Journal of Microbiology

Volume 50(4); August 2012

Review

REVIEW] Cell Migration: Regulation of Cytoskeleton by Rap1 in Dictyostelium discoideum: Mi-Rae Lee , Taeck J. Jeon; J. Microbiol. 2012;50(4):555-561. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2246-7

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31 Citations

Abstract: Cell movement involves a coordinated regulation of the cytoskeleton, F-actin-mediated protrusions at the front and myosin-mediated contraction of the posterior of a cell. The small GTPase Rap1 functions as a key regulator in the spatial and temporal control of cytoskeleton reorganization for cell migration. This review outlines the establishment of cell polarity by differential localizations of the cytoskeleton and discusses the spatial and temporal regulation of cytoskeleton
reorganization via the Rap1 signaling pathway during chemotaxis with a focus on recent advances in the study of chemotaxis using a simple eukaryotic model organism, Dictyostelium discoideum.

Journal Article

Flavobacterium cheonhonense sp. nov., Isolated from a Freshwater Reservoir: Siwon Lee , Jung-Hwan Oh , Hang-Yeon Weon , Tae-Young Ahn; J. Microbiol. 2012;50(4):562-566. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-1229-z

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12 Citations

Abstract: A novel bacterium, designated strain ARSA-15T, was isolated from a freshwater sample collected from the Cheonho reservoir, Cheonan, Republic of Korea. The isolate was deepyellow pigment, Gram-negative, rod-shaped, non-motile, and catalase- and oxidase-positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belongs to the genus Flavobacterium, and shared less than 97% sequence similarity with recognized Flavobacterium species. The novel species was able to grow at 10–37°C, pH 6.5–10.0, and in 0–0.5% (w/v) NaCl concentrations. Chemotaxonomically, iso-C15:1, iso-C15:0, and iso-C16:0 were observed to be the predominant cellular fatty acid, and menaquinone-6 (MK-6) was the predominant respiratory quinone. The major polar lipid patterns of strain ARSA-19T was phosphatidylethanolamine, unknown aminolipid (AL1 and AL2), and unidentified polar lipids (L1, L2, and L3). The genomic DNA G+C content of the isolate was 39.2 mol%. On the basis of polyphasic approach, strain ARSA-15T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium cheonhonense sp. nov. is proposed. The type strain is ARSA-15T (=KACC 14967T =KCTC 23180T =JCM 17064T).

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

Parallel Gene Loss and Acquisition Among Strains of Different Brucella Species and Biovars: Zhijun Zhong , Yufei Wang , Jie Xu , Yanfen Chen , Yuehua Ke , Xiaoyan Zhou , Xitong Yuan , Dongsheng Zhou , Yi Yang , Ruifu Yang , Guangneng Peng , Hai Jiang , Jing Yuan , Hongbin Song , Buyun Cui , Liuyu Huang , Zeliang Chen; J. Microbiol. 2012;50(4):567-574. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2022-8

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9 Citations

Abstract: The genus Brucella is divided into six species; of these, B. melitensis and B. abortus are pathogenic to humans, and B. ovis and B. neotomae are nonpathogenic to humans. The definition of gene loss and acquisition is essential for understanding Brucella’s ecology, evolutionary history, and host relationships. A DNA microarray containing unique genes of B. melitensis Type strain 16MT and B. abortus 9-941 was constructed and used to determine the gene contents of the representative strains of Brucella. Phylogenetic relationships were inferred from sequences of housekeeping genes. Gene loss and acquisition of different Brucella species were inferred. A total of 214 genes were found to be differentially distributed, and 173 of them were clustered into 15 genomic islands (GIs). Evidence of horizontal gene transfer was observed for 10 GIs. Phylogenetic analysis indicated that the 19 strains formed five clades, and some of the GIs had been lost or acquired independently among the different lineages. The derivation of Brucella lineages is concomitant with the parallel loss or acquisition of GIs, indicating a complex interaction between various Brucella species and hosts.

