Journal of Microbiology

Volume 52(10); October 2014

Review

MINIREVIEW] The Potential Hazards of Aspergillus sp. in Foods and Feeds, and the Role of Biological Treatment: A Review: Sheikh Imranudin Sheikh-Ali , Akil Ahmad , Siti-Hamidah Mohd-Setapar , Zainul Akmal Zakaria , Norfahana Abdul-Talib , Aidee Kamal Khamis , Md Enamul Hoque; J. Microbiol. 2014;52(10):807-818. Published online October 1, 2014; DOI: https://doi.org/10.1007/s12275-014-4294-7

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Abstract: The contamination of food and feed by Aspergillus has become a global issue with a significant worldwide economic impact. The growth of Aspergillus is unfavourable to the development of food and feed industries, where the problems happen mostly due to the presence of mycotoxins, which is a toxic metabolite secreted by most Aspergillus groups. Moreover, fungi can produce spores that cause diseases, such as allergies and asthma, especially to human beings. High temperature, high moisture, retarded crops, and poor food storage conditions encourage the growth of mold, as well as the development of mycotoxins. A variety of chemical, biological, and physical strategies have been developed to control the production of mycotoxins. A biological approach, using a mixed culture comprised of Saccharomyces cerevisiae and Lactobacillus rhamnosus resulted in the inhibition of the growth of fungi when inoculated into fermented food. The
results
reveal that the mixed culture has a higher potential (37.08%) to inhibit the growth of Aspergillus flavus (producer of Aflatoxin) compared to either single culture, L. rhamnosus NRRL B-442 and S. cerevisiae, which inhibit the growth by 63.07% and 64.24%, respectively.

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

Citrimicrobium luteum gen. nov., sp. nov., Aerobic Anoxygenic Phototrophic Bacterium Isolated from the Gut of a Sea Cucumber Stichopus japonicus: Hong-Joo Jung , In-Tae Cha , Kyung June Yim , Hye Seon Song , Kichul Cho , Daekyung Kim , Hae-Won Lee , Jae Kook Lee , Myung-Ji Seo , Seong Woon Roh , Sung-Jae Lee; J. Microbiol. 2014;52(10):819-824. Published online August 27, 2014; DOI: https://doi.org/10.1007/s12275-014-4136-7

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Abstract: A Gram-stain negative, yellow-pigmented, motile, pleomorphic bacterium, designated strain CBA4602T, was isolated from the gut of the sea cucumber Stichopus japonicus, which was collected from Jeju Island in the Republic of Korea. In a phylogenetic analysis based on the 16S rRNA gene, strain CBA4602T belonged to the order Sphingomonadales in the class Alphaproteobacteria. The 16S rRNA gene sequence similarity between strain CBA4602T and ‘Citromicrobium bathyomarinum’ JF-1, the most closely related strain having nonvalidly published name, was 98.4%, followed by 95.2–96.7% identities with sequence of the other closest strains in the genus Erythrobacter. Strain CBA4602T had bacteriochlorophyll a and carotenoids. Strain CBA4602T grew in 0–10% (w/v) NaCl, at 10–42°C and pH 6.0–8.0, with optimal growth in 1–2% NaCl, at 30–37°C and pH 7.0. Strain CBA4602T was positive for catalase and oxidase activities and was able to hydrolyse gelatine and Tween 20 and 40, but not starch, Tween 80 or L-tyrosine. The G+C content of genomic DNA from strain CBA4602T was 68.0 mol% and Q-10 was the major detected isoprenoid quinone. The polar lipids were three unidentified phospholipids, three unidentified glycolipids, and two unidentified lipids. The dominant fatty acids were anteiso-C15:0, C16:0, anteiso-C17:0 and C18:0. As considering the current taxonomic status of the genus ‘Citromicrobium’ and polyphasic taxonomic analyses, strain CBA4602T represents a novel genus and species. The name Citrimicrobium luteum is proposed for the type strain CBA4602T (=KACC 17668T =JCM 19530T).

Identification of Proteolytic Bacteria from the Arctic Chukchi Sea Expedition Cruise and Characterization of Cold-active Proteases: Ha Ju Park , Yung Mi Lee , Sunghui Kim , Ah Ram Wi , Se Jong Han , Han-Woo Kim , Il-Chan Kim , Joung Han Yim , Dockyu Kim; J. Microbiol. 2014;52(10):825-833. Published online August 27, 2014; DOI: https://doi.org/10.1007/s12275-014-4226-6

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Abstract: Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127–2,130 bp, encoding 708–709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3–72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.

