Journal of Microbiology

Volume 50(2); April 2012

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

TBC: A Clustering Algorithm Based on Prokaryotic Taxonomy: Jae-Hak Lee , Hana Yi , Yoon-Seong Jeon , Sungho Won , Jongsik Chun; J. Microbiol. 2012;50(2):181-185. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1214-6

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19 Citations

Abstract: High-throughput DNA sequencing technologies have revolutionized the study of microbial ecology. Massive sequencing of PCR amplicons of the 16S rRNA gene has been widely used to understand the microbial community structure of a variety of environmental samples. The resulting sequencing reads are clustered into operational taxonomic units that are then used to calculate various statistical indices that represent the degree of species diversity in a given sample. Several algorithms have been developed to perform this task, but they tend to produce different outcomes. Herein, we propose a novel sequence clustering algorithm, namely Taxonomy-Based Clustering (TBC). This algorithm incorporates the basic concept of prokaryotic taxonomy in which only comparisons to the type strain are made and used to form species while omitting full-scale multiple sequence alignment. The clustering quality of the proposed method was compared with those of MOTHUR, BLASTClust, ESPRITTree, CD-HIT, and UCLUST. A comprehensive comparison using three different experimental datasets produced by pyrosequencing demonstrated that the clustering obtained using TBC is comparable to those obtained using MOTHUR and ESPRIT-Tree and is computationally efficient. The program was written in JAVA and is available from http://sw. ezbiocloud.net/tbc.

Identification and Enumeration of Microcystis Using a Sandwich Hybridization Assay: Jing Ping Zhu , Xian Li , Shi Du; J. Microbiol. 2012;50(2):186-190. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1418-9

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Abstract: Based on sequence analyses of phycocyanin intergenic spacers (PC-IGS) from Microcystis, Anabaena, Aphanizomenon, and Planktothrix (Oscillatoria) strains, a genus-specific probe pair TF/TR was designed, and a sandwich hybridization assay was established to quantitatively detect Microcystis. Through BLAST and cyanobacterial culture tests, TF/TR was demonstrated to be specific for Microcystis. A calibration curve for the sandwich hybridization assay was established, and the lowest detected concentration was 100 cell/ml. Laboratory and field samples were analyzed with both sandwich hybridization assay and microscopy. The biotic and abiotic components of the samples were of little disturbance to the sandwich hybridization assay. The results showed no distinct difference between the two methods. In this study, a sandwich hybridization assay was established to detect Microcystis, providing an alternative to traditional microscopic, morphology- based methods.

Isolation and Characterization of Plant Growth-Promoting Rhizobacteria from Wheat Roots by Wheat Germ Agglutinin Labeled with Fluorescein Isothiocyanate: Jian Zhang , Jingyang Liu , Liyuan Meng , Zhongyou Ma , Xinyun Tang , Yuanyuan Cao , Leni Sun; J. Microbiol. 2012;50(2):191-198. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1472-3

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40 Citations

Abstract: Thirty-two isolates were obtained from wheat rhizosphere by wheat germ agglutinin (WGA) labeled with fluorescein isothiocyanate (FITC). Most isolates were able to produce indole acetic acid (65.6%) and siderophores (59.3%), as well as exhibited phosphate solubilization (96.8%). Fourteen isolates displayed three plant growth-promoting traits. Among these strains, two phosphate-dissolving ones, WS29 and WS31, were evaluated for their beneficial effects on the early growth of wheat (Triticum aestivum Wan33). Strain WS29 and WS31 significantly promoted the development of lateral roots by 34.9% and 27.6%, as well as increased the root dry weight by 25.0% and 25.6%, respectively, compared to those of the control. Based on 16S rRNA gene sequence comparisons and phylogenetic positions, both isolates were determined to belong to the genus Bacillus. The proportion of isolates showing the properties of plant growth-promoting rhizobacteria (PGPR) was higher than in previous reports. The efficiency of the isolation of PGPR strains was also greatly increased by WGA labeled with FITC. The present study indicated that WGA could be used as an effective tool for isolating PGPR strains with high affinity to host plants from wheat roots. The proposed approach could facilitate research on biofertilizers or biocontrol agents.

