinant duck IL-2 (rduIL-2) was secretory expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks and then scaled up in a 5.0-l bioreactor. The result showed that the maximal fresh-cell-weight of 594.1 g/l and the maximal
of 408 were achieved in the bioreactor. The rduIL-2 was purified by two steps of purification procedures, and approximately 311 mg of rduIL-2/L fermentation supernatant was obtained. SDS-PAGE showed that the purified rduIL-2 constituted a homogeneous band of ~16 kDa or ~14 kDa corresponding to the glycosylated or non-glycosylated duIL-2 protein in size, respectively. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay. The result indicated that the rduIL-2 greatly promoted the proliferation of ConA-stimulated lymphocytes in vitro. The P. pastoris expression system described here could provide promising, inexpensive, and large-scale production of the rduIL-2, which lays the foundation for development of novel immunoadjuvants to enhance both the immunity of ducks against various infectious pathogens and vaccine efficacy.
Molecular and Morphological Identification of Fungal Species Isolated from Bealmijang Meju
Kim, Ji-Yeun ; Yeo, Soo-Hwan ; Baek, Sung-Yeol ; Choi, Hye-Sun ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1270~1279
DOI : 10.4014/jmb.1105.05013
Bealmijang is a short-term aged paste made from meju, which is a brick of fermented soybeans and other ingredients. Different types of bealmijang are available depending on the geographic region or ingredients used. However, no study has clarified the microbial diversity of these types. We identified 17 and 14 fungal species from black soybean meju (BSM) and buckwheat meju (BWM), respectively, on the basis of morphology, culture characteristics, and internal transcribed spacer and
-tubulin gene sequencing. In both meju, Aspergillus oryzae, Rhizopus oryzae, Penicillium polonicum, P. steckii, Cladosporium tenuissimum, C. cladosporioides, C. uredinicola, and yeast species Pichia burtonii were commonly found. Moreover, A. flavus, A. niger, P. crustosum, P. citrinum, Eurotium niveoglaucum, Absidia corymbifera, Setomelanomma holmii, Cladosporium spp. and unclassified species were identified from BSM. A. clavatus, Mucor circinelloides, M. racemosus, P. brevicompactum, Davidiella tassiana, and Cladosporium spp. were isolated from BWM. Fast growing Zygomycetous fungi is considered important for the early stage of meju fermentation, and A. oryae and A. niger might play a pivotal role in meju fermentation owing to their excellent enzyme productive activities. It is supposed that Penicillium sp. and Pichia burtonii could contribute to the flavor of the final food products. Identification of this fungal diversity will be useful for understanding the microbiota that participate in meju fermentation, and these fungal isolates can be utilized in the fermented foods and biotechnology industries.
Development of a Quantitative PCR for Detection of Lactobacillus plantarum Starters During Wine Malolactic Fermentation
Cho, Gyu-Sung ; KrauB, Sabrina ; Huch, Melanie ; Toit, Maret Du ; Franz, Charles M.A.P. ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1280~1286
DOI : 10.4014/jmb.1107.07003
A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at
CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above
/ml for approx. 10 days, after which cell numbers decreased to levels of
CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca.
CFU/ml was detected. The minimum detection level for quantitative PCR in this study was
CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.
Biochemical Characterization of the Exopolysaccharide Purified from Laetiporus sulphureus Mycelia
Seo, Min-Jeong ; Kang, Byoung-Won ; Park, Jeong-Uck ; Kim, Min-Jeong ; Lee, Hye-Hyeon ; Choi, Yung-Hyun ; Jeong, Yong-Kee ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1287~1293
DOI : 10.4014/jmb.1106.06046
The extracellular polysaccharide (EPS) was isolated from mycelial cultures of Laetiporus sulphureus var. miniatus and purified by DEAE cellulose and Sephadex G-50 column chromatography. The purified EPS (EPS-2-1) was composed of only glucose units and its molecular mass was 6.95 kDa. The chemical structure of EPS-2-1 consisted of a main chain containing (
)-Glcp units with branches at the C-6 position of the chain carrying-Glcp-(
)-linked residues. The effect of purified EPS on immunomodulatory genes and proteins of the Bcl-2 family was observed using cultured U937 human leukemia cells. Of note, the levels of Bax and Bad proteins treated with the EPS (4 mg/ml) were approximately 23- and 18-times higher than those in non-treated cells, respectively. These results may suggest that the EPS purified from the mushroom L. sulphureus is associated with the activation of immunomodulatory mediators, Bax and Bad proteins.
