Journal of Microbiology

Volume 47(1); February 2009

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

Response of Saccharomyces cerevisiae to Stress-Free Acidification: Allen Kuan-Liang Chen , Cristy Gelling , Peter L. Rogers , Ian W. Dawes , Bettina Rosche; J. Microbiol. 2009;47(1):1-8. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0167-2

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Genome-wide transcriptional analysis of a Saccharomyces cerevisiae batch culture revealed that more than 829 genes were regulated in response to an environmental shift from pH 6 to pH 3 by added sulfuric acid. This shift in pH was not detrimental to the rate of growth compared to a control culture that was maintained at pH 6 and the transcriptional changes most strikingly implicated not up- but down-regulation of stress responses. In addition, the transcriptional changes upon acid addition indicated remodeling of the cell wall and central carbon metabolism. The overall trend of changes was similar for the pH-shift experiment and the pH 6 control. However, the changes in the pH 6 control were much weaker and occurred 2.5 h later than in the pH-shift experiment. Thus, the reaction to the steep pH decrease was an immediate response within the normal repertoire of adaptation shown in later stages of fermentation at pH 6. Artificially preventing the yeast from acidifying the medium may be considered physiologically stressful under the tested conditions.

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Development of a High-Cell-Density Production Process for a Biotherapeutic Yeast, Saccharomyces cerevisiae var. boulardii, for Use as a Human Probiotic
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Isolation and Characterization of a Marine Algicidal Bacterium against the Harmful Raphidophyceae Chattonella marina: Yun Sook Kim , Dae-Sung Lee , Seong-Yun Jeong , Won Jae Lee , Myung-Suk Lee; J. Microbiol. 2009;47(1):9-18. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0141-z

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Abstract PDF

A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of Uljin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal activity by different culture filtrate volumes, the 10% (100 ?/ml) concentration showed the biggest change in algicidal activity; there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5. Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina.

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Class 1 and Class 2 Integrons and Plasmid-Mediated Antibiotic Resistance in Coliforms Isolated from Ten Rivers in Northern Turkey: Osman Birol Ozgumus , Cemal Sandalli , Ali Sevim , Elif Celik-Sevim , Nuket Sivri; J. Microbiol. 2009;47(1):19-27. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0206-z

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Abstract PDF: We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, blaOXA-30, and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens.

Incidence of Wolbachia and Cardinium Endosymbionts in the Osmia Community in Korea: Gilsang Jeong , Kyeongyong Lee , Jiyoung Choi , Seokjo Hwang , Byeongdo Park , Wontae Kim , Youngcheol Choi , Ingyun Park , Jonggill Kim; J. Microbiol. 2009;47(1):28-32. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-009-0198-3

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Sex ratio distorting endosymbionts induce reproductive anomalies in their arthropod hosts. They have recently been paid much attention as firstly texts of evolution of host-symbiont relationships and secondly potential biological control agents to control arthropod pests. Among such organisms, Wolbachia and Cardinium bacteria are well characterized. This study aims at probing such bacteria in the Osmia community to evaluate their potential utilization to control arthropod pests. Among 17 PCR tested species, Osmia cornifrons and a parasitic fly are infected with Wolbachia and a mite species is infected with Cardinium. Phylogenetic tree analyses suggest that horizontal transfer of the bacteria occurred between phylogenetically distant hosts.

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Gilsang Jeong, Taeman Han, Haechul Park, Soyeon Park, Pureum Noh
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Cardinium endosymbionts are widespread in synanthropic mite species (Acari: Astigmata)
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Vera I D Ros, Vicki M Fleming, Edward J Feil, Johannes A J Breeuwer
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Impact of cry1AC-Carrying Brassica rapa subsp. pekinensis on Leaf Bacterial Community: Young Tae Kim , Kang Seon Lee , Moon Jung Kim , Seung Bum Kim; J. Microbiol. 2009;47(1):33-39. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0254-4

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Abstract PDF: The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of transgene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.

