Journal of Microbiology

Volume 42(3); September 2004

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

Impact of Genetically Modified Enterobacter cloacae on Indigenous Endophytic Community of Citrus sinensis Seedlings: Fernando Dini Andreote , Marcelo Jose Mortatti Gullo , Andre Oliveira de Souza Lima , Walter Maccheroni Junior , Joao Lucio Azevedo , Welington Luiz Araujo; J. Microbiol. 2004;42(3):169-173.

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Abstract: Enterobacter cloacae (strain PR2/7), a genetically modified endophyte (GME) in citrus plants, carrying different plasmids (pEC3.0/18, pCelE, pEglA and pGFP), was inoculated into Citrus sinensis seedlings under greenhouse conditions. The impact of this on the indigenous bacterial endophytic community was studied by analyses of 2 different morphologic groups. The germination rates of inoculated seeds were evaluated in greenhouse, and plasmid stability under in vitro conditions. Results demonstrated a great and diverse endophytic community inside plants, and specialization in tissue colonization by some bacterial groups, in different treatments. Shifts in seed germination rate were observed among treatments: in general, the PR2/7 harboring pEglA bacterial clone significantly reduced seed germination, compared to the PR2/7 harboring pEC3.0/18 clone. This suggests that the presence of the pEglA plasmid changes bacteria-seed interactions. The endophytic community of citrus seedlings changed according to treatment. In seedlings treated with the PR2/7 with pEglA clone, the population of group II decreased significantly, within the context of the total endophytic community. These results indicate that the application of GMEs induces shifts in the endophytic bacterial community of citrus seedlings.

Journal Article

Growth and Physiological Properties of Wild Type and Mutants of Halomonas subglaciescola DH-1 in Saline Environment: Hye Jeong Ryu , Yoo Jung Jeong , Doo Hyun Park; J. Microbiol. 2004;42(3):174-180.; DOI: https://doi.org/2093 [pii]

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Abstract: A halophilic bacterium was isolated from fermented seafood. The 16S rDNA sequence identity between the isolate and Halomonas subglaciescola AJ306801 was above 95%. The isolate that did not grow in the condition without NaCl or in the condition with other sodium (Na^+) or chloride ions (Cl^-) instead of NaCl was named H. subglaciescola DH-1. Two mutants capable of growing without NaCl were obtained by random mutagenesis, of which their total soluble protein profiles were compared with those of the wild type by two-dimensional electrophoresis. The external compatible solutes (betaine and choline) and cell extract of the wild type did not function as osmoprotectants, and these parameters within the mutants did not enhance their growth in the saline environment. In the proton translocation test, rapid acidification of the reactant was not detected for the wild type, but it was detected for the mutant in the condition without NaCl. From these results, we derived the hypothesis that NaCl may be absolutely required for the energy metabolism of H. subglaciescola DH-1 but not for its osmoregulation, and the mutants may have another modified proton translocation system that is independent of NaCl, except for those mutants with an NaCl-dependent system.

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

Comparative Analysis of Cyanobacterial Communities from Polluted Reservoirs in Korea: Jin-Book Kim , Mi-Sook Moon , Dong-Hun Lee , Sung-Taik Lee , Marco Bazzicalupo , Chi-Kyung Kim; J. Microbiol. 2004;42(3):181-187.; DOI: https://doi.org/2092 [pii]

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Abstract: Cyanobacteria are the dominant phototrophic bacteria in water environments. Here, the diversity of cyanobacteria in seven Korean reservoir waters where different levels of algal blooms were observed during the summer of 2002, was examined by T-RFLP analysis. The number of T-RF bands in the HaeIII T-RFLP profiles analyzed from those water samples ranged from 20 to 44. Of these, cyanobacteria accounted for 6.1 to 27.2% of the total bacteria. The water samples could be clustered into 2 groups according to the Dice coefficient of the T-RF profiles. The eutrophic Dunpo and oligotrophic Chungju reservoirs were selected, and several representative clones from both reservoir waters analyzed for the nucleotide sequences of their 16S rDNA. The major clones were found to belong to the Microcystis and Anabaena species in the waters from the Dunpo and Chungju reservoirs, respectively, which was in agreement with the T-RFLP result. That is, the Microcystis and Anabaena species were dominant in the eutrophic and polluted Dunpo and oligotrophic Chungju reservoir waters, respectively. These results indicated that there is a correlation between prevalence of cyanobacterial species and levels of pollution in reservoir waters.

