Journal of Microbiology

Volume 48(4); August 2010

Journal Article

New Phylogenetic Lineages of the Spirochaetes Phylum Associated with Clathrina Species (Porifera): Sven C. Neulinger , Rüdiger Stöhr , Vera Thiel , Rolf Schmaljohann , Johannes F. Imhoff; J. Microbiol. 2010;48(4):411-418. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0017-x

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Abstract: Though spirochetes have been repeatedly found in marine sponges and other invertebrates, little attention has been paid to the specificity of this association. This study demonstrates that different genoand morphotypes of spirochetes can reside within the same sponge individual and develop in considerable numbers. Specimens of the calcareous sponge Clathrina clathrus collected from the Adriatic Sea off Rovinj (Croatia) were found to harbor spirochete-like bacteria, which were characterized by scanning electron microscopy (SEM), 16S rRNA gene analysis, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Two novel spirochete sequence types related to the Brachyspiraceae could be retrieved. By use of specifically designed CARD-FISH probes, the C. clathrus-associated sequences could be assigned to a linear and a helical spirochete morphotype. Both were located within the sponge mesohyl and resembled the spirochete-like cells identified by SEM. In addition, from a Clathrina sp., most likely C. coriacea, that originated from Indonesian coastal waters, four different spirochete type sequences were recovered. Two of these also affiliated with the Brachyspiraceae, the other two were found associated with the Spirochaetaceae, one with the genera Borrelia and Cristispira.

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

Development of a Latex Agglutination Test for Norovirus Detection: Heetae Lee , YoungBin Park , Misoon Kim , Youngmee Jee , Doo-sung Cheon , Hae Sook Jeong , GwangPyo Ko; J. Microbiol. 2010;48(4):419-425. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0071-4

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Abstract: Norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Currently, reverse transcription polymerase chain reaction (RT-PCR) is used commonly to detect NoVs in both clinical and environmental samples. However, RT-PCR requires expensive equipment and cannot be performed on site. In this study, a latex agglutination test (LAT) using antibody-labeled latex beads for detecting NoVs was developed. Two kinds of polyclonal antibodies, one generated from synthetic peptides and the other from E. coli-expressed NoV capsid proteins, were used to develop the LAT. Each of these polyclonal antibodies was immobilized on the surface of latex beads and tested for the ability to detect NoVs. Under optimized conditions, our LAT detected GII.4 NoV at concentrations as low as 3.3×105 RT-PCR units/ml in stool samples. The detection limit for the LAT was approximately 1.7×103 RT-PCR units. Forty-eight stool samples were tested for NoVs using this LAT. In comparison with an RT-PCR assay, the sensitivity and specificity of the LAT were 35% and 100%, respectively. With further optimization, this LAT used with appropriate antibodies could be applied for convenient detection of NoVs in clinical diagnosis and food monitoring.

Diversity of Cold-Active Protease-Producing Bacteria from Arctic Terrestrial and Marine Environments Revealed by Enrichment Culture: Eun Hye Kim , Kyeung Hee Cho , Yung Mi Lee , Joung Han Yim , Hong Kum Lee , Jang-Cheon Cho , Soon Gyu Hong; J. Microbiol. 2010;48(4):426-432. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0015-z

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25 Citations

Abstract: A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).

Aquimarina litoralis sp. nov., Isolated from a Coastal Seawater: You-Sung Oh , Hyung-Yeel Kahng , Young Sun Lee , Byoung-Jun Yoon , Sang-Bin Lim , Jae Sung Jung , Duck-Chul Oh , Dong-Heon Lee; J. Microbiol. 2010;48(4):433-437. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0088-8