Distinct Patterns of Marine Bacterial Communities in the South and North Pacific Oceans: Sung-Suk Suh , Mirye Park , Jinik Hwang , Sukchan Lee , Youngjae Chung , Taek-Kyun Lee; J. Microbiol. 2014;52(10):834-841. Published online October 1, 2014; DOI: https://doi.org/10.1007/s12275-014-4287-6

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Abstract: The study of oceanic microbial communities is crucial for our understanding of the role of microbes in terms of biomass, diversity and ecosystem function. In this study, 16S rRNA gene tag pyrosequencing was used to investigate change in bacterial community structure between summer and winter water masses from Gosung Bay in the South Sea of Korea and Chuuk in Micronesia, located in the North and South Pacific Oceans, respectively. Summer and winter sampling from each water mass revealed highly diverse bacterial communities, containing ~900 Operational Taxonomic Units (OTUs). The microbial distribution and highly heterogeneous composition observed at both sampling sites were different from those of most macroorganisms. The bacterial communities in the seawater at both sites were most abundant in Proteobacteria during the summer in Gosung and in Bacterioidetes during the winter. The proportion of Cyanobacteria was higher in summer than in winter in Chuuk and similar in Gosung. Additionally, the microbial community during summer in Gosung was significantly different from other communities observed based on the unweighted UniFrac distance. These data suggest that in both oceanic areas sampled, the bacterial communities had distinct distribution patterns with spatially- and temporally-heterogeneous distributions.

Synthetic Lethal Screen of NAA20, a Catalytic Subunit Gene of NatB N-Terminal Acetylase in Saccharomyces cerevisiae: Kang-Eun Lee , Jun-Young Ahn , Jeong-Mok Kim , Cheol-Sang Hwang; J. Microbiol. 2014;52(10):842-848. Published online August 27, 2014; DOI: https://doi.org/10.1007/s12275-014-3694-z

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Abstract: The Saccharomyces cerevisiae NatB N-terminal acetylase contains a catalytic subunit Naa20 and an auxiliary subunit Naa25. To elucidate the cellular functions of the NatB, we utilized the Synthetic Genetic Array to screen for genes that are essential for cell growth in the absence of NAA20. The genome-wide synthetic lethal screen of NAA20 identified genes encoding for serine/threonine protein kinase Vps15, 1,3-beta-glucanosyltransferase Gas5, and a catabolic repression regulator Mig3. The present study suggests that the catalytic activity of the NatB N-terminal aceytase is involved in vacuolar protein sorting and cell wall maintenance.

σB Affects Biofilm Formation under the Dual Stress Conditions Imposed by Adding Salt and Low Temperatur in Listeria monocytogenes: Jin-Ju Lee , Gilho Lee , Ji-Hyun Shin; J. Microbiol. 2014;52(10):849-855. Published online October 1, 2014; DOI: https://doi.org/10.1007/s12275-014-4369-5

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Abstract: The food-borne pathogenic bacteria Listeria monocytogenes can form biofilms on various surfaces including food-processing equipment. Biofilms offer survival benefits to the organisms entrapped against environmental insults. Moreover, the σB transcription factor of L. monocytogenes plays an important role in its survival under various stress conditions. In this study, we evaluated whether σB contributes to biofilm formation when L. monocytogenes is grown under various temperatures and media. When the wild-type strain was grown under static biofilm culture below ambient temperature (15°C) for 72 h, the difference in viable cell number (in both planktonic and biofilm cells) between the wild-type and ΔsigB mutant increased by adding NaCl to BHI broth (9% salt BHI > 6% salt BHI > BHI, w/v), and the specific activity of β-galactosidase was highly induced in the wild-type strain grown in 6% salt containing BHI broth. Furthermore, we measured surface-adhered biofilm forming ability using the crystal violet staining method. The wild-type strain formed a four times larger biofilm than that of the ΔsigB mutant in 6% salt-BHI medium at 15°C over a 72 h incubation and also showed the highest level of β-galactosidase specific activity. However, both the wild-type and ΔsigB mutant L. monocytogenes were defective for forming a biofilm in 9% salt-BHI medium at 15°C. Our results suggest that σB plays an enhanced role in surface-adhered biofilm formation when L. monocytogenes encounters dual stress conditions, such as 6% NaCl and low temperature.

