Journal of Microbiology

Volume 48(6); December 2010

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

Assessment of Genetic and Functional Relationship of Antagonistic Fluorescent Pseudomonads of Rice Rhizosphere by Repetitive Sequence, Protein Coding Sequence and Functional Gene Analyses: Jayakumar Pathma , Niraikulam Ayyadurai , Natarajan Sakthivel; J. Microbiol. 2010;48(6):715-727. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0064-3

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Abstract: Antagonistic fluorescent pseudomonads isolated from rice rhizospheric soil were characterized using biochemical, taxonomical and molecular tools. Production of cyclopropane fatty acid (CFA) was correlated with their antagonistic potential. Strains were grouped into 18 different genotypes on the basis of amplified ribosomal DNA restriction analysis (ARDRA) and repetitive (rep)-PCR based genotypic fingerprinting analyses. High phylogenetic resolution among antagonistic fluorescent pseudomonad strains was obtained based on the DNA gyrase B subunit (gyrB) and RNA polymerase sigma factor 70 (rpoD) gene sequence analyses. Combined gyrB and rpoD sequence analysis resulted in the accurate estimation of molecular phylogeny and provided a significant correlation between the genetic distances among strains. Present study demonstrated the genetic and functional relationship of fluorescent pseudomonads. The knowledge on genetic and functional potential of fluorescent pseudomonads associated with rice rhizosphere is useful to understand their ecological role and for their utilization in sustainable agriculture.

An Improved Method for Extracting Bacteria from Soil for High Molecular Weight DNA Recovery and BAC Library Construction: Juan Liu , Jingquan Li , Li Feng , Hui Cao , Zhongli Cui; J. Microbiol. 2010;48(6):728-733. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0139-1

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34 Citations

Abstract: Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb.

Differentiation of Acremonium chrysogenum M35 in Submerged Culture with Glass Beads or Silicone Rubbers: Hwan Hyo Lee , Hyun Yong Shin , Eun Ji Kim , Seung Wook Kim; J. Microbiol. 2010;48(6):734-738. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0108-8

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Abstract: In this study, we investigated the effects of glass beads and silicone rubbers on the differentiation and morphological changes of A. chrysogenum M35 in submerged culture. Differentiation in the center of the cell pellets was improved by the addition of glass beads or silicone rubbers to the primary medium. The fragmentation rate constant (kfrag), which is used to estimate the tensile strength of fungal hyphae, was increased by more than 40% in baffled flasks containing glass beads. The maximum fragmentation rate was also increased by 48% when silicone rubbers were added to a 5 L bioreactor containing the culture. During the cultivation in the main medium with 6 glass beads, the value of the fractal dimension increased by about 8% when it was compared with baffled flasks without glass beads. Additionally, the number of arthrospores and the dry cell weight were increased by more than 10% in baffled flasks containing beads. The degree of roundness, which is the ratio of the object area to the longest Feret diameter, was decreased from 0.85 at day 1 to 0.77 at day 5. The differentiation of A. chrysogenum M35 was also supposedly closely related with the enlargement of the cell surfaces. Taken together, these results indicate that complex changes in morphology resulted in increased cell growth and differentiation in the culture broth containing glass beads and silicone rubbers.

Metagenomic Assessment of a Sulfur-Oxidizing Enrichment Culture Derived from Marine Sediment: Man-Young Jung , VinhHoa Pham , Soo-Je Park , So-Jeong Kim , Jong-Chan Chae , Yul Roh , Sung-Keun Rhee; J. Microbiol. 2010;48(6):739-747. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0257-9

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Abstract: The biological oxidation of reduced sulfur compounds is a critically important process in global sulfur biogeochemistry. In this study, we enriched from marine sediments under denitrifying conditions, chemolithotrophic sulfur oxidizers that could oxidize a variety of reduced sulfur compounds: thiosulfate, tetrathionate, sulfide, and polysulfide. Two major phylotypes of 16S rRNA gene (>99% identity in each phylotype) were detected in this enrichment culture. In order to characterize sulfide oxidation, we sequenced and characterized one fosmid clone (43.6 kb) containing the group I sulfide-quinone reductase (sqr) gene. Interestingly, four putative rhodanese genes were found in this clone. Furthermore, comparative alignment with the closest genome of Thiomicrospira crunogena XCL2 revealed that three homologous genes were located within the vicinity of the sqr gene. Fosmid clones harboring carbon fixation (cbbL and cbbM) and denitrification (narG) genes were screened, and the phylogeny of the functional genes was analyzed. Along with the comparison between the sqr-containing fosmid clones and the relevant gamma-proteobacteria, our phylogenetic study based on the 16S rRNA gene and carbon fixation genes suggest the prevalence of chemolithotrophic gamma-proteobacteria in the denitrifying cultures. The findings of this study imply that a combination of cultivation and metagenomic approaches might provide us with a glimpse into the characteristics of sulfur oxidizers in marine sediments.