Journal Article

Detecting Nonculturable Bacteria in the Active Mycorrhizal Zone of the Pine Mushroom Tricholoma matsutake: Ryota Kataoka , Zaki Anwar Siddiqui , Junichi Kikuchi , Masaki Ando , Rina Sriwati , Ai Nozaki , Kazuyoshi Futai; J. Microbiol. 2012;50(2):199-206. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1371-7

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36 Citations

Abstract: The fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a ‘shiro’. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent
methods
. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.

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

Molecular Analysis of Spatial Variation of Iron-Reducing Bacteria in Riverine Alluvial Aquifers of the Mankyeong River: So-Jeong Kim , Dong-Chan Koh , Soo-Je Park , In-Tae Cha , Joong-Wook Park , Jong-Hwa Na , Yul Roh , Kyung-Seok Ko , Kangjoo Kim , Sung-Keun Rhee; J. Microbiol. 2012;50(2):207-217. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1342-z

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38 Citations

Abstract: Alluvial aquifers are one of the mainwater resources in many countries. Iron reduction in alluvial aquifers is often a major anaerobic process involved in bioremediation or causing problems, including the release of As trapped in Fe(III) oxide. We investigated the distribution of potential iron-reducing bacteria (IRB) in riverine alluvial aquifers (B1, B3, and B6 sites) at the Mankyeong River, Republic of Korea. Inactive iron reduction zones, the diversity and abundance of IRB can be examined using a clone library and quantitative PCR analysis of 16S rRNA genes. Geobacter spp. are potential IRB in the iron-reducing zone at the B6 (9 m) site, where high Fe(II) and arsenic (As) concentrations were observed. At the B3 (16 m) site, where low iron reduction activity was predicted, a dominant clone (10.6%) was 99% identical in 16S rRNA gene sequence with Rhodoferax ferrireducens. Although a major clone belonging to Clostridium spp. was found, possible IRB candidates could not be unambiguously determined at the B1 (18 m) site. Acanonical correspondence analysis demonstrated that, among potential IRB, only the Geobacteraceae were well correlated with Fe(II) and As concentrations. Our results indicate high environmental heterogeneity, and thus high spatial variability, in thedistribution of potential IRB in the riverine alluvial aquifersnear the Mankyeong River.

Microbial Fingerprinting Detects Unique Bacterial Communities in the Faecal Microbiota of Rats with Experimentally-Induced Colitis: Ashis K. Samanta , Valeria A. Torok , Nigel J. Percy , Suzanne M. Abimosleh , Gordon S. Howarth; J. Microbiol. 2012;50(2):218-225. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1362-8

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43 Citations

Abstract: An abnormal composition of the gut microbiota is believed to be associated with the pathogenesis of inflammatory bowel disease (IBD). We utilized terminal restriction fragment length polymorphism (T-RFLP) analysis to quantify faecal bacterial communities from rats with experimental colitis. Male Sprague Dawley rats (n=10/group) ingested 2% dextran sulfate sodium (DSS) or water for up to 7 days. Rats were killed and colonic tissues collected for histological analysis. Damage severity score in the distal colon was significantly greater (P<0.001) following DSS consumption compared to controls. T-RFLP faecal bacterial profiles generated with either MspI or CfoI revealed a significant difference (P<0.001) in community composition between healthy and colitic rats, with bacterial composition in healthy rats more variable than in rats with colitis. Operational taxonomic units (OTU: taxonomically related groups of bacteria) associated with either the healthy or colitic state were identified. OTU (116, 226, 360, and 948; CfoI) and (118 and 188; MspI) were strongly associated with untreated healthy rats, while OTU (94, 98, 174, and 384; CfoI) and (94 and 914; MspI) were predominantly associated with DSS-treated colitic rats. Phylogenetic OTU assignment suggested that Bacteroidales and Lactobacillus sp. were predominantly associated with the colitic and healthy rats, respectively. These
results
show that faecal bacterial profiling is a rapid, sensitive and non-invasive tool for detecting and identifying changes in gut microbiota associated with colitis. Restoring microbial homeostasis by targeting colitis-associated OTU through specific microbiological interventions could form the basis of novel therapeutic strategies for IBD.

