Journal of Microbiology

Volume 44(2); April 2006

Reviews

Computational Approaches to Gene Prediction: Jin Hwan Do , Dong-Kug Choi; J. Microbiol. 2006;44(2):137-144.; DOI: https://doi.org/2372 [pii]

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Abstract: The problems associated with gene identification and the prediction of gene structure in DNA sequences have been the focus of increased attention over the past few years with the recent acquisition by large-scale sequencing projects of an immense amount of genome data. A variety of prediction programs have been developed in order to address these problems. This paper presents a review of the computational approaches and gene-finders used commonly for gene prediction in eukaryotic genomes. Two approaches, in general, have been adopted for this purpose: similarity-based and ab initio techniques. The information gleaned from these methods is then combined via a variety of algorithms, including Dynamic Programming (DP) or the Hidden Markov Model (HMM), and then used for gene prediction from the genomic sequences.

Heterotrimeric G protein signaling and RGSs in Aspergillus nidulans: Jae-Hyuk Yu; J. Microbiol. 2006;44(2):145-154.; DOI: https://doi.org/2371 [pii]

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Abstract: Heterotrimeric G proteins (G proteins) are conserved in all eukaryotes and are crucial components sensing and relaying external cues into the cells to elicit appropriate physiological and biochemical responses. Basic units of the heterotrimeric G protein signaling system include a G protein-coupled receptor (GPCR), a G protein composed of α, β, and γ subunits, and variety of effectors. Sequential sensitization and activation of these G protein elements translates external signals into gene expression changes, resulting in appropriate cellular behaviors. Regulators of G protein signaling (RGSs) constitute a crucial element of appropriate control of the intensity and duration of G protein signaling. For the past decade, G protein signaling and its regulation have been intensively studied in a number of model and/or pathogenic fungi and outcomes of the studies provided better understanding on the upstream regulation of vegetative growth, mating, development, virulence/pathogenicity establishment, and biosynthesis of secondary metabolites in fungi. This review focuses on the characteristics of the basic upstream G protein components and RGS proteins, and their roles controlling various aspects of biological processes in the model filamentous ascomycete fungus Aspergillus nidulans. In particular, their functions in controlling hyphal proliferation, asexual spore formation, sexual fruiting, and the mycotoxin sterigmatocystin production are discussed.

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

Analysis of Microbial Communities Using Culture-dependent and Culture-independent Approaches in an Anaerobic/Aerobic SBR Reactor: Shipeng Lu , Minjeong Park , Hyeon-Su Ro , Dae Sung Lee , Woojun Park , Che Ok Jeon; J. Microbiol. 2006;44(2):155-161.; DOI: https://doi.org/2370 [pii]

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Abstract: Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63% (137 of 217) in the standard PCR method to about 34% (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: α, β, γ- Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivationbased method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.

Detection of Human Adenoviruses and Enteroviruses in Korean Oysters Using Cell Culture, Integrated Cell Culture-PCR, and Direct PCR: Yoe-Jin Choo , Sang-Jong Kim; J. Microbiol. 2006;44(2):162-170.; DOI: https://doi.org/2369 [pii]

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Abstract: Oysters are known to be carriers of food-born diseases, but research on viruses in Korean oysters is scarce despite its importance for public health. We therefore tested oysters cultivated in Goheung, Seosan, Chungmu, and Tongyeong, for viral contamination using cell culture and integrated cell culture PCR (ICC-PCR) with Buffalo green monkey kidney (BGMK) and human lung epithelial (A549) cells. Additional screens via PCR, amplifying viral nucleic acids extracted from oysters supplemented our analysis. Our methods found 23.6%, 50.9%, and 89.1% of all oysters to be positive for adenoviruses when cell culture, ICC-PCR, and direct PCR, respectively, was used to conduct the screen. The same methodology identified enteroviruses in 5.45%, 30.9%, and 10.9% of all cases. Most of the detected enteroviruses (81.3%) were similar to poliovirus type 1; the remainder resembled coxsackievirus type A1. A homology search with the adenoviral sequences revealed similarities to adenovirus subgenera C (type 2, 5, and 6), D (type 44), and F (enteric type 40 and 41). Adenovirus-positive samples were more abundant in A549 cells (47.3%) than in BGMK cells (18.2%), while the reverse was true for enteroviruses (21.8% vs. 14.5%). Our data demonstrate that Korean oysters are heavily contaminated with enteric viruses, which is readily detectable via ICC-PCR using a combination of A549 and BGMK cells.