The First Report of Two Species of Polyporus (Polyporaceae, Basidiomycota) from South Korea: Jin Sung Lee , Eun Ju Woo , Kyoung Hee Oh , Jae-Jin Kim , Young Woon Lim; J. Microbiol. 2010;48(6):748-753. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0105-y

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Abstract: Based on morphological examination, two species of Polyporus, P. dictyopus, and P. tuberaster, were identified, which constitutes the first record of these species in South Korea. To confirm their affinity within the genus Polyporus, the phylogenetic relationships of Polyporus and allied genera were established from nuclear large subunit ribosomal DNA (nLSU rDNA) sequences, and a morphological diagnostic key is presented to clarify the Korean species of Polyporus.

Acinetobacter kyonggiensis sp. nov., a beta-Glucosidase-Producing Bacterium, Isolated from Sewage Treatment Plant: Hye-Jung Lee , Sang-Seob Lee; J. Microbiol. 2010;48(6):754-759. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0355-8

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Abstract: A Gram-negative, non-motile bacterium, designated KSL5401-037T, was isolated from a sewage treatment plant in Gwangju in the Republic of Korea and was characterized using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that strain KSL5401-037T belonged to the genus Acinetobacter in the family Moraxellaceae of the Gammaproteobacteria (Brisou and Prevot, 1954). According to a 16S rRNA gene sequence analysis, it was closely related to Acinetobacter johnsonii ATCC 17909T (97.3%), A. bouvetii 4B02T (97.2%), and A. beijerinckii 58aT (96.8%). Chemotaxonomic data revealed that strain KSL5401- 037T possesses an ubiquinone system with Q-8 as the predominant compound and C16:0 (19.2%), C18:1 w9c (19.5%), and summed feature 3 (C16:1 w6c / C16:1 w7c, 34.1%) as the predominant cellular fatty acids. The major polar lipids detected in strain KSL5401-037T were diphosphatidylglycerol (DPG) and, phosphatidylethanolamine (PE), followed by phosphatidylglycerol (PG) and moderate amounts of phosphatidylcholine and phosphatidylserine. The G+C content of the genomic DNA was 41.2-42.1 mol%. Strain KSL5401-037T exhibited relatively low levels of DNA-DNA relatedness with respect to A. johnsonii DSM 6963T (17.7%) and A. bouvetii 4B02T (9.3%). The DNA-DNA relatedness values, biochemical, and physiological characteristics of strain KSL5401-037T strongly support its genotypic and phenotypic differentiation from other recognized type strains of the genus Acinetobacter. Based on these data, strain KSL5401-037T (JCM 17071T =KEMC 5401- 037T) should be classified in the genus Acinetobacter as a type strain of novel species, for which the name Acinetobacter kyonggiensis sp. nov. is proposed.

Sphingomonas ginsenosidimutans sp. nov., with Ginsenoside Converting Activity: Tae-Eun Choi , Qing-Mei Liu , Jung-Eun Yang , Siyi Sun , Se-Young Kim , Tae-Hoo Yi , Wan-Taek Im; J. Microbiol. 2010;48(6):760-766. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0469-z

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Abstract: The Gram-reaction-negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated Gsoil 1429T was isolated from the soil of ginseng cultivating field of Pocheon province in South Korea. This bacterium was characterized in order to determine its taxonomic position by using the polyphasic approach. Strain Gsoil 1429T grew well at 25-37°C and at pH 7.0 on R2A and nutrient agar without NaCl supplement. Strain Gsoil 1429T had beta-glucosidase activity, which was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to F2 via gypenoside XVII. On the basis of 16S rRNA gene sequence similarity, strain Gsoil 1429T was shown to belong to the family Sphingomonadaceae and to be related to Sphingomonas yunnanensis YIM 003T (98.2% sequence similarity), S. phyllosphaerae FA2T (97.5%), S. koreensis JSS26T (97.3%), and S. asaccharolytica IFO 15499T (97.1%). The G+C content of the genomic DNA was 65.6%. The major respiratory quinone was Q-10 and the major fatty acids were summed feature 8 (comprising C18:1 w7c/w9t/w12t), C16:0 and C14:0 2OH. DNA and chemotaxonomic data supported the affiliation of strain Gsoil 1429T to the genus Sphingomonas. The DNA-DNA relatedness values between strain Gsoil 1429T and its closest phylogenetically neighbours were below 28%. Strain Gsoil 1429T could be differentiated genotypically and phenotypically from the recognized species of the genus Sphingomonas. The isolate therefore represents a novel species, for which the name Sphingomonas ginsenosidimutans sp. nov. is proposed, with the type strain Gsoil 1429T (=KACC 14949T =JCM 17074T =LMG 25799T).

