Journal of Microbiology

Volume 46(4); August 2008

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

Rapid Phylogenetic Dissection of Prokaryotic Community Structure in Tidal Flat Using Pyrosequencing: Bong-Soo Kim , Byung Kwon Kim , Jae-Hak Lee , Myungjin Kim , Young Woon Lim , Jongsik Chun; J. Microbiol. 2008;46(4):357-363. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0071-9

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63 Citations

Abstract: Dissection of prokaryotic community structure is prerequisite to understand their ecological roles. Various methods are available for such a purpose which amplification and sequencing of 16S rRNA genes gained its popularity. However, conventional methods based on Sanger sequencing technique require cloning process prior to sequencing, and are expensive and labor-intensive. We investigated prokaryotic community structure in tidal flat sediments, Korea, using pyrosequencing and a subsequent automated bioinformatic pipeline for the rapid and accurate taxonomic assignment of each amplicon. The combination of pyrosequencing and bioinformatic analysis showed that bacterial and archaeal communities were more diverse than previously reported in clone library studies. Pyrosequencing analysis revealed 21 bacterial divisions and 37 candidate divisions. Proteobacteria was the most abundant division in the bacterial community, of which Gammaand Delta-Proteobacteria were the most abundant. Similarly, 4 archaeal divisions were found in tidal flat sediments. Euryarchaeota was the most abundant division in the archaeal sequences, which was further divided into 8 classes and 11 unclassified euryarchaeota groups. The system developed here provides a simple, in-depth and automated way of dissecting a prokaryotic community structure without extensive pretreatment such as cloning.

An Examination of the Bacteriophages and Bacteria of the Namib Desert: Eric Prestel , Sylvie Salamitou , Michael S. DuBow; J. Microbiol. 2008;46(4):364-372. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0007-4

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63 Citations

Abstract: Bacteria and their viruses (called bacteriophages, or phages), have been found in virtually every ecological niche on Earth. Arid regions, including their most extreme form called deserts, represent the single largest ecosystem type on the Earth''s terrestrial surface. The Namib desert is believed to be the oldest (80 million years) desert. We report here an initial analysis of bacteriophages isolated from the Namib desert using a combination of electron microscopy and genomic approaches. The virus-like particles observed by electron microscopy revealed 20 seemingly different phage-like morphologies and sizes belonging to the Myoviridae and Siphoviridae families of tailed phages. Pulsed-field gel electrophoresis revealed a majority of phage genomes of 55~65 kb in length, with genomes of approximately 200, 300, and 350 kb also observable. Sample sequencing of cloned phage DNA fragments revealed that approximately 50% appeared to be of bacterial origin. Of the remaining DNA sequences, approximately 50% displayed no significant match to any sequence in the databases. The majority of the 16S rDNA sequences amplified from DNA extracted from the sand displayed considerable (94~98%) homology to members of the Firmicutes, and in particular to members of the genus Bacillus, though members of the Bacteroidetes, Planctomycetes, Chloroflexi, and delta-Proteobacteria groups were also observed.

Diversity of Endophytic Enterobacteria Associated with Different Host Plants: Adalgisa Ribeiro Torres , Welington Luiz Araujo , Luciana Cursino , Mariangela Hungria , Fabio Plotegher , Fabio Luis Mostasso , Joao Lucio Azevedo; J. Microbiol. 2008;46(4):373-379. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-007-0165-9

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38 Citations

Abstract: Fifty-three endophytic enterobacteria isolates from citrus, cocoa, eucalyptus, soybean, and sugar cane were evaluated for susceptibility to the antibiotics ampicillin and kanamycin, and cellulase production. Susceptibility was found on both tested antibiotics. However, in the case of ampicillin susceptibility changed according to the host plant, while all isolates were susceptible to kanamycin. Cellulase production also changed according to host plants. The diversity of these isolates was estimated by employing BOX-PCR genomic fingerprints and 16S rDNA sequencing. In total, twenty-three distinct operational taxonomic units (OTUs) were identified by employing a criterion of 60% fingerprint similarity as a surrogate for an OTU. The 23 OTUs belong to the Pantoea and Enterobacter genera, while their high diversity could be an indication of paraphyletic classification. Isolates representing nine different OTUs belong to Pantoea agglomerans, P. ananatis, P. stewartii, Enterobacter sp., and E. homaechei. The results of this study suggest that plant species may select endophytic bacterial genotypes. It has also become apparent that a review of the Pantoea/Enterobacter genera may be necessary.