Effect of Natural Mediators on the Stability of Trametes trogii Laccase during the Decolourization of Textile Wastewaters: Rim Khlifi-Slama , Tahar Mechichi , Sami Sayadi , Abdelhafidh Dhouib; J. Microbiol. 2012;50(2):226-234. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1421-1

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26 Citations

Abstract: The purpose of the present study was to determine the effect of natural mediators on the stability of the Trametes trogii crude laccase in the process of decolourization of textile effluents. Acetosyringone allowed the highest wastewaters decolourization rate of 25%. At higher concentrations of acetosyringone, the relative activity of laccase decreased approximately by between 38% and 88% after 5 days of incubation. T. trogii laccase was strongly inactivated at 3 mM syringaldehyde, after 3 days of incubation. However, laccase activity is more stable in the presence of the vanillin and m-coumarate. The T. trogii growth on solid effluentbased- medium was examined and evaluated by measuring the colony diameter in cm. T. trogii was completely inhibited on 100:0 and 80:20 effluent:water solid medium, however, colony diameter reached 5 cm on 60:40 effluent:water solid medium after 13–14 days incubation. When the textile effluent was pre-treated with laccase and laccase-acetosyringone system, the colony diameter of 2 cm of T. trogii on 80:20 effluent:water solid medium was reached after 14 and 10 days of incubation respectively.

Purification and Structure Analysis of Mycolic Acids in Corynebacterium glutamicum: Yang Yang , Feng Shi , Guanjun Tao , Xiaoyuan Wang; J. Microbiol. 2012;50(2):235-240. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1459-0

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22 Citations

Abstract: Corynebacterium glutamicum is widely used for producing amino acids. Mycolic acids, the major components in the cell wall of C. glutamicum might be closely related to the secretion of amino acids. In this study, mycolic acids were extracted from 5 strains of C. glutamicum, including ATCC 13032, ATCC 13869, ATCC 14067, L-isoleucine producing strain IWJ-1, and L-valine producing strain VWJ-1. Structures of these mycolic acids were analyzed using thin layer chromatography and electrospray ionization mass spectrometry. More than twenty molecular species of mycolic acid were observed in all 5 strains. They differ in the length (20–40 carbons) and saturation (0–3 double bonds) of their constituent fatty acids. The dominant species of mycolic acid in every strain was different, but their two hydrocarbon chains were similar in length (14–18 carbons), and the meromycolate chain usually contained double bonds. As the growth temperature of cells increased from 30°C to 34°C, the proportion of mycolic acid species containing unsaturated and shorter hydrocarbon chains increased. These results provide new information on mycolic acids in C. glutamicum, and could be useful for modifying the cell wall to increase the production of amino acids.

Effects of Exopolysaccharide Production on Liquid Vegetative Growth, Stress Survival, and Stationary Phase Recovery in Myxococcus xanthus: Wei Hu , Jing Wang , Ian McHardy , Renate Lux , Zhe Yang , Yuezhong Li , Wenyuan Shi; J. Microbiol. 2012;50(2):241-248. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1349-5

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11 Citations

Abstract: Exopolysaccharide (EPS) of Myxococcus xanthus is a wellregulated cell surface component. In addition to its known functions for social motility and fruiting body formation on solid surfaces, EPS has also been proposed to play a role in multi-cellular clumping in liquid medium, though this phenomenon has not been well studied. In this report, we confirmed that M. xanthus clumps formed in liquid were correlated with EPS levels and demonstrated that the EPS encased cell clumps exhibited biofilm-like structures. The clumps protected the cells at physiologically relevant EPS concentrations, while cells lacking EPS exhibited significant reduction in long-term viability and resistance to stressful conditions. However, excess EPS production was counterproductive to vegetative growth and viable cell recovery declined in extended late stationary phase as cells became trapped in the matrix of clumps. Therefore, optimal EPS production by M. xanthus is important for normal physiological functions in liquid.