Thionine Increases Electricity Generation from Microbial Fuel Cell Using Saccharomyces cerevisiae and Exoelectrogenic Mixed Culture: Mostafa Rahimnejad , Ghasem Darzi Najafpour , Ali Asghar Ghoreyshi , Farid Talebnia , Giuliano C. Premier , Gholamreza Bakeri , Jung Rae Kim , Sang-Eun Oh; J. Microbiol. 2012;50(4):575-580. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2135-0

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90 Citations

Abstract: Microbial fuel cells (MFCs) have been shown to be capable of clean energy production through the oxidation of biodegradable organic waste using various bacterial species as biocatalysts. In this study we found Saccharomyces cerevisiae, previously known electrochemcially inactive or less active species, can be acclimated with an electron mediator thionine for electrogenic biofilm formation in MFC, and electricity production is improved with facilitation of electron transfer. Power generation of MFC was also significantly increased by thionine with both aerated and non-aerated cathode. With electrochemically active biofilm enriched with swine wastewater, MFC power increased more significantly by addition of thionine. The optimum mediator concentration was 500 mM of thionine with S. cerevisae in MFC with the maximum voltage and current generation in the microbial fuel cell were 420 mV and 700 mA/m2, respectively. Cyclic voltametry shows that thionine improves oxidizing and reducing capability in both pure culture and acclimated biofilm as compared to non-mediated cell. The results obtained indicated that thionine has great potential to enhance power generation from unmediated yeast or electrochemically active biofilm in MFC.

Enhancement of Anti-candidal Activity of Endophytic Fungus Phomopsis sp. ED2, Isolated from Orthosiphon stamineus Benth, by Incorporation of Host Plant Extract in Culture Medium: Tong Woei Yenn , Chong Chai Lee , Darah Ibrahim , Latiffah Zakaria; J. Microbiol. 2012;50(4):581-585. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-2083-8

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25 Citations

Abstract: This study examined the effect of host extract in the culture medium on anti-candidal activity of Phomopsis sp. ED2, previously isolated from the medicinal herb Orthosiphon stamineus Benth. Interestingly, upon addition of aqueous host extract to the culture medium, the ethyl acetate extract prepared from fermentative broth exhibited moderate anticandidal activity in a disc diffusion assay. The minimal inhibitory concentration of this extract was 62.5 μg/ml and it only exhibited fungistatic activity against C. albicans. In the time-kill study, a 50% growth reduction of C. albicans was observed at 31.4 h for extract from the culture incorporating host extract. In the bioautography assay, only one single spot (Rf 0.59) developed from the extract exhibited anti-candidal activity. A spot with the a similar Rf was not detected for the crude extract from YES broth without host extract. This indicated that the terpenoid anti-candidal compound was only produced when the host extract was introduced into the medium. The study concluded that the incorporation of aqueous extract of the host plant into the culture medium significantly enhanced the anti-candidal activity of Phomopsis sp. ED2.

Copper as an Antimicrobial Agent against Opportunistic Pathogenic and Multidrug Resistant Enterobacter Bacteria: Wen-Xiao Tian , Shi Yu , Muhammad Ibrahim , Abdul Wareth Almonaofy , Liu He , Qiu Hui , Zhu Bo , Bin Li , Guan-lin Xie; J. Microbiol. 2012;50(4):586-593. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-2067-8

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44 Citations

Abstract: Infections by Enterobacter species are common and are multidrug resistant. The use of bactericidal surface materials such as copper has lately gained attention as an effective antimicrobial agent due to its deadly effects on bacteria, yeast, and viruses. The aim of the current study was to assess the antibacterial activity of copper surfaces against Enterobacter species. The antibacterial activity of copper surfaces was tested by overlying 5×106 CFU/ml suspensions of representative Enterobacter strains and comparing bacterial survival counts on copper surfaces at room temperature. Iron, stainless steel, and polyvinylchloride (PVC) were used as controls. The mechanisms responsible for bacterial killing on copper surfaces were investigated by a mutagenicity assay of the D-cycloserin (cyclA gene), single cell gel electrophoresis, a staining technique, and inductively coupled plasma mass spectroscopy. Copper yielded a significant decrease in the viable bacterial counts at 2 h exposure and a highly significant decrease at 4 h. Loss of cell integrity and a significantly higher influx of copper into bacterial cells exposed to copper surfaces, as compared to those exposed to the controls, were documented. There was no increase in mutation rate and DNA damage indicating that copper contributes to bacterial killing by adversely affecting cellular structure without directly targeting the genomic DNA. These findings suggest that copper’s antibacterial activity against Enterobacter species could be utilized in health care facilities and in food processing plants to reduce the bioburden, which would increase protection for susceptible members of the community.