High Infectivity of an Endoparasitic Fungus Strain, Esteya vermicola, against Nematodes: Chun Yan Wang , Zhe Ming Fang , Bai Shen Sun , Li Juan Gu , Ke Qin Zhang , Chang-Keun Sung; J. Microbiol. 2008;46(4):380-389. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-007-0122-7

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49 Citations

Abstract: Esteya vermicola, as the first recorded endoparasitic fungus of pinewood nematodes, exhibits great potential as a biological agent against nematodes. However, only two strains of this species have been described so far. In this study, we identified a novel endoparasitic fungal strain, CNU 120806, isolated from infected nematodes in forest soil samples during a survey of nematophagous fungi in Korea. This strain showed similar morphological characteristics and infection mode with the two previously described strains of E. vermicola. All strains are characterized by the ability to produce two types of conidiogenous cells and conidia, and to parasitize nematodes with lunate adhesive conidia. Moreover, the CNU 120806 strain showed 100% identity with E. vermicola CBS 115803 when their partial sequences of 28S rRNA gene were compared. Molecular phylogenetic analysis further identified CNU 120806 as a strain of E. vermicola, by clustering CNU 120806 and E. vermicola CBS 115803 into a single subclade. Culture medium influenced the proportion of dimorphic CNU 120806 conidia, and further changed the adhesive and mortality rates of nematodes. The CNU 120806 strain exhibits high infection activity against nematodes on nutrient-rich PDA medium. Almost all tested nematodes were killed within 8~10 days after inoculation. This study provides justification for further research of E. vermicola, and the application and formulation of this fungus as a bio-control agent against nematodes.

Comamonas granuli sp. nov., Isolated from Granules Used in a Wastewater Treatment Plant: Kyoung-Ho Kim , Leonid N. Ten , Qing-Mei Liu , Wan-Taek Im , Sung-Taik Lee; J. Microbiol. 2008;46(4):390-395. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0019-0

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28 Citations

Abstract: A Gram-negative, motile, rod-shaped, non-spore-forming bacterial strain, designated as Ko03T, was isolated from microbial granules, and was characterized, using a polyphasic approach, in order to determine its taxonomic position. The isolate was positive for catalase and oxidase, but negative for gelatinase and β-galactosidase. Phylogenetic analyses using the 16S rRNA gene sequence showed that the strain formed a monophyletic branch towards the periphery of the evoluionary radiation occupied by the genus Comamonas, its closest neighbors being Comamonas koreensis KCTC 12005T (95.9% sequence similarity), Comamonas nitrativorans DSM 13191T (95.7%), and Comamonas odontotermitis LMG 23579T (95.7%). Strain Ko03T had a genomic DNA G+C content of 68.4 mol% and the predominant respiratory quinone was Q-8. The major fatty acids were C16:1 ω7c (44.7%), C16:0 (28.1%), C18:1 (16.1%), and C10:0 3-OH (3.5%). These chemotaxonomic results supported the affiliation of strain Ko03T to the genus Comamonas. However, low 16S rRNA gene sequence similarity values and distinguishing phenotypic characteristics allowed genotypic and phenotypic differentiation of strain Ko03T from recognized Comamonas species. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Ko03T represents a novel species of the genus Comamonas, for which the name Comamonas granuli sp. nov. is proposed. The type strain is Ko03T (= KCTC 12199T= NBRC 101663T).

Acinetobacter soli sp. nov., Isolated from Forest Soil: Duwoon Kim , Keun Sik Baik , Mi Sun Kim , Seong Chan Park , Seon Suk Kim , Moon Soo Rhee , Young Se Kwak , Chi Nam Seong; J. Microbiol. 2008;46(4):396-401. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0118-y

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64 Citations

Abstract: A non-motile and rod shaped bacterium, designated strain B1T, was isolated from forest soil at Mt. Baekwoon, Republic of Korea. Cells were Gram-negative, catalase-positive, and oxidase-negative. The major fatty acids were 9-octadecenoic acid (C18:1 ω9c; 42%) and hexadecanoic acid (C16:0; 25.9%) and summed feature 3 (comprising iso-C15:0 2-OH and/or C16:1 ω7c; 10.0%). The DNA G+C content was 44.1 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain B1T formed a lineage within the genus Acinetobacter and was closely related to A. baylyi DSM 14961T (98.6% sequence similarity), followed by A. baumannii DSM 30007T (97.4%), A. calcoaceticus DSM 30006T (97.0%), and 3 genomic species (96.8~7.6%). Phenotypic characteristics, gyrB gene sequence analysis and DNA-DNA relatedness data distinguished strain B1T from type strains of A. baylyi, A. baumannii, and A. calcoaceticus. On the basis of the evidence presented in this study, strain B1T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter soli sp. nov. is proposed. The type strain is B1T (= KCTC 22184T= JCM 15062T).