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Abstract: A strictly aerobic, red-pigmented, non-motile, catalase- and oxidase-positive, Gram-staining-negative bacterium, designated strain CNURIC011T, was isolated from seawater off the coast of Jeju Island in Korea. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain CNURIC011T belongs to the genus Aquimarina in the family Flavobacteriaceae. 16S rRNA gene sequence analysis revealed that the close relatives of the novel strain are Aquimarina latercula ATCC 23177T, Aquimarina marcrocephali JAMB N27T, Aquimarina intermedia KMM 6258T, Aquimarina muelleri KMM 6020T, and Aquimarina brevivitae SMK-19T, with sequence similarities of 97.6, 96.6, 96.0, 95.6, and 94.2%, respectively. DNA-DNA hybridization revealed that the level of relatedness between strain CNURIC011T and Aquimarina latercula ATCC 23177T (=KCTC 2912T) was 4.9%. The DNA G+C content was 35.8 mol% and the major respiratory quinone was MK-6. The major fatty acids were iso-C15:0 (14.9%), C15:0 (13.9%), iso-C17:0 3-OH (12.6%), iso-C15:1 G (7.3%), and iso-C17:1 ω9c (7.2%). On the basis of phenotypic, phylogenetic, and genotypic data, strain CNURIC011T represents a novel species within the genus Aquimarina, for which the name Aquimarina litoralis sp. nov. is proposed. The type strain is CNURIC011T (=KCTC 22614T =JCM 15974T).

Glycosylation and Production Characteristics of Epothilones in Alkali-Tolerant Sorangium cellulosum Strain So0157-2: Lin Zhao , Peng-fei Li , Chun-hua Lu , Shu-guang Li , Yue-mao Shen , Yue-zhong Li; J. Microbiol. 2010;48(4):438-444. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0048-3

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Abstract: 3-O-α-D-ribofuranosyl epothilone A (epothiloneoside A) is a major component of glycosylated epothilones in Sorangium cellulosum strain So0157-2. The production and glycosylation ratios of epothiloneoside A in both solid and liquid culture conditions with various pH values and carbon sources were studied. The results showed that glycosylation occurs whenever epothilones are produced, regardless of changes in pH values, production time curves, and different carbon sources. We suggest that glycosylation is a stable process, paralleling the biosynthesis of epothilones in the So0157-2 strain.

Patterns of Survival and Volatile Metabolites of Selected Lactobacillus Strains During Long-Term Incubation in Milk: Łucja Łaniewska-Trokenheim , Magdalena Olszewska , Marta Mik&# , Anna Zadernowska; J. Microbiol. 2010;48(4):445-451. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0056-3

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Abstract: The focus of this study was to monitor the survival of populations and the volatile compound profiles of selected Lactobacillus strains during long-term incubation in milk. The enumeration of cells was determined by both the Direct Epifluorescent Filter Technique using carboxyfluorescein diacetate (CFDA) staining and the plate method. Volatile compounds were analysed by the gas-chromatography technique. All strains exhibited good survival in cultured milks, but Lactobacillus crispatus L800 was the only strain with comparable growth and viability in milk, assessed by plate and epifluorescence methods. The significant differences in cell numbers between plate and microscopic counts were obtained for L. acidophilus strains. The investigated strains exhibited different metabolic profiles. Depending on the strain used, 3 to 8 compounds were produced. The strains produced significantly higher concentrations of acetic acid, compared to other volatiles. Lactobacillus strains differed from one another in number and contents of the volatile compounds.

Purification and Characterization of the α-Glucosidase Produced by Thermophilic Fungus Thermoascus aurantiacus CBMAI 756: Ana Flávia Azevedo Carvalho , Maurício Boscolo , Roberto da Silva , Henrique Ferreira , Eleni Gomes; J. Microbiol. 2010;48(4):452-459. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-9319-2

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Abstract: Αn α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0-9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na+, Ba2+, Co2+, Ni2+, Mg2+, Mn2+, Al3+, Zn2+, Ca2+ this enzyme maintained 90-105% of its maximum activity and was inhibited by Cr3+, Ag+, and Hg2+. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The Km measured for the α-glucosidase was 0.07 μM, with a Vmax of 318.0 μmol/min/mg.