Genetic Organization of the dhlA Gene Encoding 1,2-Dichloroethane Dechlorinase from Xanthobacter flavus UE15: Ji-Sook Song , Dong-Hun Lee , Kyoung Lee , Chi-Kyung Kim; J. Microbiol. 2004;42(3):188-193.; DOI: https://doi.org/2091 [pii]

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Abstract: Xanthobacter flavus strain UE15 was isolated in wastewater obtained from the Ulsan industrial complex, Korea. This strain functions as a 1,2-dichloroethane (1,2-DCA) degrader, via a mechanism of hydrolytic dechlorination, under aerobic conditions. The UE15 strain was also capable of dechlorinating other chloroaliphatics, such as 2-chloroacetic acid and 2-chloropropionic acid. The dhlA gene encoding 1,2-DCA dechlorinase was cloned from the genomic DNA of the UE15 strain, and its nucleotide sequence was determined to consist of 933 base pairs. The deduced amino acid sequence of the DhlA dechlorinase exhibited 100% homology with the corresponding enzyme from X. autotrophicus GJ10, but only 27 to 29% homology with the corresponding enzymes from Rhodococcus rhodochrous, Pseudomonas pavonaceae, and Mycobacterium sp. strain GP1, which all dechlorinate haloalkane compounds. The UE15 strain has an ORF1 (1,356 bp) downstream from the dhlA gene. The OFR1 shows 99% amino acid sequence homology with the transposase reported from X. autotrophicus GJ10. The transposase gene was not found in the vicinity of the dhlA in the GJ10 strain, but rather beside the dhlB gene coding for haloacid dechlorinase. The dhlA and dhlB genes were confirmed to be located at separate chromosomal loci in the Xanthobacter flavus UE15 strain as well as in X. autotrophicus GJ10. The dhlA and transposase genes of the UE15 strain were found to be parenthesized by a pair of insertion sequences, IS1247, which were also found on both sides of the transposase gene in the GJ10 strain. This unique structure of the dhlA gene organization in X. flavus strain UE15 suggested that the dechlorinase gene, dhlA, is transferred with the help of the transposase gene.

Coregulation of lux Genes and Riboflavin Genes in Bioluminescent Bacteria of Photobacterium phosphoreum: Nack-Do Sung , Chan Yong Lee; J. Microbiol. 2004;42(3):194-199.; DOI: https://doi.org/2090 [pii]

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Abstract: Investigation of the expression of the riboflavin (rib) genes, which are found immediately downstream of luxG in the lux operon in Photobacterium phosphoreum, provides more information relevant to the evolution of bioluminescence, as well as to the regulation of supply of flavin substrate for bacterial bioluminescence reactions. In order to answer the question of whether or not the transcriptions of lux and rib genes are integrated, a transcriptional termination assay was performed with P. phosphoreum DNA, containing the possible stem-loop structures, located in the intergenic region of luxF and luxE ([omega]_A), of luxG and ribE ([omega]_B), and downstream of ribA ([omega]_C). The expression of the CAT (Chloramphenicol Acetyl Transferase) reporter gene was remarkably decreased upon the insertion of the stem-loop structure ([omega]_C) into the strong lux promoter and the reporter gene. However, the insertion of the structure ([omega]_B) into the intergenic region of the lux and the rib genes caused no significant change in expression from the CAT gene. In addition, the single stranded DNA in the same region was protected by the P. phosphoreum mRNA from the S1 nuclease protection assay. These results suggest that lux genes and rib genes are part of the same operon in P. phosphoreum

A Proteomic Approach to Study msDNA Function in Escherichia coli: Mi-Ae Jeong , Dongbin Lim; J. Microbiol. 2004;42(3):200-204.; DOI: https://doi.org/2089 [pii]

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Abstract: Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase. It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation. In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis. Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS. Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources. One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation. The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive.

Molecular Cloning and Characterization of CMCase gene (celC) from Salmonella typhimurium UR: Ju-Soon Yoo , Youn-Ju Jung , Soo-Yeol Chung , Young-Choon Lee , Yong-Lark Choi; J. Microbiol. 2004;42(3):205-210.; DOI: https://doi.org/2088 [pii]

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Abstract: The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium UR1. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50^oC and pH 6.5, respectively.