Enhanced Expression of Laccase during the Degradation of Endocrine Disrupting Chemicals in Trametes versicolor: Yunjung Kim , Sumin Yeo , Hong-Gyu Song , Hyoung T. Choi; J. Microbiol. 2008;46(4):402-407. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-007-0236-y

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24 Citations

Abstract: A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57~97% homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.

The Role and Regulation of Trx1, a Cytosolic Thioredoxin in Schizosaccharomyces pombe: Ji-Yoon Song , Jung-Hye Roe; J. Microbiol. 2008;46(4):408-414. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0076-4

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35 Citations

Abstract: The genome of fission yeast Schizosaccharomyces pombe harbors two genes for thioredoxins, trx1+ and trx2+, which encode cytosolic and mitochondrial thioredoxins, respectively. The Δtrx1 mutant was found sensitive to diverse external stressors such as various oxidants, heat, and salt, whereas Δtrx2 mutant was not sensitive except to paraquat, a superoxide generator. Both Δtrx1 and Δtrx2 mutants were more resistant to diamide, a thiol-specific oxidant, than the wild type. The trx1+ gene expression was induced by H2O2 and menadione, being mediated through a stress-responsive transcription factor Pap1. In Δtrx1 cells, the basal expression of Pap1-regulated genes were elevated, suggesting a role for Trx1 as a reducer for oxidized (activated) Pap1. The Δtrx1 mutant exhibited cysteine auxotrophy, which can be overcome by adding sulfite. This suggests that Trx1 serves as a primary electron donor for 3’-phosphoadenosine-5’-phosphosulfate (PAPS) reductase and thus is an essential protein for sulfur assimilation in S. pombe. These results suggest that, in contrast to Trx2 whose role is more confined to mitochondrial functions, Trx1 plays a major role in protecting S. pombe against various stressful conditions and enables proper sulfur metabolism.

Cloning and Characterization of a Na+/H+ Antiporter Gene of the Moderately Halophilic Bacterium Halobacillus aidingensis AD-6T: Ya Jie Zou , Li Fu Yang , Lei Wang , Su Sheng Yang; J. Microbiol. 2008;46(4):415-421. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0009-2

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14 Citations

Abstract: A gene encoding a Na+/H+ antiporter was obtained from the genome of Halobacillus aidingensis AD-6T, which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na+/H+ antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na+/H+ as well as Li+/H+ antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K+ /H+ antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na+/H+ antiporter activity.

The Fission Yeast Homologue of Gle1 is Essential for Growth and Involved in mRNA Export: DongGeRaMi Moon , Jin-Ah Bae , Hyun Jin Cho , Jin Ho Yoon; J. Microbiol. 2008;46(4):422-428. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0177-0

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Abstract: We have isolated Gle1 homologue (named as spgle1) as a partial multicopy suppressor of the synthetic lethality of rae1-167 elf1-21 in fission yeast Schizosaccharomyces pombe. The spgle1 is also able to complement partially temperature-sensitive phenotype of rae1-167 only at a lower restrictive temperature. The spgle1 gene contains one intron and encodes a 480 amino-acid protein with predicted molecular weight of 56.2 kDa. We showed that spgle1 gene is essential for vegetative growth and functional Gle1-GFP protein is localized mainly in NPC. The accumulation of poly(A)+ RNA in the nucleus is exhibited when expression of spgle1 is repressed or over-expressed. These results suggest that the spGle1 protein is also involved in mRNA export in fission yeast.

Promoter Analysis of Bombyx mori Nucleopolyhedrovirus Ubiquitin Gene: Xu’ai Lin , Yin Chen , Yongzhu Yi , Jie Yan , Zhifang Zhang; J. Microbiol. 2008;46(4):429-435. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-007-0163-y

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Abstract: The aim of this study was to analyze the characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) ubiquitin gene promoter and the effects of conserved motifs, such as TAAG, TATA, and CAAT, along with baculovirus enhancer homologous region 3 (hr3), on promoter activity. Ubiquitin gene of BmNPV was expressed during the late phase of virus infection. In the presence of viral factors, significant reduction of promoter activity was observed by deletion of -382 to -124 bp upstream of ATG. The fragment between -187 and -383 bp upstream of ATG, including distal TAAG, CAAT motif, and TATA box, could also drive expression of the reporter gene. The mutation of cis-elements TATA boxes and TAAG motifs significantly decreased the promoter’s activity, while CAAT mutations enhanced promoter activity by 2- or 3-fold, as compared with the native promoter. In the presence of BmNPV, hr3, both located downstream of the reporter gene of the same vector, and separate vector, could significantly enhance transcription activity of ubiquitin promoter as compared to the control. We concluded that BmNPV ubiquitin gene might be regulated by dual sets of promoter elements, where TAAG and TATA box may positively regulate the expression of ubiquitin, while CAAT motif functions as a negative regulator. Viral factor(s) play an important role in the co-activation of hr3 and promoter.