Isolation and Identification of an Anticancer Drug, Taxol from Phyllosticta tabernaemontanae, a Leaf Spot Fungus of an Angiosperm, Wrightia tinctoria: Rangarajulu Senthil Kumaran , Johnpaul Muthumary , Byung-Ki Hur; J. Microbiol. 2009;47(1):40-49. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0127-x

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Phyllosticta tabernaemontanae, a leaf spot fungus isolated from the diseased leaves of Wrightia tinctoria, showed the production of taxol, an anticancer drug, on modified liquid medium (M1D) and potato dextrose broth (PDB) medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The amount of taxol produced by this fungus was quantified using high performance liquid chromatography (HPLC). The maximum amount of taxol production was recorded in the fungus grown on M1D medium (461 ug/L) followed by PDB medium (150 ug/L). The production rate was increased to 9.2x103 fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The results designate that P. tabernaemontanae is an excellent candidate for taxol production. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay.

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Induction of Growth Phase-Specific Autolysis in Bacillus subtilis 168 by Growth Inhibitors: Jin-Kyo Chung , Hyun Ee Yoon , Ha Chul Shin , Eun-Young Choi , Woo-Hyeon Byeon; J. Microbiol. 2009;47(1):50-59. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0256-2

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Abstract PDF: Growth phase-specific autolysis of Bacillus subtilis by inhibitors of membrane permeability, inhibitors of macromolecule biosynthesis, inhibitors of cell wall biosynthesis and detergents were tested and characterized in glucose limited liquid medium. The minimum autolysis induction concentration (MAIC) of test compounds, which was at least 1/20th lower than the conventional autolysis induction concentration, induced autolysis only for cells at the glucose exhaustion point (diauxic point) of the growth phase, while it was not induced for cells at pre- and post-diauxic points. Inhibitors of macromolecule synthesis that are not known for inducing autolysis, such as chloramphenicol, rifampicin, nalidixic acid, and detergents, also induced specific autolysis. Two types of autolysis corresponding to the concentrations of compounds are distinguished: concentration-sensitive and concentration-insensitive types.

Overexpression of Bacterioferritin Comigratory Protein (Bcp) Enhances Viability and Reduced Glutathione Level in the Fission Yeast Under Stress: Ga-Young Kang , Eun-Hee Park , Kyunghoon Kim , Chang-Jin Lim; J. Microbiol. 2009;47(1):60-67. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0077-3

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The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCP10. The bcp+ mRNA level in the pBCP10-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCP10 exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels.

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A unique thioredoxin reductase plays defensive roles against oxidative, nitrosative and nutritional stresses in Schizosaccharomyces pombe
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Thermoresistant properties of bacterioferritin comigratory protein against high temperature stress in Schizosaccharomyces pombe
In Wang Ryu, Su Hee Lee, Hye-Won Lim, Kisup Ahn, Kwanghark Park, Jae-Hoon Sa, Kyung Jin Jeong, Chang-Jin Lim, Kyunghoon Kim
The Korean Journal of Microbiology.2016; 52(4): 398. CrossRef
Role of bacterioferritin comigratory protein and glutathione peroxidase-reductase system in promoting bentazone tolerance in a mutant of Synechococcus elongatus PCC7942
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Characterization of Conjugative Plasmids Carrying Antibiotic Resistance Genes Encoding 16S rRNA Methylase, Extended-Spectrum Beta-Lactamase, and/or Plasmid-Mediated AmpC Beta-Lactamase: Hee Young Kang , Jungmin Kim , Sung Yong Seol , Yoo Chul Lee , Je Chul Lee , Dong Taek Cho; J. Microbiol. 2009;47(1):68-75. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0158-3