Development of a Suicidal Vector-Cloning System Based on Butanal Susceptibility Due to an Expression of YqhD Aldehyde Reductase: Changhan Lee , Chankyu Park; J. Microbiol. 2012;50(2):249-255. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1438-5

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4 Citations

Abstract: Previously, we observed butanal/propanal sensitivity of Escherichia coli K-12 when cells overexpress YqhD protein, a NADPH dependent aldehyde reductase, possibly due to an accumulation of butanol/propanol in vivo as the reaction products. Based on this finding, we developed a suicidal vector-cloning system derived from pUC19, in which lacZ was substituted with the yqhD gene. As a result, when foreign DNA was inserted into its multiple cloning sites by disrupting an expression of YqhD, the recombinants survived on butanal/propanal containing plate, whereas cells containing the YqhD vector died because of the alcohol production by YqhD. The cloning efficiency, estimated based on colony PCR and enzyme digestion, was achieved more than 90% when the suicidal vector system was used. Moreover, the plasmid vector itself was stably maintained in the cell, presumably due to its ability to remove toxic aldehydes being accumulated in E. coli cell by metabolic stress.

Effects of Mutations in the WD40 Domain of α-COP on Its Interaction with the COPI Coatomer in Saccharomyces cerevisiae: Ki-Hyun Kim , Eun Kyung Kim , Ki Young Jeong , Yun-Hee Park , Hee-Moon Park; J. Microbiol. 2012;50(2):256-262. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1326-z

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4 Citations

Abstract: Replacement of glycine 227 in the fifth WD40 motif of α-COP/Ret1p/Soo1p by charged or aromatic amino acids is responsible for the temperature-dependent osmo-sensitivity of Saccharomyces cerevisiae, while truncations of WD40 motifs exerted a reduction in cell growth rate and impairment in assembly of cell-wall associated proteins such as enolase and Gas1p. Yeast two-hybrid analysis revealed that the ret1-1/soo1-1 mutation of α-COP abolished the interaction with β- and ε-COP, respectively, and that the interaction between α-COP and β-COP relied on the WD40 domain of α-COP. Furthermore, although the WD40 domain is dispensable for interaction of α-COP with ε-COP, structural alterations in the WD40 domain could impair the interaction.

Morphological Structure of Propagules and Electrophoretic Karyotype Analysis of False Smut Villosiclava virens in Rice: Rongtao Fu , Lei Ding , Jun Zhu , Ping Li , Ai-ping Zheng; J. Microbiol. 2012;50(2):263-269. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1456-3

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23 Citations

Abstract: The target pathogen Villosiclava virens (teleomorph: claviceps oryzae-sativae) was isolated from the infected rice, where it caused false smut. In our study, the forming processes of the chlamydospores, chlamydospore balls, conidiospores, and secondary conidiospores during the asexual reproduction were observed more precisely and in greater detail than previous descriptions. The microstructure of the infected rice kernel showed that the outer dense chlamydospores piled around the false smut balls grown on XBZ medium; moreover the sclerotia consisting of dense mycelium were found. The different morphology was observed across the different growing conditions. In addition, we observed the nuclear numbers of both the conidiospores and hyphae using 4′,6-diamidino-2-phenylindole (DAPI) staining. Because the fungus has small chromosomes and the numbers were not previously known, we analyzed the electrophoretic karyotype using a pulsed field gel electrophoresis (PFGE) technique. The results showed that V. virens has at least 10 chromosomes ranging in size from 0.6 kb to 6 Mb. The V. virens genome size is estimated to be 23 Mb. Here, we report the morphological characteristics of the fungus and the process of asexual spores forming asexual propagules, along with the first analyze the molecular karyotype of V. virens. These
results
supply a foundation for further study of the pathogenicity and biology of this devastating pathogen.

