Journal of Microbiology

Volume 52(5); May 2014

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

Variations in 16S rRNA-based Microbiome Profiling between Pyrosequencing Runs and between Pyrosequencing Facilities: Minseok Kim , Zhongtang Yu; J. Microbiol. 2014;52(5):355-365. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3443-3

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32 Citations

Abstract: Pyrosequencing of 16S rRNA gene amplicons on the 454 FLX Titanium platform has been widely used to analyze microbiomes in various environments. However, different results may stem from variations among sequencing runs or among sequencing facilities. This study aimed to evaluate these variations between different pyrosequencing runs by sequencing 16S rRNA gene amplicon libraries generated from three sets of rumen samples twice each on the 454 FLX Titanium system at two independent sequencing facilities. Similar relative abundances were found for predominant taxa represented by large numbers of sequence reads but not for minor taxa represented by small numbers of sequence reads. The two sequencing facilities revealed different bac-terial profiles with respect to both predominant taxa and minor taxa, including the most predominant genus Prevo-tella, the family Lachnospiraceae, and the phylum Proteo-bacteria. Differences in primers used to generate amplicon libraries may be a major source of variations in microbiome profiling. Because different primers and regions of 16S rRNA genes are often used by different researchers, significant variations likely exist among studies. Quantitative interpre-tation for relative abundance of taxa, especially minor taxa, from prevalence of sequence reads and comparisons of re-sults from different studies should be done with caution.

Journal Article

Comparative Analysis of Superantigen Genes in Staphylococcus xylosus and Staphylococcus aureus Isolates Collected from a Single Mammary Quarter of Cows with Mastitis: Karol Fijałkowski , Magdalena Struk , Jolanta Karakulska , Aleksandra Paszkowska , Stefania Giedrys-Kalemba , Helena Masiuk , Danuta Czernomysy-Furowicz , Paweł Nawrotek; J. Microbiol. 2014;52(5):366-372. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3436-2

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Abstract: The purpose of this study was to analyze and compare genes encoding superantigens (SAgs) in Staphylococcus xylosus and Staphylococcus aureus isolates collected simultaneously from milk of the same cows with clinical mastitis. Genes encoding staphylococcal enterotoxins and enterotoxin-like proteins (sea-selu), toxic shock syndrome toxin 1 (tst-1) and exfolia-tive toxins (eta and etd) were investigated. It was found that among 30 isolates of S. xylosus, 16 (53.3%) harbored from 1 to 10 SAg genes. In total, in 16 SAg positive S. xylosus, 11 different enterotoxin genes were detected: sec, sed, seg, seh, sei, selm, seln, selo, selp, ser, selu and one etd gene encoding exfoliative toxin D. The most prevalent genes were ser, selu, and selo. Among all the positive isolates of S. xylosus, a total of 14 different SAg gene combinations were detected. One combination was repeated in 3 isolates, whereas the rest were detected only once. However, in the case of S. aureus all the 30 isolates harbored the same combination of SAg genes: seg, sei, selm, seln, selo and on the basis of PFGE analysis all belonged to the same clonal type. Also noteworthy was the observation that SAg genes detected in S. aureus have also been found in S. xylosus. The findings of this study further extend previous observations that SAg genes are present not only in S. aureus but also in coagulase-negative staphy-lococci, including S. xylosus. Therefore, taking into account that the SAg genes are encoded on mobile genetic elements it is possible that these genes can be transferred between different species of coexisting staphylococci.

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

Dyella jejuensis sp. nov., Isolated from Soil of Hallasan Mountain in Jeju Island: Min-Soo Kim , Dong-Wook Hyun , Joon Yong Kim , Soyeon Kim , Jin-Woo Bae , Eun-Jin Park; J. Microbiol. 2014;52(5):373-377. Published online May 9, 2014; DOI: https://doi.org/10.1007/s12275-014-3670-7

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Abstract: A novel bacterium, designated JP1T, was isolated from soil of Hallasan Mountain in Jeju Island. The isolate was a Gram- negative, aerobic, motile and rod-shaped (0.2–0.4 × 1.2–2.0 μm) bacterium. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain JP1T was closely related to Dyella koreensis with 97.6% similarity. Growth of strain JP1T occurred at 10–37°C, pH 5–7 and 0–1% (w/v) NaCl. The genomic DNA G+C content of strain JP1T was 62.1 mol%. The major fatty acids were iso-C16:0, iso-C17:1 ω9c, and iso- C15:0. The predominant quinone was ubiquinone-8. The major polar lipids of strain JP1T were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, uniden-tified aminolipids and unidentified aminophospholipids. The DNA-DNA relatedness values between strain JP1T and pre-viously reported Dyella species were <10%. Based on pheno-typic, genotypic, and phylogenetic distinctness, strain JP1T represents a novel species in the genus Dyella, for which the name Dyella jejuensis sp. nov. is proposed. The type strain is JP1T (=KACC 17701T =JCM 19615T).