Bacillus megaterium Strain XTBG34 Promotes Plant Growth by Producing 2-Pentylfuran: Changsong Zou , Zhifang Li , Diqiu Yu; J. Microbiol. 2010;48(4):460-466. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0068-z

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Abstract: Several chemical changes in soil are associated with plant growth-promoting rhizobacteria. An endosporeforming bacterium, strain XTBG34, was isolated from a Xishuangbanna Tropical Botanical Garden soil sample and identified as Bacillus megaterium. The strain’s volatiles had remarkable plant growth promotion activity in Arabidopsis thaliana plants; after 15 days treatment, the fresh weight of plants inoculated with XTBG34 was almost 2-fold compared with those inoculated with DH5α. Head space volatile compounds produced by XTBG34, trapped with headspace solid phase microextraction and identified by gas chromatography–mass spectrometry, included aldehydes, alkanes, ketones and aroma components. Of the 11 compounds assayed for plant growth promotion activity in divided Petri plates, only 2-pentylfuran increased plant growth. We have therefore identified a new plant growth promotion volatile of B. megaterium XTBG34, which deserves further study in the mechanisms of interaction between plant growth-promoting rhizobacteria and plants.

Isolation and Characterization of Lactic Acid Bacteria Strains with Ornithine Producing Capacity from Natural Sea Salt: Jin-Ju Yu , Suk-Heung Oh; J. Microbiol. 2010;48(4):467-472. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0204-9

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Abstract: Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO2 production. The isolates grew well on MRS broth at 30-37°C and a pH of 6.5-8.0. The optimum temperature and pH for growth are 37°C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0-6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.

DRA0336, Another OxyR Homolog, Involved in the Antioxidation Mechanisms in Deinococcus radiodurans: Longfei Yin , Liangyan Wang , Huiming Lu , Guangzhi Xu , Huan Chen , Hongdan Zhan , Bing Tian , Yuejin Hua; J. Microbiol. 2010;48(4):473-479. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0043-8

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Abstract: A novel OxyR (DR0615) with one conserved cysteine that senses hydrogen peroxide in Deinococcus radiodurans had been identified in our previous work. Comparative genomics revealed that D. radiodurans possesses another OxyR homolog, OxyR2 (DRA0336). In this study, we constructed the deletion mutant of oxyR2 and the double mutant of both the OxyR homologs to investigate the role of OxyR in response to oxidative stress in D. radiodurans. Deletion of oxyR2 resulted in an obviously increased sensitivity to hydrogen peroxide, and the double mutant for oxyR and oxyR2 was significantly more sensitive than any of the two single mutants. The total catalase activity of the double mutant was lower than that of any of the single mutants, and reactive oxygen species (ROS) accumulated to a greater extent. DNA microarray analysis further suggested that oxyR2 was involved in antioxidation mechanisms. Site-direct mutagenesis and complementation analysis revealed that C228 in OxyR2 was essential. This is the first report of the presence of two OxyR in one organism. These results suggest that D. radiodurans OxyR and OxyR2 function together to protect the cell against oxidative stress.

Differential Expression Profiles of Alternaria alternate Genes in Response to Carbonyl Sulfide Fumigation: Tao Liu , Li Li , Yuejin Wang , Guoping Zhan , Bo Liu; J. Microbiol. 2010;48(4):480-485. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-9301-z

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Abstract: Carbonyl sulfide (COS) is a new fumigant used in phytosanitary treatments. It was developed as a potential alternative to methyl bromide, which is being phased out because of its ozone-depletion properties. To understand the molecular and cellular mechanisms occurring in fungal pathogens in response to COS fumigation, we cloned 510 cDNA fragments of Alternaria alternata (Fr.) Keissler genes that are differentially expressed; these genes were cloned using suppression subtractive hybridization. Changes in the levels of transcripts of 79 fragments were confirmed by microarray analysis and qRT-PCR. Further homology search revealed that they are highly homologous to 41 genes of other fungi, which were related to general metabolism, growth and division, defense, cellular transport, and signal transduction. These results provide an overview of differential expression profiles of A. alternata genes following COS treatment and some new clues about the mechanism of COS fungitoxicity.

Immunological Responses Induced by asd and wzy/asd Mutant Strains of Salmonella enterica serovar Typhimurium in BALB/c Mice: Hong Hua Piao , Vo Thi Minh Tam , Hee Sam Na , Hyun Ju Kim , Phil Youl Ryu , Soo Young Kim , Joon Haeng Rhee , Hyon E. Choy , Suhng Wook Kim , Yeongjin Hong; J. Microbiol. 2010;48(4):486-495. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0023-z

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Abstract: Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate ß- semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer’s patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 106 higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1×1010 CFU) or i.p. (1×107 CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD50 (5×106 CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.

