Journal of Microbiology

Volume 39(3); September 2001

Regulation Mechanism of Redox Reaction in Rubredoxin: Tongpil Min , Marly K. Eidsness , Toshiko Ichiye , ChulHee Kang; J. Microbiol. 2001;39(3):149-153.

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Abstract: The electron transfer reaction is one of the most essential processes of life. Not only does it provide the means of transforming solar and chemical energy into a utilizable form for all living organisms, it also extends into a range of metabolic processes that support the life of a cell. Thus, it is of great interest to understand the physical basis of the rates and reduction potentials of these reactions. To identify the major determinants of reduction potentials in redox proteins, we have chosen the simplest electron transfer protein, rubredoxin, a small (52-54 residue) iron-sulfur protein family, widely distributed in bacteria and archaea. Rubredoxins can be grouped into two classes based on the correlation of their reduction potentials with the identity of residue 44; those with Ala44 (ex: Pyrococcus furiosus) have reduction potentials that are ~50 mV higher than those with Val44 (ex: Clostridium pasteurianum). Based on the crystal structures of rubredoxins from C. pasteurianum and P. furiosus , we propose the identity of residue 44 alone determines the reduction potential by the orientation of the electric dipole moment of the peptide bond between 43 and 44. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins from C. pasteurianum, the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated.

Effects of Lactic Acid Bacteria on Intestinal Microbial Enzyme Activity and Composition in Rats Treated with Azoxymethane: Sang-Myeong Lee , Wan-Kyu Lee; J. Microbiol. 2001;39(3):154-161.

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Abstract: In recent years, colon cancer has been reported to be one of the most important causes of cancer morbidity and mortality in Korea. Epidemiological and experimental studies suggest that lactic acid bacteria (LAB) used to ferment dairy products inhibits colon carcinogenesis. The present study was designed to determine whether the colon cancer inhibitory effect of LAB (Bifidobacterium longum HY8001; Bif and Lactobacillus acidophilus HY2104; Lac) of Korean origin, is associated with intestinal microflora composition and certain enzyme activity in rats treated with azoxymethane (AOM). At five weeks of age, SD rats were divided at random into four (AOM alone, Bif, Lac, and Bif+Lac) groups. Oral administration of lactic acid bacteria cultures were performed daily until the termination of the study. Two weeks later, all animals were given a subcutaneous injection of AOM dissolved in normal saline at a dose of 15 mg/kg of body weight once weekly for 2 weeks. Every two weeks for 10 weeks, five of the rats in each group were randomly chosen for fecal specimen collection. The fecal specimens were used for assay of [beta]-glucuronidase and nitroreductase, and analysis of intestinal microflora composition. The activity of [beta]-glucuronidase which plays an important role in the production of the carcinogenic metabolite of azoxymethane was remarkably increased in the AOM alone group after AOM injection and maintained the high level during the experiment. However, LAB inhibited the AOM-induced increase in [beta]-glucuronidase activity. Nitroreductase activity decreased by 30-40% in LAB treated groups in comparison with that of the AOM alone group. The results of the present study suggest that LAB inhibits colon carcinogenesis by modulating the metabolic activity of intestinal microflora and improving the composition of intestinal microflora.

Stress-shock Response of a Methylotrophic Bacterium Methylovorus sp. strain SS1 DSM 11726: Jong H. Park , Si W. Kim , Eungbin Kim , Young T. Ro , Young M. Kim; J. Microbiol. 2001;39(3):162-167.