Massilia kyonggiensis sp. nov., Isolated from Forest Soil in Korea: Jaisoo Kim; J. Microbiol. 2014;52(5):378-383. Published online May 9, 2014; DOI: https://doi.org/10.1007/s12275-014-4010-7

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Abstract: A Gram-negative, short, rod-shaped bacterium, TSA1T, was isolated from forest soil collected at Kyonggi University, South Korea. Assessment of 16S rRNA gene sequence sim-ilarity indicated that the strain is related to Massilia niastensis 5516S-1T (98.3%), M. haematophila CCUG 38318T (97.9%), M. aerilata 5516S-11T (97.9%), M. tieshanensis TS3T (97.6%), and M. varians CCUG 3529T (97.1%). Colonies grown on Reasoner’s 2A agar at 30°C for 2 days were transparent, white, round, smooth, and glossy. The cells grew at 10–42°C (optimum: 25–37°C) and pH 5–9 (optimum: 5–9) and in 0–2% NaCl (optimum: 0–1%). TSA1T was able to grow on trypticase soy and nutrient agar, but not on Luria-Bertani or MacConkey agar. The strain was catalase- and oxidase- positive and able to degrade starch and casein, but not car-boxymethyl cellulose. The predominant quinone of TSA1T was Q-8, the major fatty acids were summed feature 3 and C16:0, and the DNA G+C content was 66.7 mol%. Given these findings, we propose that this strain is a novel species of the genus Massilia. We suggest the name Massilia kyonggiensis sp. nov. (type strain, KACC 17471T =KEMB 9005-031T =JCM 19189T).

Isolation and Characterization of Novel Lipase Gene LipHim1 from the DNA Isolated from Soil Samples: Pavan Kumar Pindi , Raja Srinath Rebba , Theetha L. Pavankumar; J. Microbiol. 2014;52(5):384-388. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3302-2

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12 Citations

Abstract: Metagenomics is a magnificent tool to isolate genes from unknown/uncharacterized species and also from organisms that cannot be cultured. In this study, we constructed a meta-genomic library from isolated DNA of soil samples collected from Palamuru University campus premises, in Mahabub-nagar district of Andhra Pradesh, India. We isolated a novel lipase gene LipHim1, which has an open reading frame of 591 base pairs and encodes ~23 kDa protein consisting of 196 amino acids. The Lipase LipHim1 showed maximum 32% homology at the protein level with the extracellular Aeromonas hydrophila lipase (Class II, GDSL family) and was significantly different from all other known lipases. The isolated lipase catalyzed the hydrolysis of fatty acid esters of polyoxyethylene sorbitan such as Tween 60. Our results in-dicate that the isolated lipase gene is novel.

Journal Article

Production of an Endoinulinase from Aspergillus niger AUMC 9375, by Solid State Fermentation of Agricultural Wastes, with Purification and Characterization of the Free and Immobilized Enzyme: Manal M. Housseiny; J. Microbiol. 2014;52(5):389-398. Published online May 9, 2014; DOI: https://doi.org/10.1007/s12275-014-3561-y

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24 Citations

Abstract: Two different substrates, sunflower (Helianthus annuus L.) tubers and lettuce (Lactuca sativa) roots, were tested. Using a mixture of both wastes resulted in higher production of endoinulinase than either waste alone. Also, ten fungal spe-cies grown on these substrates as inexpensive, carbon sour-ces were screened for the best production of endoinulinase activities. Of these, Aspergillus niger AUMC 9375 was the most productive, when grown on the mixture using a 6:1 w/w ratio of sun flower: lettuce, and yielded the highest levels of inulinase at 50% moisture, 30°C, pH 5.0, with seven days of incubation, and with yeast extract as the best nitrogen source. Inulinase was purified to homogeneity by ion-exchange chro-matography and gel-filtration giving a 51.11 fold purification. The mixture of sunflower tubers and lettuce roots has poten-tial to be an effective and economical substrate for inulinase production. Inulinase was successfully immobilized with an immobilization yield of 71.28%. After incubation for 2 h at 60°C, the free enzyme activity decreased markedly to 10%, whereas that of the immobilized form decreased only to 87%. A reusability test demonstrated the durability of the immo-bilized inulinase for 10 cycles and in addition, that it could be stored for 32 days at 4°C. These results indicate that this inulinase, in the immobilized form, is a potential candidate for large-scale production of high purity fructose syrups.