Inhibitory Effect of the Essential Oil from Chamaecyparis obtusa on the Growth of Food-Borne Pathogens: Mi-Jin Park , Won-Sil Choi , Ha-Young Kang , Ki-Seob Gwak , Geun-Shik Lee , Eui-Bae Jeung , In-Gyu Choi; J. Microbiol. 2010;48(4):496-501. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-9327-2

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Abstract: In this study, the antibacterial activity of essential oil from Chamaecyparis obtusa (Sieb. et Zucc) leaves and twigs was investigated. The test strains were Klebsiella pneumoniae, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, Legionella pneumophila, and Methicilline-resistant Staphylococcus aureus. Antibacterial activity was estimated by measuring bacterial growth inhibition. Histopathological examination was also performed. C. obtusa oil distinctly inhibited the growth of all test strains and exhibited the strongest antibacterial activity against L. monocytogenes. It was chromatographically divided into several fractions. The fractions were further tested against antibacterial activity and their chemical compositions were analyzed. The fraction containing terpinen-4-ol (TA) showed high antibacterial activity toward all strains tested. Tests with authentic samples showed that TA played a major role in the antibacterial activity of C. obtusa oil, and in a mice test, the oil actively minimized inflammation by S. aureus.

Characterization and Identification of Distinct Mycobacterium massiliense Extracellular Proteins from Those of Mycobacterium abscessus: A-Rum Shin , Hosung Sohn , Choul Jae Won , Byungsoo Lee , Woo Sik Kim , Hyun Bae Kang , Hwa-Jung Kim , Sang Nae Cho , Sung Jae Shin; J. Microbiol. 2010;48(4):502-511. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0038-5

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Abstract: Mycobacterium massiliense is an emerging pathogen and very similar to Mycobacterium abscessus of rapidly growing mycobacteria in the phenotype and genotype. Pathogenic bacteria secrete a diversity of factors into extracellular medium which contribute to the bacterial pathogenicity. In the present study, we performed the comparative proteome analysis of culture filtrate proteins from a clinical isolate of M. massiliense and M. abscessus strains using two-dimensional gel electrophoresis and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Interestingly, 9 proteins of M. massiliense were distinctly expressed from those of M. abscessus. Bioinformatic analysis of the identified proteins revealed that 3 unique proteins corresponded to serine/arginine rich protein, membrane protein from Streptomyces coelicolor, and one hypothetical protein from Corynebacterium efficiens YS-314, respectively. Culture filtrate proteins from M. massiliense induced the release of pro-inflammatory cytokines from macrophages in a dose-dependent manner but not that from M. abscessus. Taken together, the functional study on the identified proteins uniquely produced from M. massiliense may provide not only the clues for the different pathogensis, but also help develop the diagnostic tools for the differentiation between two mycobacterial species.

Newly Identified CpG ODNs, M5-30 and M6-395, Stimulate MouseNewly Identified CpG ODNs, M5-30 and M6-395, Stimulate Mouse Immune Cells to Secrete TNF-α and Enhance Th1-Mediated Immunity: Sun-Shim Choi , Eunkyung Chung , Yu-Jin Jung; J. Microbiol. 2010;48(4):512-517. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-0053-6

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Abstract: Bacterial CpG motifs are known to induce both innate and adaptive immunity in infected hosts via toll-like receptor 9 (TLR9). Because small oligonucleotides (ODNs) mimicking bacterial CpG motifs are easily synthesized, they have found use as immunomodulatory agents in a number of disease models. We have developed a novel bioinformatics approach to identify effective CpG ODN sequences and evaluate their function as TLR9 ligands in a murine system. Among the CpG ODNs we identified, M5-30 and M6-395 showed significant ability to stimulate TNF-α and IFN-γ production in a mouse macrophage cell line and mouse splenocytes, respectively. We also found that these CpG ODNs activated cells through the canonical NF-κB signaling pathway. Moreover, both CpG ODNs were able to induce Th1-mediated immunity in Mycobacterium tuberculosis (Mtb)-infected mice. Our results demonstrate that M5-30 and M6-395 function as TLR9-specific ligands, making them useful in the study of TLR9 functionality and signaling in mice.

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