Thalassobius aestuarii sp. nov., Isolated from Tidal Flat Sediment: Hana Yi , Jongsik Chun; J. Microbiol. 2006;44(2):171-176.; DOI: https://doi.org/2368 [pii]

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Abstract: A strictly aerobic, non-motile, ovoid-shaped Alphaproteobacteria, designated strain JC2049T,was isolated from a tidal flat sediment sample. The results of 16S rRNA gene sequence analysis indicated that this isolate belonged to the genus Thalassobius, with a sequence similarity of 96.9-97.3% to other valid Thalassobius spp. The cells required 1-7% NaCl for growth (optimum 2%) and accumulated poly-β-hydroxybutyrate. Nitrite was reduced to nitrogen, but nitrate was not reduced to nitrite. No genetic potential for aerobic anoxygenic photosynthesis was detected. The primary isoprenoid quinone (Ubiquinone-10), predominant cellular fatty acids (C18:1ω7c, 11 methyl C18:1ω7c and C16:0) and DNA G+C content (61 mol%) were all consistent with the assignment of this isolate to the genus Thalassobius. Several phenotypic characteristics clearly distinguished our isolate from other Thalassobius species. The degree of genomic relatedness between strain JC2049T and other Thalassobius species was in a range of 20-43%. The polyphasic data presented in this study indicates that our isolate should be classified as a novel species within the genus Thalassobius. The name Thalassobius aestuarii sp. nov. is therefore proposed for this isolate; the type strain is JC2049T (= IMSNU 14011T = KCTC 12049T = DSM 15283T).

Estrogenic Reduction of Styrene Monomer Degraded by Phanerochaete chrysosporium KFRI 20742: Jae-Won Lee , Soo-Min Lee , Eui-Ju Hong , Eui-Bae Jeung , Ha-Young Kang , Myung-Kil Kim , In-Gyu Choi; J. Microbiol. 2006;44(2):177-184.; DOI: https://doi.org/2367 [pii]

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Abstract: The characteristic biodegradation of monomeric styrene by Phanerochaete chrysosporium KFRI 20742, Trametes versicolor KFRI 20251 and Daldinia concentrica KFRI 40-1 was carried out to examine the resistance, its degradation efficiency and metabolites analysis. The estrogenic reduction effect of styrene by the fungi was also evaluated. The mycelium growth of fungi differentiated depending on the concentration levels of styrene. Additionally P. chrysosporium KFRI 20742 showed superior mycelium growth at less than 200 mg/l, while D. concentrica KFRI 40-1 was more than 200 mg/l. The degradation efficiency reached 99% during one day of incubation for all the fungi. Both manganese-dependent peroxidase and laccase activities in liquid medium were the highest at the initial stage of incubation, whereas the lowest was after the addition of styrene. However, both activities were gradually recovered after. The major metabolites of styrene by P. chrysosporium KFRI 20742 were 2-phenyl ethanol, benzoic acid, cyclohexadiene-1,4-dione, butanol and succinic acid. From one to seven days of incubating the fungi, the expression of pS2 mRNA widely known as an estrogen response gene was decreased down to the level of baseline after one day. Also, the estrogenic effect of styrene completely disappeared after treatment with supernatant of P. chrysosporium KFRI 20742 from one week of culture down to the levels of vehicle.

Journal Article

Purification and Characterization of a Catalase from Photosynthetic Bacterium Rhodospirillum rubrum S1 Grown under Anaerobic Conditions: Yoon-Suk Kang , Dong-Heon Lee , Byoung-Jun Yoon , Duck-Chul Oh; J. Microbiol. 2006;44(2):185-191.; DOI: https://doi.org/2366 [pii]