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Abstract PDF: In this study, we identified extended-spectrum ß-lactamase (ESBL) and plasmid-mediated AmpC ß-lactamase which were associated with 16S rRNA methylase gene on the conjugative plasmid. Among 82 clinical isolates of Enterobacteriaceae that carry 16S rRNA methylase gene (64 strains, armA, and 18 strains, rmtB), blaSHV-12 was detected either alone or combined with blaDHA-1, blaCTX-M-3, and blaCTX-M-14 in 30 strains carrying armA and 6 strains carrying rmtB. The blaCTX-M-3 was detected in 13 of 64 strains carrying armA but no strains carrying rmtB. Whereas blaCTX-M-14 was detected in 15 of 18 strains carrying rmtB but only 2 of 64 strains carrying armA. Overall, blaSHV-12 and blaCTX-M-14 was the most common ESBL gene which was associated with armA and rmtB, respectively. In addition, we found that blaCTX-M-3 localized with armA on the same IncL/M plasmid and blaCTX-M-14 localized with rmtB on the same IncA/C plasmid. Restriction fragment length polymorphism of conjugative plasmids and pulsed-field gel electrophoresis of genomic DNAs revealed that intercellular horizontal transfer of conjugative plasmid and clonal transmission have been occurred at the same time.

Distribution of Marine Birnavirus (MABV) in Marine Organisms from Okinawa, Japan, and a Unique Sequence Variation of the VP2/NS Region: Manami Inaba , Satoru Suzuki , Shin-Ichi Kitamura , Norichika Kumazawa , Hiroshi Kodama; J. Microbiol. 2009;47(1):76-84. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0250-8

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Distribution of marine type of Aquabirnavirus (MABV) was examined in shellfish and fish from Okinawa and Ishigaki Islands, Japan, where water temperature is higher than 25°C through the year. Genome detection and virus isolation were performed for shellfish and fish samples, and the results revealed the prevalent distribution of MABV in diverse species in the area, although isolation was not frequently. Detection rate of MABV genome in bivalves was higher than gastropods, which was similar result to former report in mainland of Japan. Furthermore, the unique five-nucleotide deletion was found with a high rate of occurrence in the MABV genome from shellfish and fish. This study showed distribution status of MABV in organisms in subtropical waters by wide monitoring, and discovered new genome variation in VP2/NS region of this virus.

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Molecular phylogenetic studies of the HIV-1 isolated from Koreans have suggested the presence of the so- called “Korean clade”, which can be defined as a cluster free of foreign isolates. The Korean clade accounts for more than 60% of Korean isolates and exerts characteristic amino acid sequences. Thus, it is merited to estimate when this Korean clade first emerged in order to understand the evolutionary pattern of the Korean clade. We analyzed and reconstructed the most recent common ancestor (MRCA) sequences from nef (n=229) and vif (n=179) Korean clade sequences. Linear regression analyses of sequence divergence estimates were plotted against sampling years to infer the year in which there was zero divergence from the MRCA sequences. MRCA sequences suggested the Korean clade was first emerged around 1984, before the first detection of HIV-1 in Korea in 1985. Further studies on synonymous and nonsynonymous substitution rates suggested positive selection event for the Korean clade, while other subtype B had undergone negative to neutral evolution.

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To investigate the genetic characteristics of human influenza viruses circulating in Chungbuk province, we tested 510 clinical samples of nasopharyngeal suction from pediatric patients diagnosed with respiratory illness between June 2007 and June 2008. Genetic characterization of the HA genes of H3N2 isolates indicated the relative higher similarity to A/Virginia/04/07 (99.6%) rather than that of A/Wisconsin/67/2005 (98.4%), a Northern Hemisphere 2007~2008 vaccine strain, based on amino acid sequences. We found several altered amino acids at the H3 HA1 antigenic sites compared with the vaccine strain; K140I at site A, K158R at site B, and K173N (H471) or K173Q, and S262N at site E, but there was no antigenic shift among the H3N2 viruses. Interestingly, A/Cheongju/H383/08 and A/Cheongju/H407/08 isolates had single amino acid substitution at D151G on the catalytic site of the N2 NA while A/Cheongju/H412/08 and A/Cheongju/H398/07 isolates had one amino acid deletion at residue 146. Furthermore, we found that 25% (3 out of 12 isolates) of the H3N2 subtype viruses had the amino acid substitution at position 31 on the M2 protein (Aspartic acid to Asparagine) and confirmed their drug-resistance by biological assays. Taken together, the results of this study demonstrated continuous evolutions of human H3N2 viruses by antigenic drift and also highlighted the need to closely monitor antigenic drug resistance in influenza A viruses to aid in the early detection of potentially pandemic strains, as well as underscore the need for new therapeutics.