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

Characterization of Recombinant β-Glucosidase from Arthrobacter chlorophenolicus and Biotransformation of Ginsenosides Rb1, Rb2, Rc, and Rd: Myung Keun Park , Chang-Hao Cui , Sung Chul Park , Seul-Ki Park , Jin-Kwang Kim , Mi-Sun Jung , Suk-Chae Jung , Mi-Sun Jung , Suk-Chae Jung , Sun-Chang Kim , Wan-Taek Im; J. Microbiol. 2014;52(5):399-406. Published online May 9, 2014; DOI: https://doi.org/10.1007/s12275-014-3601-7

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Abstract: The focus of this study was the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing β-glucosidase from Arthrobacter chlorophenolicus with an ultimate objective to more efficiently bio-transform ginse-nosides. The gene bglAch, consisting of 1,260 bp (419 amino acid residues) was cloned and the recombinant enzyme, over-expressed in Escherichia coli BL21 (DE3), was characterized. The GST-fused BglAch was purified using GST·Bind agarose resin and characterized. Under optimal conditions (pH 6.0 and 37°C) BglAch hydrolyzed the outer glucose and arabino-pyranose moieties of ginsenosides Rb1 and Rb2 at the C20 position of the aglycone into ginsenoside Rd. This was fol-lowed by hydrolysis into F2 of the outer glucose moiety of ginsenoside Rd at the C3 position of the aglycone. Additio-nally, BglAch more slowly transformed Rc to F2 via C-Mc1 (compared to hydrolysis of Rb1 or Rb2). These results in-dicate that the recombinant BglAch could be useful for the production of ginsenoside F2 for use in the pharmaceutical and cosmetic industries.

Biodegradation of C5-C8 Fatty Acids and Production of Aroma Volatiles by Myroides sp. ZB35 Isolated from Activated Sludge: Zijun Xiao , Xiankun Zhu , Lijun Xi , Xiaoyuan Hou , Li Fang , Jian R. Lu; J. Microbiol. 2014;52(5):407-412. Published online May 9, 2014; DOI: https://doi.org/10.1007/s12275-014-4109-x

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Abstract: In the effluents of a biologically treated wastewater from a heavy oil-refining plant, C5-C8 fatty acids including penta-noic acid, hexanoic acid, heptanoic acid, octanoic acid, and 2-methylbutanoic acid are often detected. As these residual fatty acids can cause further air and water pollution, a new Myroides isolate ZB35 from activated sludge was explored to degrade these C5-C8 fatty acids in this study. It was found that the biodegradation process involved a lag phase that became prolonged with increasing acyl chain length when the fatty acids were individually fed to this strain. However, when fed as a mixture, the ones with longer acyl chains were found to become more quickly assimilated. The branched 2- methylbutanoic acid was always the last one to be depleted among the five fatty acids under both conditions. Metabolite analysis revealed one possible origin of short chain fatty acids in the biologically treated wastewater. Aroma volatiles inclu-ding 2-methylbutyl isovalerate, isoamyl 2-methylbutanoate, isoamyl isovalerate, and 2-methylbutyl 2-methylbutanoate were subsequently identified from ZB35 extracts, linking the source of the fruity odor to these esters excreted by Myroides species. To our best knowledge, this is the first finding of these aroma esters in bacteria. From a biotechnological viewpoint, this study has revealed the potential of Myroides species as a promising source of aroma esters attractive for food and fragrance industries.

Low Cell Density Regulator AphA Upregulates the Expression of Vibrio vulnificus iscR Gene Encoding the Fe-S Cluster Regulator IscR: Jong Gyu Lim , Jin Hwan Park , Sang Ho Choi; J. Microbiol. 2014;52(5):413-421. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3592-4

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Abstract: IscR is a global transcriptional regulator that contributes to the pathogenesis of Vibrio vulnificus, a food-borne pathogen. In the present study, the regulatory mechanism for the iscR expression of V. vulnificus was evaluated. The expression of iscR was found to be upregulated by a transcriptional regu-lator AphA, a homologue of the low cell density regulator AphA of the Vibrio species, in the exponential phase of growth. The promoter activity of iscR appeared to be acti-vated and repressed by AphA and IscR, respectively. EMSA and DNase I protection assay showed that both AphA and IscR bind to the iscR regulatory region and the binding site for AphA overlapped with part of the binding site for IscR. Further mutational analysis suggested that AphA upregu-lates the iscR expression only in the presence of functional IscR. An examination of the roles of AphA and the binding sites revealed that the binding of AphA would hinder the IscR-mediated repression of the iscR transcription. The com-bined results show that V. vulnificus AphA upregulates iscR expression by antagonizing its negative autoregulation.