Involvement of Alternative Oxidase in the Regulation of Growth, Development, and Resistance to Oxidative Stress of Sclerotinia sclerotiorum: Ting Xu , Fei Yao , Wu-Sheng Liang , Yong-Hong Li , Dian-Rong Li , Hao Wang , Zheng-Yi Wang; J. Microbiol. 2012;50(4):594-602. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2015-7

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37 Citations

Abstract: Sclerotinia sclerotiorum is a cosmopolitan, filamentous, fungal pathogen that can cause serious disease in many kinds of crops. Alternative oxidase is the terminal oxidase of the alternative mitochondrial respiratory pathway in fungi and higher plants. We report the presence of this alternative pathway respiration and demonstrate its expression in two isolates of S. sclerotiorum under unstressed, normal culture conditions. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, severely inhibited the mycelial growth of S. sclerotiorum both on potato dextrose agar plates and in liquid culture media. Inhibition of alternative oxidase could influence the growth pattern of S. sclerotiorum, as salicylhydroxamic acid treatment induced obvious aerial mycelia growing on potato dextrose agar plates. Under the treatment with salicylhydroxamic acid, S. sclerotiorum formed sclerotia much more slowly than the control. Treatment with hydrogen peroxide in millimolar concentrations greatly decreased the growth rate of mycelia and delayed the formation of sclerotia in both tested S. sclerotiorum isolates. As well, this treatment obviously increased their alternative pathway respiration and the levels of both mRNA and protein of the alternative oxidase. These results indicate that alternative oxidase is involved in the regulation of growth, development, and resistance to oxidative stress of S. sclerotiorum.

Journal Article

Screening for Probiotic Properties of Strains Isolated from Feces of Various Human Groups: Sathyaseelan Sathyabama , Rajendran Vijayabharathi , Palanisamy Bruntha devi , Manohar Ranjith kumar , Venkatesan Brindha Priyadarisini; J. Microbiol. 2012;50(4):603-612. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-2045-1

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49 Citations

Abstract: The present study searched for potential probiotic strains from various human fecal samples. A total of 67 aerobic and 38 anaerobic strains were isolated from 5 different categories of human feces. Systematic procedures were used to evaluate the probiotic properties of the isolated strains. These showed about 75–97% survivability in acidic and bile salt environments. Adhesion to intestinal cell line Caco-2 was also high. The isolates exhibited hydrophobic properties in hexadecane. The culture supernatants of these strains showed antagonistic effects against pathogens. The isolates were resistant to a simulated gastrointestinal environment in vitro. Of the 4 best isolates, MAbB4 (Staphylococcus succinus) and FIdM3 (Enterococcus fecium), were promising candidates for a potential probiotic. S. succinus was found to be a probiotic strain, which is the second such species reported to date in this particular genus. A substantial zone of inhibition was found against Salmonella spp., which adds further support to the suggestion that the probiotic strain could help prevent intestinal infection. This study suggested that the human flora itself is a potential source of probiotics.

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

Lactobacillus delbrueckii ssp. lactis R4 Prevents Salmonella typhimurium SL1344-Induced Damage to Tight Junctions and Adherens Junctions: Qinghua Yu , Liqi Zhu , Zhisheng Wang , Pengcheng Li , Qian Yang; J. Microbiol. 2012;50(4):613-617. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-1596-5

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17 Citations

Abstract: Cell junctions are the gatekeepers of the paracellular route and defend the mucosal barrier. Several enteropathogenic bacteria can invade intestinal epithelial cells by targeting and damaging cell junctions. It is not well understood how Salmonella typhimurium is able to overcome the intestinal barrier and gain access to the circulation, nor is it understood how Lactobacillus prevents the invasion of S. typhimurium. Therefore, we sought to determine whether infection with S. typhimurium SL1344 could regulate the molecular composition of cell junctions and whether Lactobacillus delbrueckii ssp. lactis R4 could affect this modification. Our data demonstrated that infection of Caco-2 cells with S. typhimurium over 2 h resulted in a redistribution of claudin-1, ZO-1, occluding, and E-cadherin. Western blot analysis of epithelial cell lysates demonstrated that S. typhimurium could decrease the expression of cell junction proteins. However, L. delbrueckii ssp. lactis R4 ameliorated this destruction and induced increased expression of ZO-1, occludin, and E-cadherin relative to the levels in the control group. The results of these experiments implied that S. typhimurium may facilitate its uptake and distribution within the host by regulating the molecular composition of cell junctions. Furthermore, Lactobacillus may prevent the adhesion and invasion of pathogenic bacteria by maintaining cell junctions and the mucosal barrier.