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Randomized Controlled Trial

Antimicrobial Activity of Enterocins from Enterococcus faecalis SL-5 against Propionibacterium acnes, the Causative Agent in Acne Vulgaris, and Its Therapeutic Effect: Bong Seon Kang , Jae-Gu Seo , Gwa-Su Lee , Jung-Hwa Kim , Sei Yeon Kim , Ye Won Han , Hoon Kang , Hyung Ok Kim , Ji Hwan Rhee , Myung-Jun Chung , Young Min Park; J. Microbiol. 2009;47(1):101-109. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0179-y

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Abstract PDF: A lactic acid bacterial strain was isolated from human fecal specimen and identified as Enterococcus faecalis SL-5. The isolated strain showed antimicrobial activity against Gram-positive pathogens assayed, especially the highest activity against Propionibacterium acnes. The antimicrobial substance was purified and verified as a bacteriocin (named ESL5) of E. faecalis SL-5 by activity-staining using P. acnes as an indicator. N-terminal sequence of ESL5 was determined (MGAIAKLVAK) and sequence analysis revealed that it is almost identical to the some of enterocins including L50A/B of E. faecium L50 and MR10A/B of E. faecalis MRR 10-3. From the sequencing data of L50A/B structural genes, the nucleotide sequence showed 100% identity with that of the MR10A/B structural genes, implying that ESL5 is an equivalent of enterocin MR10. Meanwhile, we also tested the therapeutic effect of anti-P. acnes activity in patients with mild to moderate acne because of its pathogenic role to acne vulgaris. For this purpose, a concentrated powder of CBT SL-5 was prepared using cell-free culture supernatant (CFCS) of E. faecalis SL-5 and included in a lotion for application in the patients. The study showed that CBT SL-5 lotion significantly reduced the inflammatory lesions like pustules compared to the placebo lotion. Therefore our results indicate that the anti-P. acnes activity produced by E. faecalis SL-5 has potential role to the treatment of acne as an alternative to topical antibiotics.

Journal Article

Molecular Characterization of Vibrio cholerae Isolates from Cholera Outbreaks in North India: Joseph J. Kingston , Kuruvilla Zachariah , Urmil Tuteja , Sanjay Kumar , Harsh Vardhan Batra; J. Microbiol. 2009;47(1):110-115. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0162-7

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Abstract PDF

Vibrio cholerae isolates recovered from cholera outbreaks in Bhind district of Madhya Pradesh and Delhi, Northern India were characterized. The O1 serogroup isolates from Bhind outbreak were of Inaba serotype whereas both Ogawa and Inaba serotypes were recovered from Delhi. PCR analysis revealed that only O1 serogroup V. cholerae isolates carried the virulence-associated genes like ctxA, tcpA, ace, and zot. Molecular typing by repetitive sequence based ERIC, VCR1, and VC1 PCR’s revealed similar DNA profile for both Inaba and Ogawa serotypes. A discrete VC1-PCR band identified among the El Tor strains had greater similarity (>97%) to the V. cholerae genome sequence and therefore has the potential to be used as a marker for the identification of the V. cholerae strains. Non-O1 strains recovered from Bhind region differed among themselves as well as from that of the O1 isolates. All the O1 serogroup isolates possessed SXT element and were uniformly resistant to the antibiotics nalidixic acid, polymyxin-B, furazolidone, cloxacilin, trimethoprim-sulfamethaxazole, and vibriostatic agent O129. Inaba strains from both Delhi and Bhind differed from Ogawa strains by their resistance to streptomycin despite sharing similar DNA patterns in all the three rep-PCRs. Though Delhi and Bhind are separate geographical regions in Northern India, Inaba strains from both these places appear to be closely related owing to their similarity in antibiogram and genetic profile.

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

Molecular Cloning of the Phospholipase D Gene from Streptomyces sp. YU100 and Its Expression in Escherichia coli: Ji-Seon Lee , Munkhtsetseg Bat-Ochir , Atanas V. Demirev , Doo Hyun Nam; J. Microbiol. 2009;47(1):116-122. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0161-8

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Abstract PDF

The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and heterologously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.

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