Surface Display Expression of Bacillus licheniformis Lipase in Escherichia coli Using Lpp’OmpA Chimera: Jae-Hyung Jo , Chan-Wook Han , Seung-Hwan Kim , Hyuk-Jin Kwon , Hyune-Hwan Lee; J. Microbiol. 2014;52(10):856-862. Published online August 27, 2014; DOI: https://doi.org/10.1007/s12275-014-4217-7

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Abstract: The lipase from Bacillus licheniformis ATCC14580 was displayed on the cell surface of Escherichia coli using Lpp’OmpA as the anchoring protein. The expressed Lpp’OmpA-lipase fusion protein has a molecular weight of approximately 35 kDa, which was confirmed by SDS-PAGE and western blot analysis. The Lpp’OmpA-lipase fusion protein was located on the cell surface, as determined by immunofluorescence confocal microscopy and flow cytometry. The enzyme activity of the surface-displayed lipase showed clear halo around the colony. The cell surface-displayed lipase showed the highest activity of 248.12 ± 9.42 U/g (lyophilized cell) at the optimal temperature of 37°C and pH 8.0. The enzyme exhibited the highest activity toward the substrate p-nitrophenyl caprylate (C8). These results suggest that E. coli, which displayed the lipase on its surface, could be used as a whole cell biocatalyst.

Biocatalytic Properties and Substrate-binding Ability of a Modular GH10 β-1,4-Xylanase from an Insect-symbiotic Bacterium, Streptomyces mexicanus HY-14: Do Young Kim , Dong-Ha Shin , Sora Jung , Jong Suk Lee , Han-Young Cho , Kyung Sook Bae , Chang-Keun Sung , Young Ha Rhee , Kwang-Hee Son , Ho-Yong Park; J. Microbiol. 2014;52(10):863-870. Published online October 1, 2014; DOI: https://doi.org/10.1007/s12275-014-4390-8

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Abstract: The gene (1350-bp) encoding a modular β-1,4-xylanase (XylU), which consists of an N-terminal catalytic GH10 domain and a C-terminal carbohydrate-binding module 2 (CBM 2), from Streptomyces mexicanus HY-14 was cloned and functionally characterized. The purified His-tagged recombinant enzyme (rXylU, 44.0 kDa) was capable of efficiently hydrolyze diverse xylosidic compounds, p-nitrophenyl-cellobioside, and pnitrophenyl- xylopyranoside when incubated at pH 5.5 and 65°C. Especially, the specific activities (649.8 U/mg and 587.0 U/mg, respectively) of rXylU toward oat spelts xylan and beechwood xylan were relatively higher than those (<500.0 U/mg) of many other GH10 homologs toward the same substrates. The results of enzymatic degradation of birchwood xylan and xylooligosaccharides (xylotriose to xylohexaose) revealed that rXylU preferentially hydrolyzed the substrates to xylobiose (>75%) as the primary degradation product. Moreover, a small amount (4%<) of xylose was detected as the degradation product of the evaluated xylosidic substrates, indicating that rXylU was a peculiar GH10 β-1,4- xylanase with substrate specificity, which was different from its retaining homologs. A significant reduction of the binding ability of rXylU caused by deletion of the C-terminal CBM 2 to various insoluble substrates strongly suggested that the additional domain might considerably contribute to the enzyme-substrate interaction.

Characterization of a Novel Antigen of Mycobacterium tuberculosis K Strain and Its Use in Immunodiagnosis of Tuberculosis: Paul J. Park , Ah Reum Kim , Yangkyo P. Salch , Taeksun Song , Sung Jae Shin , Seung Jung Han , Sang-Nae Cho; J. Microbiol. 2014;52(10):871-878. Published online August 27, 2014; DOI: https://doi.org/10.1007/s12275-014-4235-5