Cloning of metK from Actinoplanes teichomyceticus ATCC31121 and Effect of Its High Expression on Antibiotic Production
Kim, Du-Yeong ; Hwang, Yong-Il ; Choi, Sun-Uk ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1294~1298
DOI : 10.4014/jmb.1101.01018
A metK gene encoding S-adenosyl-L-methionine synthetase was cloned from the non-Streptomyces actinomycetes, Actinoplanes teichomyceticus ATCC31121. In order to evaluate the effect of the metK expression on antibiotic production in actinomycetes, an expression vector harboring the metK gene was constructed and introduced into Streptomyces lividans TK24 and A. teichomyceticus, and the antibiotic production of the exconjugants was assessed. As a result, it was determined that the expression of metK induced 17-fold and 2.2-fold increases in actinorhodin production from S. lividans TK24 and teicoplanin production from A. teichomyceticus, respectively, compared with the control strains.
Combined TGE-SGE Expression of Novel PAI-1-Resistant t-PA in CHO DG44 Cells Using Orbitally Shaking Disposable Bioreactors
Davami, Fatemeh ; Barkhordari, Farzaneh ; Alebouyeh, Mahmoud ; Adeli, Ahmad ; Mahboudi, Fereidoun ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1299~1305
DOI : 10.4014/jmb.1106.05060
An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncated-mutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncated-mutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.
Monitoring of Environmental Arsenic by Cultures of the Photosynthetic Bacterial Sensor Illuminated with a Near-Infrared Light Emitting Diode Array
Maeda, Isamu ; Sakurai, Hirokazu ; Yoshida, Kazuyuki ; Siddiki, Mohammad Shohel Rana ; Shimizu, Tokuo ; Fukami, Motohiro ; Ueda, Shunsaku ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1306~1311
DOI : 10.4014/jmb.1105.05017
Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5
/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.
Chitinolytic and Chitosanolytic Activities from Crude Cellulase Extract Produced by A. niger Grown on Apple Pomace Through Koji Fermentation
Dhillon, Gurpreet Singh ; Brar, Satinder Kaur ; Kaur, Surinder ; Valero, Jose R. ; Verma, Mausam ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1312~1321
DOI : 10.4014/jmb.1106.06036
Enzyme extracts of cellulase [filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase)], chitinase, and chitosanase produced by Aspergillus niger NRRL-567 were evaluated. The interactive effects of initial moisture and different inducers for FP cellulase and CMCase production were optimized using response surface methodology. Higher enzyme activities [FPase
IU/gram fermented substrate (gfs) and CMCase
IU/gfs] were achieved after 48 h fermentation in solid-state medium containing apple pomace supplemented with rice husk [1% (w/w)] under optimized conditions [pH 4.5, moisture 55% (v/w), and inducers veratryl alcohol (2 mM/kg), copper sulfate (1.5 mM/kg), and lactose 2% (w/w)] (p<0.05). Koji fermentation in trays was carried out and higher enzyme activities (FPase
IU/gfs and CMCase
IU/gfs) were achieved. The nonspecific chitinase and chitosanase activities of cellulase enzyme extract were analyzed using chitin and chitosan substrates with different physicochemical characteristics, such as degree of deacetylation, molecular weight, and viscosity. Higher chitinase and chitosanase activities of
IU/gfs, respectively, were achieved. Moreover, the enzyme was stable and retained 92-94% activity even after one month. Cellulase enzyme extract obtained from A. niger with chitinolytic and chitosanolytic activities could be potentially used for making low-molecular-weight chitin and chitosan oligomers, having promising applications in biomedicine, pharmaceuticals, food, and agricultural industries, and in biocontrol formulations.
Rice Straw-Decomposing Fungi and Their Cellulolytic and Xylanolytic Enzymes
Lee, Sang-Joon ; Jang, Yeong-Seon ; Lee, Young-Min ; Lee, Jae-Jung ; Lee, Han-Byul ; Kim, Gyu-Hyeok ; Kim, Jae-Jin ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1322~1329
DOI : 10.4014/jmb.1107.07022
Filamentous fungi colonizing rice straw were collected from 11 different sites in Korea and were identified based on characterization of their morphology and molecular properties. The fungi were divided into 25 species belonging to 16 genera, including 14 ascomycetes, one zygomycete, and one basidiomycete. Fungal cellulolytic and xylanolytic enzymes were assessed through a two-step process, wherein highly active cellulase- and/or hemicellulase-producing fungi were selected in a first screening step followed by a second step to isolate the best enzyme-producer. Twenty-five fungal species were first screened for the production of total cellulase (TC), endo-
-1,4 glucanase (EG), and endo-
-1,4 xylanase (XYL) using solid-state fermentation with rice straw as substrate. From this screening, six species, namely, Aspergillus niger KUC5183, A. ochraceus KUC5204, A. versicolor KUC5201, Mucor circinelloides KUC6014, Trichoderma harzianum 1 KUC5182, and an unknown basidiomycete species, KUC8721, were selected. These six species were then incubated in liquid Mandels' media containing cellulose, glucose, rice straw, or xylan as the sole carbon source and the activities of six different enzymes were measured. Enzyme production was highly influenced by media conditions and in some cases significantly increased. Through this screening process, Trichoderma harzianum 1 KUC5182 was selected as the best enzyme producer. Rice straw and xylan were good carbon sources for the screening of cellulolytic and xylanolytic enzymes.