Journal Article

Performance of the Immunoglobulin G Avidity and Enzyme Immunoassay IgG/IgM Screening Tests for Differentiation of the Clinical Spectrum of Toxoplasmosis: Mehmet Tanyuksel , Cakir Guney , Engin Araz , M.Ali Saracli , Levent Doganci; J. Microbiol. 2004;42(3):211-215.; DOI: https://doi.org/2087 [pii]

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Abstract: Toxoplasmosis has been well known as an important human infection to consider especially in pregnant women. Although many serologic methods are available, the diagnosis of toxoplasmosis can be extremely difficult. The presence of increased levels of Toxoplasma-specific IgG antibodies indicates an infection, but it does not differentiate between a recent and past infection. The purpose of our study was to compare the performance of the ELISA T. gondii IgG/IgM test, a widely used enzyme-linked immunosorbent assay, to the ELISA IgG avidity method. One hundred and four serum samples (from 38 males and 66 females) were tested and evaluated from symptomatic patients (chorioretinitis, lymphadenopathy), and from women in their first trimester of pregnancy who were suspected of having toxoplasmosis. The high IgG avidity and ELISA IgG antibody levels were in agreement for 51 of the specimens (49.0%). Thirty-eight discrepant (borderline) results from the IgG avidity method were positive for IgM (3 specimens) and IgG (37 specimens). Interestingly, out of the eight serum samples that were positive for both IgG and IgM antibodies, two samples were low IgG avidity, and three samples were borderline. There was no statistically significant relation observed between the results of the IgG avidity method and the ELISA IgG test, and the IgG avidity method and ELISA IgM test (c^2=1.987; p=0.370 and c^2=2.152; p=0.341, respectively). The IgG avidity method was considered easy to perform and an acceptable approach for the differentiation of discrepant results (recent/chronic) and for the current detection of T. gondii antibodies. We concluded that the determination of IgG avidity is a helpful tool for the diagnosis of the ocular form of toxoplasmosis and it is a safe method for screening this disease in the first trimester of pregnancy.

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

PCR Method Based on the ogdH Gene for the Detection of Salmonella spp. from Chicken Meat Samples: Un-Ho Jin , Sung-Hak Cho , Min-Gon Kim , Sang-Do Ha , Keun-Sung Kim , Kyu-Ho Lee , Kwang-Yup Kim , Duck Hwa Chung , Young-Choon Lee , Cheorl-Ho Kim; J. Microbiol. 2004;42(3):216-222.; DOI: https://doi.org/2086 [pii]

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Abstract: In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.

Journal Article

Isolation and Characterization of a-Glucosidase Inhibitor from the Fungus Ganoderma lucidum: Shin-Duk Kim , Hong Joon Nho; J. Microbiol. 2004;42(3):223-227.; DOI: https://doi.org/2085 [pii]

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Abstract: An [alpha]-glucosidase inhibitor, SKG-3, was isolated from the fruiting bodies of Ganoderma lucidum and its physico-chemical properties were characterized. It was a highly specific and effective reversible inhibitor of [alpha]-glucosidase. It showed very potent inhibitory activity against [alpha]-glucosidase with an IC_50 value of 4.6 mg/ml, but no activity for any other glycosidases tested. Enzyme activity could be recovered upon dialysis, thus providing evidence for the reversibility of the inhibition. A Lineweaver-Burk plot indicated that the SKG-3 inhibition of [alpha]-glucosidase was competitive.

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

Purification of Filamentous Bacteriophage M13 by Expanded Bed Anion Exchange Chromatography: Tau Chuan Ling , Chee Kin Loong , Wen Siang Tan , Beng Ti Tey , Wan Mohammad Wan Abdullah , Arbakariya Ariff; J. Microbiol. 2004;42(3):228-232.; DOI: https://doi.org/2084 [pii]

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Abstract: In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine^TM 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (H_o=15cm) of STREAMLINE DEAE (r=1.2 g/cm^3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.