The Role as Inoculum Sources of Xanthomonas citri pv. citri Surviving on the Infected Satsuma mandarin Fruits: So Young Kang , Ki Deok Kim , Jeum Kyu Hong , He Nam Hyun , Yong Chull Jeun; J. Microbiol. 2014;52(5):422-426. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3366-z

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Abstract: Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solu-tion II). It indicated that the pathogenicity of the citrus can-ker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solu-tion directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result supports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.

Genetic Analysis of the Capsid Region of Norovirus GII.4 Variants Isolated in South Korea: Ju-Eun Kim , Sung-Geun Lee , Han-Gil Cho , Sang-Ha Han , Lae-Hyung Kang , Youn-Mi Lee , Chul-Jong Park , Soon-Young Paik; J. Microbiol. 2014;52(5):427-434. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3538-x

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Abstract: Norovirus is one of the major causes of non-bacterial gas-troenteritis in humans. The aim of this study was to analyze the amino acid variation of open reading frame 2 of GII.4 variants in South Korea during the period from November 2006 to December 2012. Sixty-nine complete nucleotide se-quences of open reading frame 2 were obtained from 113 GII.4 strains. The GII.4 2006b variants were detected pre-dominantly between 2006 and 2009; however, new GII.4 variants, which were termed the 2010 variant and the 2012 variant, emerged in 2010 and 2012, respectively. The num-ber of GII.4 2006b variants steadily decreased until 2012, whereas the number of gastroenteritis cases caused by the new variants increased between 2010 and 2012. The amino acid sequence in the ORF2 region obtained in this study was compared with other GII.4 variants isolated in various countries. Amino acid variations were observed primarily at epitope sites and the surrounding regions. Amino acids 294, 359, 393, and 413 of the P2 subdomain were the most variable sites among the GII.4 variants. The information in this study can be useful in basic research to predict the emergence and determine the genetic functions of new GII.4 variants.

Validation Study

Comparison of JEV Neutralization Assay Using Pseudotyped JEV with the Conventional Plaque-Reduction Neutralization Test: Hee-Jung Lee , Kyung-Il Min , Ki Hoon Park , Hyo Jung Choi , Min-Kyoung Kim , Chi-Young Ahn , Young-Jin Hong , Young Bong Kim; J. Microbiol. 2014;52(5):435-440. Published online March 7, 2014; DOI: https://doi.org/10.1007/s12275-014-3529-y

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Abstract: We previously reported the development of a neutralization assay system for evaluating Japanese Encephalitis Virus (JEV) neutralizing antibody (NAb) using pseudotyped-JEV (JEV- PV). JEV-PV-based neutralization assay offers several advan-tages compared with the current standard plaque-reduc-tion neutralization test (PRNT), including simplicity, safety, and speed. To evaluate the suitability of the JEV-PV assay as new replacement neutralization assay, we compared its repeatability, reproducibility, specificity, and correlated its results with those obtained using the PRNT. These analyses showed a close correlation between the results obtained with the JEV-PV assay and the PRNT, using the 50% plaque re-duction method as a standard for measuring NAb titers to JEV. The validation results met all analytical acceptance criteria. These results suggest that the JEV-PV assay could serve as a safe and simple method for measuring NAb titer against JEV and could be used as an alternative approach for assaying the potency of JEV neutralization.

Journal Article

Note] Antifungal Chitinase against Human Pathogenic Yeasts from Coprinellus congregatus: Yeeun Yoo Hyoung T. Choi; J. Microbiol. 2014;52(5):441-443. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3257-3

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Abstract: The inky cap, Coprinellus congregatus, produces mushrooms which become autolyzed rapidly to generate black liquid droplets, in which no cell wall is detected by microscopy. A chitinase (Chi2) which is synthesized during the autolytic phase of C. congregatus inhibits the growths of Candida al-bicans and Cryptococcus neoformans up to 10% at the con-centration of 10 μg/ml, about 50% at concentration of 20 μg/ml, and up to 95% at the concentration of 70 μg/ml. Upon treatment these yeast cells are observed to be severely de-formed, with the formation of large holes in the cell wall. The two yeast species show no growth inhibition at the concen-tration of 5 μg/ml, which means the minimum inhibitory concentrations for both yeast species are 10 μg/ml under these experimental conditions.

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