Suppressing Erwinia carotovora Pathogenicity by Projecting N-Acyl Homoserine Lactonase onto the Surface of Pseudomonas putida Cells
Li, Qianqian ; Ni, Hong ; Meng, Shan ; He, Yan ; Yu, Ziniu ; Li, Lin ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1330~1335
DOI : 10.4014/jmb.1107.07011
N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect.
The New LM-PCR/Shifter Method for the Genotyping of Microorganisms Based on the Use of a Class IIS Restriction Enzyme and Ligation-Mediated PCR
Krawczyk, Beata ; Leibner-Ciszak, Justyna ; Stojowska, Karolina ; Kur, Jozef ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1336~1344
DOI : 10.4014/jmb.1104.04019
This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.
and lytA in Vancomycin-Tolerant Pneumococci
Olivares, Alma ; Trejo, Jose Olivares ; Arellano-Galindo, Jose ; Zuniga, Gerardo ; Escalona, Gerardo ; Vigueras, Juan Carlos ; Marin, Paula ; Xicohtencatl, Juan ; Valencia, Pedro ; Velazquez-Guadarrama, Norma ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1345~1351
DOI : 10.4014/jmb.1105.05045
Vancomycin therapy failure due to the emergence of tolerance in pneumococci is increasing. The molecular mechanism of tolerance is not clear, but lytA and
are known to be involved. Our aim was to evaluate the expression of both genes in vancomycin-tolerant Streptococcus pneumoniae (VTSP) strains. Eleven VTSP strains from a total of 309 clinical isolates of S. pneumoniae from 1997 to 2006 were classified according to the criteria of Liu and Tomasz. All VTSP strains were evaluated for susceptibility according to CLSI criteria, serotype by the Quellung test, and clonality by PFGE. The expressions of lytA and
were analyzed in different growth phases by RT-PCR with and without vancomycin. Eighty-two percent of VTSP strains showed resistance to penicillin, and 100% were sensitive to vancomycin and cefotaxime. The most frequent serotypes of VTSP strains were 23F (4/11) and 6B (3/11). Clonal relationship was observed in only two strains. No significant changes were observed in
expression in the three phases of growth in VTSP strains with and without vancomycin. Interestingly,
expression in the stationary phase in the non-tolerant reference strain R6 was significantly higher. However, no significant differences in lytA expression were observed between VTSP and R6 strains during the phases of growth analyzed. The absence of changes in
expression in VTSP strains in the stationary phase may be related to their ability to tolerate high antibiotic concentrations, and thus, they survive and remain in the host under the antibiotic selective pressure reflected in therapeutic failure.
Occurrence of Virulence Determinants in Fecal Enterococcus faecalis Isolated from Pigs and Chickens in Korea
Hwang, In-Yeong ; Lim, Suk-Kyung ; Ku, Hyun-Ok ; Park, Choi-Kyu ; Jung, Suk-Chan ; Park, Yong-Ho ; Nam, Hyang-Mi ;
Journal of Microbiology and Biotechnology, volume 21, issue 12, 2011, Pages 1352~1355
DOI : 10.4014/jmb.1107.07002
Forty-one Enterococcus faecalis (E. faecalis) isolates from feces of pigs and chickens in Korea were screened for the presence of virulence factors. Gelatinase activity (85.4%, 35/41) was the more commonly observed phenotype of virulence in E. faecalis, compared with hemolytic activity (12.2%, 5/41). Thirty-one of 35 (88.6%) gelatinase-positive E. faecalis isolates harbored the gelE and fsrABC genes. A gene encoding for the enterococcal surface protein (Esp) was detected in 24.4% (10/41) of the isolates. All beta-hemolysin-producing isolates harbored the esp gene.