The Role of a Dark Septate Endophytic Fungus, Veronaeopsis simplex Y34, in Fusarium Disease Suppression in Chinese Cabbage: Rida O. Khastini , Hiroyuki Ohta , Kazuhiko Narisawa; J. Microbiol. 2012;50(4):618-624. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2105-6

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35 Citations

Abstract: The soil-inhabiting fungal pathogen Fusarium oxysporum has been an increasing threat to Chinese cabbage (Brassica campestris L.). A dark septate endophytic fungus, Veronaeopsis simplex Y34, isolated from Yaku Island, Japan, was evaluated in vitro for the ability to suppress Fusarium disease. Seedlings grown in the presence of the endophyte showed a 71% reduction in Fusarium wilt disease and still had good growth. The disease control was achieved through a synergetic effect involving a mechanical resistance created by a dense network of V. simplex Y34 hyphae, which colonized the host root, and siderophore production acting indirectly to induce a resistance mechanism in the plant. Changes in the relative abundance of the fungal communities in the soil as determined by fluorescently labelled T-RFs (terminal restriction fragments), appeared 3 weeks after application of the fungus. Results showed the dominance of V. simplex Y34, which became established in the rhizosphere and out-competed F. oxysporum.

cDNA Cloning of Korean Human Norovirus and Nucleotidylylation of VPg by Norovirus RNA-Dependent RNA Polymerase: Byung Sup Min , Kang Rok Han , Jung Ihn Lee , Jai Myung Yang; J. Microbiol. 2012;50(4):625-630. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2087-4

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4 Citations

Abstract: Norovirus, a member of the Caliciviridae family, is a major causative agent of gastroenteritis worldwide. The cDNA of the entire genome of human norovirus (HuNV) was cloned using the RNA extracted from the stool sample of a Korean patient. The RNA genome consists of 7,559 nucleotides, carries 3 open reading frames (ORFs), 5' and 3' noncoding regions, and a poly(A) tail at the 3' end. Phylogenic analysis of the nucleotide sequence indicated that it belongs to GII.4, the most dominant genogroup. To analyze RNA synthesis and nucleotidylylation of VPg by RNA-dependent RNA polymerase (RdRp), recombinant RdRp and VPg were expressed in Escherichia coli as His-tagged forms. The HuNV RdRp exhibited template and divalent cation-dependent RNA synthesis in vitro. The HuNV RdRp nucleotidylylated HuNV VPg but not murine norovirus (MNV) VPg, whereas MNV RdRp nucleotidylylated both MNV and HuNV VPg more efficiently than HuNV RdRp.

Structural and Functional Importance of Outer Membrane Proteins in Vibrio cholerae Flagellum: Wasimul Bari , Kang-Mu Lee , Sang Sun Yoon; J. Microbiol. 2012;50(4):631-637. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2116-3

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16 Citations

Abstract: Vibrio cholerae has a sheath-covered monotrichous flagellum that is known to contribute to virulence. Although the structural organization of the V. cholerae flagellum has been extensively studied, the involvement of outer membrane proteins as integral components in the flagellum still remains elusive. Here we show that flagella produced by V. cholerae O1 El Tor strain C6706 were two times thicker than those from two other Gram-negative bacteria. A C6706 mutant strain (SSY11) devoid of two outer membrane proteins (OMPs), OmpU and OmpT, produced thinner flagella. SSY11 showed significant defects in the flagella-mediated motility as compared to its parental strain. Moreover, increased shedding of the flagella-associated proteins was observed in the culture supernatant of SSY11. This finding was also supported by the observation that culture supernatants of the SSY11 strain induced the production of a significantly higher level of IL-8 in human colon carcinoma HT29 and alveolar epithelial A549 cells than those of the wild-type C6706 strain. These results further suggest a definite role of these two OMPs in providing the structural integrity of the V. cholerae flagellum as part of the surrounding sheath.