Sporulation of Several Biocontrol Fungi as Affected by Carbon and Nitrogen Sources in a Two-Stage Cultivation System: Li Gao , Xingzhong Liu; J. Microbiol. 2010;48(6):767-770. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0049-2

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Abstract: The development of fungal biopesticides requires the efficient production of large numbers spores or other propagules. The current study used published information concerning carbon concentrations and C:N ratios to evaluate the effects of carbon and nitrogen sources on sporulation of Paecilomyces lilacinus (IPC-P and M- 14) and Metarhizium anisopliae (SQZ-1-21 and RS-4-1) in a two-stage cultivation system. For P. lilacinus IPCP, the optimal sporulation medium contained urea as the nitrogen source, dextrin as the carbon source at 1 g/L, a C:N ratio of 5:1, with ZnSO4·7H2O at 10 mg/L and CaCl2 at 3 g/L. The optimal sporulation medium for P. lilacinus M-14 contained soy peptone as the nitrogen source and maltose as the carbon source at 2 g/L, a C:N ratio of 10:1, with ZnSO4·7H2O at 250 mg/L, CuSO4·5H2O at 10 mg/L, H3BO4 at 5 mg/L, and Na2MoO4·2H2O at 5 mg/L. The optimum sporulation medium for M. anisopliae SQZ-1-21 contained urea as the nitrogen source, sucrose as the carbon source at 16 g/ L, a C:N ratio of 80:1, with ZnSO4·7H2O at 50 mg/L, CuSO4·5H2O at 50 mg/L, H3BO4 at 5 mg/L, and MnSO4·H2O at 10 mg/L. The optimum sporulation medium for M. anisopliae RS-4-1 contained soy peptone as the nitrogen source, sucrose as the carbon source at 4 g/L, a C:N ratio of 5:1, with ZnSO4·7H2O at 50 mg/L and H3BO4 at 50 mg/L. All sporulation media contained 17 g/L agar. While these results were empirically derived, they provide a first step toward low-cost mass production of these biocontrol agents.

Comparative Proteome Analysis of Bacillus anthracis with pXO1 Plasmid Content: Sudipto Shahid , Ji Hyun Park , Hyung Tae Lee , Seong-Joo Kim , Ji Cheon Kim , Sang Hoon Kim , Dal Mu Ri Han , Dong In Jeon , Kyoung Hwa Jung , Young Gyu Chai; J. Microbiol. 2010;48(6):771-777. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0136-4

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Abstract: Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4-7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.

Role of Hydrogen Generation by Klebsiella pneumoniae in the Oral Cavity: Tomoko Kanazuru , Eisuke F. Sato , Kumiko Nagata , Hiroshi Matsui , Kunihiko Watanabe , Emiko Kasahara , Mika Jikumaru , June Inoue , Masayasu Inoue; J. Microbiol. 2010;48(6):778-783. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0149-z

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Abstract: Some gastrointestinal bacteria synthesize hydrogen (H2) by fermentation. Despite the presence of bactericidal factors in human saliva, a large number of bacteria also live in the oral cavity. It has never been shown that oral bacteria also produce H2 or what role H2 might play in the oral cavity. It was found that a significant amount of H2 is synthesized in the oral cavity of healthy human subjects, and that its generation is enhanced by the presence of glucose but inhibited by either teeth brushing or sterilization with povidone iodine. These observations suggest the presence of H2-generating bacteria in the oral cavity. The screening of commensal bacteria in the oral cavity revealed that a variety of anaerobic bacteria generate H2. Among them, Klebsiella pneumoniae (K. pneumoniae) generated significantly large amounts of H2 in the presence of glucose. Biochemical analysis revealed that various proteins in K. pneumoniae are carbonylated under standard culture conditions, and that oxidative stress induced by the presence of Fe++ and H2O2 increases the number of carbonylated proteins, particularly when their hydrogenase activity is inhibited by KCN. Inhibition of H2 generation markedly suppresses the growth of K. pneumoniae. These observations suggest that H2 generation and/or the reduction of oxidative stress is important for the survival and growth of K. pneumoniae in the oral cavity.

Endophytic Fungus Trichothecium roseum LZ93 Antagonizing Pathogenic Fungi In Vitro and Its Secondary Metabolites: XiaoMei Zhang , GuoHong Li , Juan Ma , Ying Zeng , WeiGuang Ma , PeiJi Zhao; J. Microbiol. 2010;48(6):784-790. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0173-z

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Abstract: The endophytic fungus Trichothecium roseum LZ93 from Maytenus hookeri was found to antagonize other pathogenic fungi in vitro. To identify which compound contributed substantially to the antagonism, we fermented the strain and purified its fermentation products. Eleven compounds were obtained, including two trichothecenes, five rosenonolactones, two cardiotonic cyclodepsipeptides, and two sterols. Compound 11β-hydroxyrosenonolactone (1) was assigned according to 1D and 2D-NMR data for the first time. At the same time, the 1H and 13C-NMR assignments for 6β-hydroxyrosenonolactone (2) were revised. Of all of them, only trichothecin (6) showed strong antifungal activity. Based on our observations of the antagonistic activity and the other experimental results, we suggest that the antifungal compound trichothecin was the main contributor to the antagonistic action of T. roseum LZ93.