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Abstract: Methylovorus sp. strain SS1 DSM 11726 was found to grow continuously when it was transferred from 30 C to 40 C and 43 C. A shift in growth temperature from 30 C to 45 C, 47 C and 50 C reduced the viability of the cell population by more than 10^2 , 10^3 and 10^5 folds, respectively, after 1 h cultivation. Cells transferred to 47 C and 50 C after preincubation for 15 min at 43 C, however, exhibited 10-fold increase in viability. It was found that incubation for 15 min at 40 C of Methylovorus sp. strain SS1 grown at 30 C was sufficient to accelerate the synthesis of a specific subset of proteins. The major heat shock proteins had apparent molecular masses of 90, 70, 66, 60, and 58 kDa. The 60 and 58 kDa proteins were found to cross-react with the antiserum raised against GroEL protein. The heat shock response persisted for over 1 h. The shock proteins were stable for 90 min in the cell. Exposure of the cells to methanol induced proteins identical to the heat shock proteins. Addition of ethanol induced a unique protein with a molecular mass of about 40 kDa in addition to the heat-induced proteins. The proteins induced in paraquat-treated cells were different from the heat shock proteins, except the 70 and 60 kDa proteins.

Analysis of Catalases from Photosynthetic Bacterium Rhodospirillum rubrum S1: Hee-Kyoung Lim , Young-Mi Kim , Dong-Heon Lee , Hyung-Yeel Kahng , Duck-Chul Oh; J. Microbiol. 2001;39(3):168-176.

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Abstract: Five different types of catalases from photosynthetic bacterium Rhodospirillum rubrum S1 grown aerobically in the dark were found in this study, and designated Cat1 (350 kDa), Cat2 (323 kDa), Cat3 (266 kDa), Cat4 (246 kDa), and Cat5 (238 kDa). Analysis of native PAGE revealed that Cat2, Cat3, and Cat4 were also produced in the cells anaerobically grown in the light. It is notable that only Cat2 was expressed much more strongly in response to the anaerobic condition. Enzyme activity staining demonstrated that Cat3 and Cat4 had bifunctional catalase-peroxidase activities, while Cat1, Cat2, and Cat5 were typical monofunctional catalases. S1 cells grown aerobically in the presence of malate as the sole source of carbon exhibited an apparent catalase Km value of 10 mM and a Vmax of about 705 U/mg protein at late stationary growth phase. The catalase activity of S1 cells grown in the anaerobic environment exhibited a much lower Vmax of about 109 U/mg protein at late logarithmic growth phase. The catalytic activity was stable in the broad range of temperatures (30 C-60 C), and pH (6.0-10.0). R. rubrum S1 was much more resistant to H_2 O_2 in the stationary growth phase than in the exponential growth phase regardless of growth conditions. Cells of stationary growth phase treated with 15 mM H 2 O 2 for 1 h showed 3-fold higher catalase activities than the untreated cells. In addition, L-glutamate induced an 80-fold increase in total catalase activity of R. rubrum S1 compared with malic acid. Through fraction analyses of S1 cells, Cat2, Cat3, Cat4 and Cat5 were found in both cytoplasm and periplasm, while Cat1 was localized only in the cytoplasm.

Improvement in the Stability of Glycinecin A through Protein Fusion of the Two Structural Components: Youngmee Kim , Somi K. Cho , Moonjae Cho; J. Microbiol. 2001;39(3):177-180.

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Abstract: Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. c. pv. vesicatoria. We have reported that purified glycinecin A is composed of two polypeptides, is active over a wide range of pH (6 to 9), and is stable at temperatures up to 60 C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits; the two encoding genes, glyA and glyB, respectively, have been cloned (Heu et al. 2001. Appl. Environ. Microbiol. 67, 4105-4110). Co-expression of glyA and glyB in the same cell is essential for bacteriocin activity. We constructed and produced a chimeric glycinecin A connecting glyA and glyB in one open reading frame. The chimeric glycinecin A has the same bactericidal activity as the wild-type glycinecin A. However, the chimeric glycinecin A is more stable in a wider range of pH and temperature.

Cytokine-Inducing and T Cell Mitogenic Effects of Cordyceps hepialidicola: Jong-Soon Lim , Seung-Hyung Kim , Jeong-Youl Choi , Jin-Seo Park , Seong Joo Park , Kwang-Soo Shin; J. Microbiol. 2001;39(3):181-185.