Transcriptional Regulation of the Gene Encoding g-Glutamylcysteine Synthetase from the Fission Yeast Schizosaccharomyces pombe: Su-Jung Kim , Hong-Gyum Kim , Byung-Chul Kim , Kyunghoon Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2004;42(3):233-238.; DOI: https://doi.org/2083 [pii]

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Abstract: Transcriptional regulation of the Schizosaccharomyces pombe [gamma]-glutamylcysteine synthetase (GCS) gene was examined using the two GCS-lacZ fusion plasmids pUGCS101 and pUGCS102, which harbor 607 bp and 447 bp upstream regions, respectively. The negatively-acting sequence was located in the -607 ~ -447 bp upstream region of the GCS gene. The upstream sequence responsible for induction by menadione (MD) and L-buthionine-(S, R)-sulfoximine (BSO) resides in the -607 ~ -447 bp region, whereas the sequence which codes for nitric oxide induction is located within the -447 bp region, measured from the translational initiation point. Carbon source-dependent regulation of the GCS gene appeared to be dependent on the nucleotide sequence within -447 bp region. The transcription factor Pap1 is involved in the induction of the GCS gene by MD and BSO, but not by nitric oxide. Induction of the GCS gene occurring due to low glucose concentration does not depend on the presence of Pap1. These data imply that induction by MD and BSO may be mediated by the Pap1 binding site, probably located in the -607 ~ -447 region, and also that the nitric oxide-mediated regulation of the S. pombe GCS gene may share a similar mechanism with its carbon-dependent induction.

Journal Article

Detection of Hepatitis B Virus and Mycobacterium tuberculosis in Korean Dental Patients: Sun-A Lee , So Young Yoo , Kee-Sung Kay , Joong-Ki Kook; J. Microbiol. 2004;42(3):239-242.; DOI: https://doi.org/2082 [pii]

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Abstract: This study examined the detection rate of the hepatitis B virus (HBV) and Mycobacterium tuberculosis (Mtb) in serum and saliva samples, respectively, from 120 dental patients who were unaware if they have or had either hepatitis or tuberculosis. The frequencies of HBsAg and anti-HBs were determined using an immunochromatic assay. Mtb positivity was determined by the PCR method. Of the 120 patients, 7 (5.8%) were HBV positive and 30 (25.0%) were Mtb positive. This highlights the fact that dental health care workers (DHCWs) can be exposed to the risk of infection from blood- or saliva-borne pathogens as a consequence of their work. Therefore, it is very important to prevent cross infection between patients and dental personnel. Accordingly, laboratory tests prior to surgical treatment are needed to determine the infectious state of dental patients in order to prevent the transmission of infectious diseases in dental clinics.

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

Green Fluorescent Protein as a Marker for Monitoring a Pentachlorophenol Degrader Sphingomonas chlorophenolica ATCC39723: Eun-Taex Oh , Jae-Seong So , Byung-Hyuk Kim , Jong-Sul Kim , Sung-Cheol Koh; J. Microbiol. 2004;42(3):243-247.; DOI: https://doi.org/2081 [pii]

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Abstract: Sphingomonas chlorophenolica ATCC39723 was successfully labeled with the gfp (green fluorescent protein) gene inserted into the pcpB gene by homologous recombination. As the gfp recombinant was easily distinguished from other indigenous organisms, the population of gfp recombinant was monitored after being released into the soil microcosms. Their population density dropped from 108 to 106 (cfu/ml) in the non-sterilized soil microcosms during the first 6 days. Moreover, the gfp recombinant was not detected even at lower dilution rates after a certain time period. The recombinant, however, survived for at least 28 days in the sterilized soil microcosms. Although the gfp recombinant did not degrade pentachlorophenol (PCP), this experiment showed the possibility of using gfp as a monitoring reporter system for S. chlorophenolica ATCC39723 and potentially other species of Sphingomonas.

Enhanced Secretion of Cell Wall Bound Enolase into Culture Medium by the soo1-1 Mutation of Saccharomyces cerevisiae: Ki-Hyun Kim , Hee-Moon Park; J. Microbiol. 2004;42(3):248-252.; DOI: https://doi.org/2080 [pii]

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Abstract: In order to identify the protein(s) secreted into culture medium by the soo1-1/ret1-1 mutation of Saccharomyces cerevisiae, proteins from the culture medium of cells grown at permissive (28^oC) and non-permissive temperatures (37^oC), were analyzed. Comparison of protein bands separated by SDS-PAGE identified a prominent band of 47-kDa band from a mutant grown at 37^oC. N-terminal amino acid sequencing of this 47-kDa protein showed high identity with enolases 1 and 2. Western blot analysis revealed that most of the cell wall-bound enolase was released into the culture medium of the mutant grown at 37^oC, some of which were separated as those with lower molecular weights. Our results, presented here, indicate the impairment of cell wall enolase biogenesis and assembly by the soo1-1/ret1-1 mutation of S. cerevisiae.

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