Protein-Protein Interactions between Histidine Kinases and Response Regulators of Mycobacterium tuberculosis H37Rv: Ha-Na Lee , Kwang-Eun Jung , In-Jeong Ko , Hyung Suk Baik , Jeong-Il Oh; J. Microbiol. 2012;50(2):270-277. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-2050-4

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25 Citations

Abstract: Using yeast two-hybrid assay, we investigated protein-protein interactions between all orthologous histidine kinase (HK)/response regulator (RR) pairs of M. tuberculosis H37Rv and identified potential protein-protein interactions between a noncognate HK/RR pair, DosT/NarL. The protein interaction between DosT and NarL was verified by phosphotransfer reaction from DosT to NarL. Furthermore, we found that the DosT and DosS HKs, which share considerable sequence similarities to each other and form a twocomponent system with the DosR RR, have different crossinteraction capabilities with NarL: DosT interacted with NarL, while DosS did not. The dimerization domains of DosT and DosS were shown to be sufficient to confer specificity for DosR, and the different cross-interaction abilities of DosS and DosT with NarL were demonstrated to be attributable to variations in the amino acid sequences of the α2-helices of their dimerization domains.

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

Shedding of Viral Hemorrhagic Septicemia Virus (Genotype IVb) by Experimentally Infected Muskellunge (Esox masquinongy): Robert K. Kim , Mohamed Faisal; J. Microbiol. 2012;50(2):278-284. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1145-2

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26 Citations

Abstract: Previous experimental infection demonstrated that juvenile muskellunge (Esox masquinongy) can survive experimental infection of viral hemorrhagic septicemia virus, Genotype IVb (VHSV IVb) at a low concentration of exposure. Herein we report that survivors of experimental infection with VHSV IVb shed the virus into the surrounding environment for an extended period of time. When muskellunge were exposed to VHSV IVb by immersion at a concentration of 1,400 plaque forming units (PFU)/ml, VHSV IVb was detected in the water of surviving fish for up to 15 weeks postexposure (p.e.) with the highest levels of shedding occurring between weeks 1 and 5 p.e. We estimated that each juvenile muskellunge can shed upwards of 1.36×105 PFU/fish/h after initial exposure signifying the uptake and amplification of VHSV to several orders of magnitude above the original exposure concentration. Muskellunge surviving low concentration exposure were re-infected with VHSV IVb by immersion at week 22 p.e. at concentrations ranging from 0 to 106 PFU/ml. Viral shedding was detected in all re-exposed fish, including mock rechallenged controls up to 15 consecutive weeks. Rates of viral shedding were substantially higher following rechallenge in the first 5 weeks. The highest rate of viral shedding was approximately 4.6×106 PFU/fish/h and shedding did not necessarily correspond to the re-exposure VHSV concentration. The results of this study shed new light into the dynamics of VHSV IVb shedding in a highly susceptible host and provide useful insights to fishery managers to design effective control strategies to this deadly virus.

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

KSHV Infection of B-Cell Lymphoma Using a Modified KSHV BAC36 and Coculturing System: Hyosun Cho , Hyojeung Kang; J. Microbiol. 2012;50(2):285-292. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1495-9

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6 Citations

Abstract: Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of two B cell lymphoproliferative diseases, namely primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV infection of B cell lymphoma in vitro has been a long-standing battle in advancing human KSHV biology. In this study, a modified form of KSHV BAC36 named BAC36A significantly increased the fidelity of gene-targeted site-directed mutagenesis in the KSHV genome. This modification eliminates tedious screening steps required to obtain mutant clones when a KSHV BAC36 reverse genetic system is used. Coculturing B-cell lymphoma BJAB cells with KSHV BAC36A stably transfected 293T cells enabled us to infect BJAB cells with a KSHV virion derived from the KSHV BAC36A. The coculture system produced substantial amounts of KSHV infection to BJAB, meaning that KSHV virions were released from 293T cells and then infected neighboring BJAB cells. Owing to our success with the KSHV BAC36A and coculture system, we propose a new genetic system for the study of KSHV gene expression and regulation in B-cell lymphoma.

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