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Abstract: The morphological characteristics of newly isolated Cordyceps hepialidicola were characterized, and the phylogenetic relationships with other Cordyceps species were investigated using a sequence analysis of the internal transcribed spacer (ITS). The PCR product of 592 bp showed a homology of 92 and 91% with C. militaris and C. nutans, respectively. In an in vitro model using mouse peripheral blood mononuclear cells (PBMC), a methanol extract of C. hepialidicola induced multiple cytokines, including IFN-[gamma], IL-4, and IL-18. The extract also enhanced the percentages of the CD4^+ and CD8^+ T cells in the healthy murine PBMCs to 56.1% and 13.0%, respectively. The percentages of CD4^+ and CD8^+ in the untreated controls were 28.4 and 7.3%, and concanavalin A-treated positive controls were 62.4 and 18.3%, respectively.

Expression of Chemokine and Tumor Necrosis Factor Alpha Genes in Murine Peritoneal Macrophages Infected with Orientia tsutsugamushi: Young-Sang Koh; J. Microbiol. 2001;39(3):186-194.

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Abstract: Scrub typhus, caused by Orientia tsutsugamushi infection, is clinically and histopathologically characterized by local as well as systemic inflammatory reactions, indicating that orientiae induce mechanisms that amplify the inflammatory response. To reveal underlying mechanisms of chemoattraction and activation of responding leukocytes, expression of chemokine and tumor necrosis factor alpha (TNF-[alpha]) genes in murine peritoneal macrophages after infection with the obligate intracellular bacterium O. tsutsugamushi was investigated. The genes that were upregulated included macrophage inflammatory proteins 1[alpha]/[beta] (MIP-1[alpha]/[beta]), MIP-2, monocyte chemoattractant protein 1 (MCP-1), RANTES (regulated upon activation, normal T-cell expressed and secreted), gamma-interferon-inducible protein 10 (IP-10), and TNF-[alpha]. Peak expression of these chemokines and TNF-[alpha] was observed between 1 and 3 h after infection. These responses returned to or approached baseline preinfection levels 6 h after challenge. Semiquantitative reverse transcription (RT)-PCR analysis revealed dramatic increases during infection in the steady-state levels of mRNA coding for the inhibitory subunit of NF-[kappa]B (I[kappa]B[alpha]), whose transcription is enhanced by binding of NF-[kappa]B within the I[kappa]B[alpha] promoter region. Thus, O. tsutsugamushi appears to be a strong inducer of chemokines and TNF-[alpha] which may significantly contribute to inflammation and tissue damage observed in scrub typhus by attracting and activating phagocytic leukocytes.

Effect of Moisture Content on Reductive Dechlorination of Polychlorinated Biphenyls and Population Dynamics of Dechlorinating Microorganisms: O-Seob Kwon , Young Eui Kim , Jong Gyu Park; J. Microbiol. 2001;39(3):195-201.

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Abstract: The effect of moisture content on the reductive dechlorination of polychlorinated biphenyls and population dynamics of dechlorinating microorganisms was investigated in sediments spiked with Aroclor 1248. In sediment slurry with an overlying water layer, dechlorination ensued after a 4-week lag period and reduced the average number of chlorines per biphenyl from 3.91 to 3.15 after 48 weeks. In the sediments of reduced moisture content, however, dechlorination occurred after a lag period of 12 weeks and decreased the average number of chlorines per biphenyl to only 3.62, and the dechlorination rate was also slower. When the population size of dechlorinators, methanogens, and sulfate-reducing bacteria was determined by the most probable number techniques, however, no difference was found between the slurry and the low-moisture sediments, except for methanogens. The growth of dechlorinating populations coincided with the end of the lag period and they then increased by 3 orders of magnitude in two conditions. Specific growth rate of dechlorinators showed little difference between the slurry and the low-moisture sediments; however, growth yield was high in the sediments of reduced moisture content. The reduction of sediment moisture decreased the dechlorination rate and extent of PCBs but did not inhibit the growth of PCB dechlorinators.