Detection of Hepatitis A Virus from Oyster by Nested PCR Using Efficient Extraction and Concentration Method: Duwoon Kim , Seok-Ryel Kim , Ki-Sung Kwon , Ji-Won Lee , Myung-Joo Oh; J. Microbiol. 2008;46(4):436-440. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0131-1

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23 Citations

Abstract: The molecular methods using polymerase chain reaction have been proposed as useful tools for the identification of viral pathogens in food and water. However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification due to the low sensitivity of PCR in the presence of PCR inhibitors. We developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol in order to detect hepatitis A virus (HAV) inoculated in oyster digestive glands. The detection limit of HAV precipitated with zirconium hydroxide was 105 fold less sensitive in a nested PCR than that precipitated the HAV supernatant twice with PEG/NaCl (16% polyethylene glycol 6,000, 0.525 M NaCl) in a 1:2 (v/v) ratio, which provided an efficient detection of 0.0148 PFU/g from approximately 0.05 g of oyster homogenate. This method is efficient for potential use in the detection of HAV from shellfish and is more sensitive than most currently published tests.

Modulation of Secreted Virulence Factor Genes by Subinhibitory Concentrations of Antibiotics in Pseudomonas aeruginosa: Lixin Shen , Ying Shi , Dan Zhang , Jinhua Wei , Michael G. Surette , Kangmin Duan; J. Microbiol. 2008;46(4):441-447. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0054-x

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46 Citations

Abstract: Recent studies have shown that subinhibitory antibiotics play important roles in regulating bacterial genes including virulence factor genes. In this study, the expression of 13 secreted virulence related gene clusters of Pseudomonas aeruginosa, an important opportunistic pathogen, was examined in the presence of subinhibitory concentrations of 4 antibiotics: vancomycin, tetracycline, ampicilin and azithromycin. Activation of gene expression was observed with phzA1, rhlAB, phzA2, lasB, exoY, and exoS. Subinhibitory concentrations of vancomycin resulted in more than 10-fold increase of rhlAB and phzA2 transcription. Both rhamnolipid production and pyocyanin production were significantly elevated, correlating phenotypes with the increased transcription. P. aeruginosa swarming and swimming motility also increased. Similar results were observed with subinhibitory tetracycline, azithromycin and ampicillin. These results indicate that the antibiotics at low concentrations can up-regulate virulence factors and therefore influence bacterial pathogenesis.

Application of Free-Flow Electrophoresis/2-Dimentional Gel Electrophoresis for Fractionation and Characterization of Native Proteome of Pseudomonas putida KT2440: Chi-Won Choi , Young S. Hong , Seung Il Kim; J. Microbiol. 2008;46(4):448-455. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0063-9

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Abstract: Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8~6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4~10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72~5.89, indicating that observed pI values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes.

A Circularly Permuted β-Lactamase as a Novel Reporter for Evaluation of Protein Cyclization Efficiency: Jeong Seon Kwon , Jyotiranjan Bal , Hai Min Hwang , Jeong-Yoon Kim; J. Microbiol. 2008;46(4):456-461. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0106-2

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Abstract: Split inteins have been used as a versatile tool in protein engineering to mediate efficient in vivo and in vitro trans-splicing of a protein. The trans-splicing ability of split inteins was also applied to the in vivo cyclization of a protein. However, cyclization efficiency is dependent upon the type of split inteins employed and the conditions under which cyclization occur. In this study, a novel reporter system that easily measures the cyclization efficiency of split inteins was developed. For this purpose TEM-1 β-lactamase was divided into two fragments (24~215 and 216~286 amino acids) and circularly permuted. The circularly permuted β-lactamase expressed in Escherichia coli showed little β-lactamase activity, most likely due to the structural modification of the protein. However, when the circularly permuted β-lactamase was cyclized by the Synechocystis sp. PCC6803 DnaB split mini-intein, β-lactamase activity both in vitro and in vivo was recovered. These results suggest that the novel reporter system can be exploited to develop new inteins with high efficiency of in vivo protein cyclization.

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