Isolation and Characterization of Antifungal Peptides Produced by Bacillus amyloliquefaciens LBM5006: Lisianne Brittes Benitez , Renata Voltolini Velho , Marcia Pagno Lisboa , Luis Fernando da Costa Medina , Adriano Brandelli; J. Microbiol. 2010;48(6):791-797. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0164-0

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Abstract: Bacillus amyloliquefaciens LBM 5006 produces antagonistic activity against pathogenic bacteria and phytopathogenic fungi, including Aspergillus spp., Fusarium spp., and Bipolaris sorokiniana. PCR analysis revealed the presence of ituD, but not sfp genes, coding for iturin and surfactin, respectively. The antimicrobial substance produced by this strain was isolated by ammonium sulfate precipitation, gel filtration chromatography and 1-butanol extraction. The ultraviolet spectrum was typical of a polypeptide and the infrared spectrum indicates the presence of peptide bonds and acyl group(s). The antimicrobial substance was resistant to proteolytic enzymes and heat treatment, and was reactive with ninhydrin. Mass spectroscopy analysis indicated that B. amyloliquefaciens LBM 5006 produces two antimicrobial peptides, with main peaks at m/z 1,058 Da and 1,464 Da, corresponding to iturin-like and fengycin-like peptides, respectively. B. amyloliquefaciens LBM 5006 showed significant activity against phytopatogenic fungi, showing potential for use as a biocontrol agent or production of antifungal preparations.

Rescue of a Cold-Sensitive Mutant at Low Temperatures by Cold Shock Proteins from Polaribacter irgensii KOPRI 22228: Ji-hyun Uh , Youn Hong Jung , Yoo Kyung Lee , Hong Kum Lee , Hana Im; J. Microbiol. 2010;48(6):798-802. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0402-5

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Abstract: Exposure to low temperatures induces the biosynthesis of specific sets of proteins, including cold shock proteins (Csps). Since many of the specific functions of pychrophilic Csps are unknown, the roles of Csps from an Arctic bacterium, Polaribacter irgensii KOPRI 22228, were examined. The genes encoding CspA and CspC of P. irgensii were cloned in this study. Sequence analysis showed that these proteins have cold shock domains containing two RNA-binding motifs, RNP1 and RNP2. Both proteins bound oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. When the P. irgensii Csps were overexpressed in Escherichia coli, the cold-resistance of the host was increased by more than five-fold. The P. irgensii Csps also rescued a cold-sensitive E. coli csp-quadruple deletion strain, BX04, at low temperatures. These results suggest that Csps from P. irgensii play a role in survival in polar environments.

Characterization of Hyperthermostable Fructose-1,6-Bisphosphatase from Thermococcus onnurineus NA1: Yeol Gyun Lee , Sung Gyun Kang , Jung-Hyun Lee , Seung Il Kim , Young-Ho Chung; J. Microbiol. 2010;48(6):803-807. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0377-2

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Abstract: To understand the physiological functions of thermostable fructose-1,6-bisphosphatase (TNA1-Fbp) from Thermococcus onnurineus NA1, its recombinant enzyme was overexpressed in Escherichia coli, purified, and the enzymatic properties were characterized. The enzyme showed maximal activity for fructose-1,6- bisphosphate at 95°C and pH 8.0 with a half-life (t1/2) of about 8 h. TNA1-Fbp had broad substrate specificities for fructose-1,6-bisphosphate and its analogues including fructose-1-phosphate, glucose-1-phosphate, and phosphoenolpyruvate. In addition, its enzyme activity was increased five-fold by addition of 1 mM Mg2+, while Li+ did not enhance enzymatic activity. TNA1-Fbp activity was inhibited by ATP, ADP, and phosphoenolpyruvate, but AMP up to 100 mM did not have any effect. TNA1-Fbp is currently defined as a class V fructose-1,6-bisphosphatase (FBPase) because it is very similar to FBPase of Thermococcus kodakaraensis KOD1 based on sequence homology. However, this enzyme shows a different range of substrate specificities. These results suggest that TNA1-Fbp can establish new criterion for class V FBPases.

Identification and Functional Analysis of a Gene Encoding β-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris: Hwang-Woo Ji , Chang-Jun Cha; J. Microbiol. 2010;48(6):808-813. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0482-2

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Abstract: The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes.

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