Evaluation of the EF-18 Agar-Hydrophobic Grid Membrane Filter (HGMF) Method to Isolate Salmonella from Poultry Products: Rosa Capita , Maite Alvarez-Astorga , Carlos Alonso-Calleja , Maria del Camino , Garcia-Fernandez , Benito Moreno; J. Microbiol. 2001;39(3):202-205.

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Abstract: The EF-18 agar/hydrophobic grid membrane filter (EF18/HGMF) method was evaluated for the isolation of Salmonella in naturally contaminated chicken carcasses, chicken parts (legs, wings and giblets) and processed chicken products (sausages and hamburgers). Percentages of false positive results for Salmonella (colonies with a similar morphology to those of Salmonella) were 78.75, 81.67 and 80% for carcasses, chicken parts and processed chicken products, respectively. The bacterial isolates that caused false positive reactions using this method were identified as Proteus mirabilis (70.85%), Citrobacter freundii (15.25%), Klebsiella ozaenae (5.83%), Hafnia alvei (4.48%), Escherichia coli (2.69%) and Enterobacter aerogenes (0.90%). The data obtained in this study suggest that the EF-18/HGMF method is not sufficiently selective or specific for isolating Salmonella from meat and chicken products.

Toxic Effects of Catechol and 4-Chlorobenzoate Stresses on Bacterial Cells: Sang-Ho Park , Yeon-Ja Ko , Chi-Kyung Kim; J. Microbiol. 2001;39(3):206-212.

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Abstract: Catechol and 4-chlorobenzoate (4CBA) which are produced from the biodegradation of a variety of aromatic and chloroaromatics have been recognized as toxic to living organisms. In this study, the toxic effects of catechol and 4-chlorobenzoate on gram-positive and -negative bacteria were examined in terms of survival, morphology, change in fatty acids and membrane protein composition. The survival rate of the organisms during treatment for 6 h was decreased, as the concentration of each aromatic was increased. Escherichia coli and Pseudomonas cells treated with catechol and 4CBA at concentrations causing a significant decrease in their viability, showed destructive openings in their cell envelopes. Bacillus subtilis treated with the aromatics were reduced in cell size and Staphylococcus aureus cells displayed irregular rod shapes with wrinkled surfaces. The bacterial cells treated with 20 mM catechol showed increases in unsaturated fatty acids, but several saturated fatty acids were decreased. In the E. coli cells treated with 20 mM catechol, inner membrane proteins of 150 kDa and 105 kDa were decreased. But several kinds of the inner and outer membrane proteins were increased. In B. subtilis treated with 20 mM catechol, several kinds of proteins were increased or decreased in membrane proteins.

Rapid Detection of Bacteria from Blood Culture by an Electronic Nose: Peter Lykos , Pravin H. Patel , Christopher Morong , Asha Joseph; J. Microbiol. 2001;39(3):213-218.

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Abstract: The treatment of patients with bacteraemia and septicemia requires accurate and rapid identification of the pathogen so that the physician can be guided regarding the selection of the proper antimicrobial therapy. The usual procedure is to withdraw an aliquot of the positive blood culture sample for gram staining and subculturing on the media for the growth and subsequent identification, and susceptibility determinations. It was noticed that during the process some microbiologists would sniff the effluent gases that are products of metabolism and in some cases guess the identity of the bacterium. That prompted us to engage in systematic investigation of two gram positive and two gram negative bacteria using an electronic nose that had been proven successful in distinguishing the aroma of coffee beans from different sources. The investigation was successful in illustrating the efficacy of such a device in this clinical setting to distinguish Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis. A representative set of patterns obtained with this apparatus is displayed as well. No effort was made to determine an optimal set of sensors for some specific set of bacterial metabolism gaseous products.