Novel Bifidobacterium Promoters Selected Through Microarray Analysis Lead to Constitutive High-Level Gene Expression: Yan Wang , Jin Yong Kim , Myeong Soo Park , Geun Eog Ji; J. Microbiol. 2012;50(4):638-643. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-1591-x

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13 Citations

Abstract: For the development of a food-grade expression system for Bifidobacterium, a strong promoter leading to high-level expression of cloned gene is a prerequisite. For this purpose, a promoter screening host-vector system for Bifidobacterium has been established using β-glucosidase from Bifidobacterium lactis as a reporter and Bifidobacterium bifidum BGN4 as a host, which is β-glucosidase negative strain. Seven putative promoters showing constitutive high-level expression were selected through microarray analysis based on the genome sequence of B. bifidum BGN4. They were cloned into upstream of β-glucosidase gene and transformed into Escherichia coli DH5α and B. bifidum BGN4. Promoter activities were analyzed both in E. coli and B. bifidum BGN4 by measuring β-glucosidase activity. β-Glucosidase activities in all of the transformants showed growth-associated characteristics. Among them, P919 was the strongest in B. bifidum BGN4 and showed maximum activity at 18 h, while P895 was the strongest in E. coli DH5α at 7 h. This study shows that novel strong promoters such as P919 can be used for high-level expression of foreign genes in Bifidobacterium and will be useful for the construction of an efficient food-grade expression system.

Phospholipase A2 Inhibitors in Bacterial Culture Broth Enhance Pathogenicity of a Fungus Nomuraea rileyi: Jung-A Park , Yonggyun Kim; J. Microbiol. 2012;50(4):644-651. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-2108-3

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10 Citations

Abstract: An entomopathogenic fungus, Nomuraea rileyi, was isolated and its identity was confirmed by its internal transcribed spacer DNA sequence. The isolated N. rileyi exhibited a specific pathogenicity to lepidopteran species. This study was focused on enhancing the fungal pathogenicity by using immunosuppressive agents. In response to infection of N. rileyi, Spodoptera exigua larvae significantly induced catalytic activity of phospholipase A2 (PLA2) in three immune-associated tissues, namely hemocytes, fat body, and hemolymph plasma. Furthermore, the infected S. exigua larvae induced transcription of several antimicrobial peptide (AMP) genes. Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata subsp. temperata (Ptt), possessed specific PLA2-inhibitory activities and their culture broths significantly inhibited the enzyme activities in hemocytes, fat body, and plasma of S. exigua. In addition, the bacterial metabolites inhibited transcription of AMP genes in S. exigua that would normally respond to the immune challenge by N. rileyi. The immunosuppressive effect of Xn or Ptt bacterial broth resulted in significant enhancement of the fungal pathogenicity against late instar larvae of S. exigua and Plutella xylostella. The effect of such a mixture was confirmed by field assay against two lepidopteran species. These results suggest that the bacterial and fungal mixture can be applied to develop a novel biopesticide to control lepidopteran species.

Screening, Purification, and Characterization of an Extracellular Prolyl Oligopeptidase from Coprinopsis clastophylla: Jen-Tao Chen , Mei-Li Chao , Chiou-Yen Wen , Wen-Shen Chu; J. Microbiol. 2012;50(4):652-659. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2099-0

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7 Citations

Abstract: Culture filtrates of 22 mushrooms were screened for extracellular prolyl oligopeptidase activity. Four strains with relatively high enzyme activity were all from inky cap mushrooms. The production of Coprinopsis clastophylla prolyl oligopeptidase was associated with the growth of the fungus and the enzyme was not released by cell lysis. The enzyme was purified 285-fold to a specific activity of 52.05 U/mg. It was purified to a single band on a native polyacrylamide gel. However, the enzyme separated into three bands on a sodium dodecyl sulfate-polyacrylamide gel with mobility corresponding to molecular weights of approximately 84, 60, and 26 kDa. The results of tandem mass spectrometric analysis revealed that the 60 kDa protein was likely a degradation product of the 84 kDa protein. The isoelectric point of the purified enzyme was 5.2. The purified enzyme had an optimal pH and temperature of 8.0 and 37°C, respectively. Diisopropyl fluorophosphate (DFP), p-chloromercuribenzoaic acid (PCMB), Hg2+, and Cu2+ strongly inhibited C. clastophylla prolyl oligopeptidase. This enzyme is a serine peptidase and one or more cysteine residues of the enzyme are close to the active site.

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