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Abstract: *For correspondence. (S.J. Han) E-mail: hansjung@yuhs.ac / (S.N. Cho) E-mail: raycho@yuhs.ac Paul J. Park, Ah Reum Kim, Yangkyo P. Salch, Taeksun Song, Sung Jae Shin, Seung Jung Han*, and Sang-Nae Cho* Department of Microbiology and Institute for Immunology and Immunological Diseases, Brain Korea 21 Plus Project for the Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea (Received Apr 16, 2014 / Revised Jul 14, 2014 / Accepted Jul 16, 2014) Journal of Microbiology (2014) Vol. 52, No. 10, pp. 871–878 Copyright 􎨰􀁇2014, The Microbiological Society of Korea DOI 10.1007/s12275-014-4235-5 Characterization of a Novel Antigen of Mycobacterium tuberculosis K strain and Its Use in Immunodiagnosis of Tuberculosis Mycobacterium tuberculosis-specific antigens would be of great value in developing immunodiagnostic tests for tuberculosis (TB), but regional differences in molecular types of the organism may result in antigenic variation, which in turn affects the outcome of the tests. For example, the Beijing strains of M. tuberculosis are prevalent in East Asia, and in particular, the K strain and related strains of the Beijing family, are most frequently isolated during school outbreaks of TB in South Korea. From comparison of genome sequences between M. tuberculosis K strain and the H37Rv strain, a non-Beijing type, we identified a K strain-specific gene, InsB, which has substantial homology with the ESAT-6-like proteins. This study was, therefore, initiated to characterize the InsB protein for its immunogenicity in mice and to confirm its expression in TB patients by detecting antibodies to the protein. The InsB gene was cloned from M. tuberculosis K strain and expressed in Escherichia coli. The recombinant InsB protein was used for immunization of mice. All mice showed strong antibody responses to the InsB protein, and splenocytes stimulated with InsB showed strong IFN-γ and IL-17 responses and a weak IL-2 response, all of which have been implicated in disease expression and used for the immunodiagnosis of TB. Serum samples from TB patients also showed significant antibody responses to the InsB protein as compared to healthy control samples. These results indicate that the InsB protein is an M. tuberculosis K-strain-specific antigen that could further improve the current immunodiagnostic
methods
, especially for the South Korean population.

Microscopical Observation of Inhibition-behaviors against Diaporthe citri by Pre-treated with Pseudomonas putida Strain THJ609-3 on the Leaves of Citrus Plants: Yun Jung Ko , Ju Sung Kim , Ki Deok Kim , Yong Chull Jeun; J. Microbiol. 2014;52(10):879-883. Published online October 1, 2014; DOI: https://doi.org/10.1007/s12275-014-4399-z

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Abstract: Citrus melanose is one of the most important diseases in orchards cultivating citrus in the world. Although the disease does not cause yield loss, the profitability of the infected fruits is often reduced in the fresh-market, resulting in economic loss. In this study, disease reduction was proven by pre-treatment with Pseudomonas putida strain THJ609-3. In order to illustrate mechanism of the disease reduction by the bacterial strain, the infection behaviors of Diaporthe citri and necrosis deposit of plant tissue were observed using a fluorescence microscope. On the leaves pre-treated with the strain THJ609-3, germination rates of D. citri conidia were significantly decreased compared to those of the untreated control. Scanning electron microscopical observations showed that bacterial cells were attached to the surface of fungal hyphae. Furthermore, morphological change of germ tubes of the conidia was detected. These results suggest that the disease reduction may be caused by the direct antifungal activity of the bacterial strain on the leaf surfaces.

NOTE] Alicyclobacillus tengchongensis sp. nov., a Thermo-Acidophilic Bacterium Isolated from Hot Spring Soil: Min Goo Kim , Jae-Chan Lee , Dong-Jin Park , Wen-Jun Li , Chang-Jin Kim Kim; J. Microbiol. 2014;52(10):884-889. Published online July 18, 2014; DOI: https://doi.org/10.1007/s12275-014-3625-z

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Abstract: A thermo-acidophilic bacterium, designated strain ACK006T, was isolated from the soil of a hot spring at Tengchong in China. Cells were Gram-staining-positive, motile, catalasepositive and oxidase-negative, spore-forming rods. The isolate grew aerobically at 30–50°C (optimum at 45°C), pH 2.0–6.0 (optimum pH 3.2) and 0–5.0% (w/v) NaCl (optimum 1% NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain ACK006T belongs to the genus Alicyclobacillus with the sequence similarity of 92.3, 92.4, 92.5, and 92.8% to Alicyclobacillus cycloheptanicus SCHT, Alicyclobacillus ferrooxydans TC-34T, Alicyclobacillus contaminans 3-A191T and Alicyclobacillus disulfidooxidans SD-11T, respectively. Similarity to other species of the genus Alicyclobacillus was 90.3–92.8% and similarity to species of the genus Tumebacillus was 85.9–87.8%. The genomic DNA G+C content was 53.7 mol%. The predominant menaquinone was MK-7. Major fatty acids were ω-cycloheptane C18:0, iso-C17:0 and anteiso-C17:0. The cell-wall peptidoglycan was the A1γ type; containing meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of polyphasic analysis from this study, strain ACK006T represents a novel species of the genus Alicyclobacillus for which the name Alicyclobacillus tengchongensis sp. nov. is proposed. The type strain is ACK006T (=KCTC 33022T =DSM 25924T).

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