Phage Typing of Staphylococcus aureus Isolates from Poultry Meat in Spain: Rosa Capita , Maite Alvarez-Astorga , Carlos Alonso-Calleja , Benito Moreno , Maria del Camino Garcia-Fernandez; J. Microbiol. 2001;39(3):219-225.

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Abstract: Phage typing is currently used for typing of Staphylococcus aureus strains beyond the species level in epidemiological studies. A total of 168 Staphylococcus aureus isolates from chicken meat and chicken by-products were phage-typed using the international bacteriophage set for typing Staphylococcus aureus of human origin. One hundred and forty-eight (88.09%) strains were phage-typeable (at least one phage produced 20 or more plaques of lysis). Lysis by phages of group III was the most frequent with 99 (58.93%) sensitive strains. This fact coincides with results of other authors. Twenty-nine different phage patterns were observed and three (95, 75/84 and 6/1030/W57) were most common. One hundred and thirty-two (89.19% of typeable strains) showed these or indistinguishable (only one phage reaction difference) patterns. Twenty-six out of seventy chicken samples (37.14%) harboured more than one phage type of Staphylococcus aureus. This fact emphasizes the convenience of subtyping several Staphylococcus aureus isolates from the same sample in epidemiological studies. 80% of sausages and hamburgers contained the same Staphylococcus aureus phage types, which were not found in any of the other food types. This fact suggests a cross contamination during the processing of these foods. Phages 6, 75, 84, 1030 and W57 showed the greatest activity. None of the Staphylococcus aureus strains were sensitive to phages 47, 81 and 94.

Detection of Lymphotropic Herpesviruses by Multiplex Polymerase Chain Reaction: Sang-Tae Park , Seung-Han Kim , Dong-Gun Lee , Jung-Hyun Choi , Wan-Shik Shin , Tai - Gyu Ki m , Soon-Young Paik , Chun-Choo Kim; J. Microbiol. 2001;39(3):226-228.

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Abstract: Human lymphotropic herpesvirus is known to be a major pathogen associated with various diseases in bone marrow transplantation (BMT) recipients. A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection of human lymphotropic herpesviruses, including Ebstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 variants A and B (HHV6-A, HHV6-B). To demonstrate the usefulness of multiplex PCR for the analysis of clinical samples, peripheral blood mononuclear cells and serum from BMT recipients were analysed. The results showed that a clear detection could be made between EBV, HCMV and HHV-6. This multiplex PCR assay is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of EBV, HCMV and HHV-6.

Role of Amino Acids in Production of D-amino Acid Oxidase: Puneet Singh , Satwinder Singh Marwaha , Neelam Verma; J. Microbiol. 2001;39(3):229-231.

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Abstract: Different DL-amino acids were studied as inducers of D-amino acid oxidase (DAAO) and for their influence on the growth of Trigonopsis variabilis. DL-amino acids with non-polar side groups were found to be the best inducers of DAAO. Maximum increase in the growth of Trigonopsis variabilis (gram dry weight per liter culfure) was observed with DL-methionine (2.39 g/l) followed by DL-serine (2.22 g/l) and DL-alanine (2.21 g/l).

Superoxide Dismutase Profiles in the Mesophilic Deinococcus Species: Young Sun Yun , Young Nam Lee; J. Microbiol. 2001;39(3):232-235.

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Abstract: Electrophoretic resolution of superoxide dismutase (SOD) from the highly UV-resistant bacteria, Deinococcus species revealed multiple forms of superoxide dismutases (SODs) in D. radiodurans, D. grandis, and D. proteolyticus, as judged from electrophoretic properties and metal cofactors. A single SOD occurred in both D. radiophilus and D. radiopugnans. Deinococcal SODs were either MnSOD, FeSOD or cambialistic Mn/FeSOD. The unique SOD profile of each mesophilic Deinococcus species, multiplicity and metal cofactors, would be valuable in identifying Deinococcus species.

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