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Abstract: The photosynthetic bacterium, Rhodospirillum rubrum S1, when grown under anaerobic conditions, generated three different types of catalases. In this study, we purified and characterized the highest molecular weight catalase from the three catalases. The total specific catalase activity of the crude cell extracts was 88 U/mg. After the completion of the final purification step, the specific activity of the purified catalase was 1,256 U/mg. The purified catalase evidenced an estimated molecular mass of 318 kDa, consisting of four identical subunits, each of 79 kDa. The purified enzyme exhibited an apparent Km value of 30.4 mM and a Vmax of 2,564 U against hydrogen peroxide. The enzyme also exhibited a broad optimal pH (5.0~9.0), and remained stable over a broad temperature range (20°C~60°C). It maintained 90% activity against organic solvents (ethanol/chloroform) known hydroperoxidase inhibitors, and exhibited no detectable peroxidase activity. The catalase activity of the purified enzyme was reduced to 19% of full activity as the result of the administration of 10 mM 3-amino-1,2,4-triazole, a heme-containing catalase inhibitor. Sodium cyanide, sodium azide, and hydroxylamine, all of which are known heme protein inhibitors, inhibited catalase activity by 50% at concentrations of 11.5 μM, 0.52 μM, and 0.11 μM, respectively. In accordance with these findings, the enzyme was identified as a type of monofunctional catalase.

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

Identification and Expression of the cym, cmt, and tod Catabolic Genes from Pseudomonas putida KL47: Expression of the Regulatory todST Genes as a Factor for Catabolic Adaptation: Kyoung Lee , Eun Kyeong Ryu , Kyung Soon Choi , Min Chul Cho , Jae Jun Jeong , Eun Na Choi , Soo O Lee , Do-Young Yoon , Ingyu Hwang , Chi-Kyung Kim; J. Microbiol. 2006;44(2):192-199.; DOI: https://doi.org/2365 [pii]

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Abstract: Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene (C1-C4), biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99%) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.

Microarray-Mediated Transcriptome Analysis of the Tributyltin (TBT)-Resistant Bacterium Pseudomonas aeruginosa 25W in the Presence of TBT: Santosh K. Dubey , Tsutomu Tokashiki , Satoru Suzuki; J. Microbiol. 2006;44(2):200-205.; DOI: https://doi.org/2364 [pii]

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Abstract: The tributyltin (TBT)-resistant bacterium, Pseudomonas aeruginosa 25W, which was isolated in seawater from the Arabian Sea, was subjected to transcriptome analysis in the presence of high concentrations of TBT. Only slight effects were observed at TBT concentration of 50 μM, but exposure to 500 μM resulted in the upregulation of 6 genes and the downregulation of 75. Among the 75 downregulated genes, 53% (40 out of 75) were of hypothetical function, followed by 14 transcriptional regulation- and translationassociated genes. The results of this study indicated that although the 25W strain was highly resistant to TBT, high concentrations of TBT result in toxic effect on the transcriptional and translational levels. The target genes likely belong to a specific category of transcription- and translation-associated genes rather than to other gene categories.

YlaC is an Extracytoplasmic Function (ECF) Sigma Factor Contributing to Hydrogen Peroxide Resistance in Bacillus subtilis: Han-Bong Ryu , Inji Shin , Hyung-Soon Yim , Sa-Ouk Kang; J. Microbiol. 2006;44(2):206-216.; DOI: https://doi.org/2363 [pii]

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Abstract: In this study, we have attempted to characterize the functions of YlaC and YlaD encoded by ylaC and ylaD genes in Bacillus subtilis. The GUS reporter gene, driven by the yla operon promoter, was expressed primarily during the late exponential and early stationary phase, and its expression increased as the result of hydrogen peroxide treatment. Northern and Western blot analyses revealed that the level of ylaC transcripts and YlaC increased as the result of challenge with hydrogen peroxide. A YlaC-overexpressing strain evidenced hydrogen peroxide resistance and a three-fold higher peroxidase activity as compared with a deletion mutant. YlaC-overexpressing and YlaD-disrupted strains evidenced higher sporulation rates than were observed in the YlaC-disrupted and YlaD-overexpressing strains. Analyses of the results of native polyacrylamide gel electrophoresis of recombinant YlaC and YlaD indicated that interaction between YlaC and YlaD was regulated by the redox state of YlaD in vitro. Collectively, the results of this study appear to suggest that YlaC regulated by the YlaD redox state, contribute to oxidative stress resistance in B. subtilis.

Journal Article

Notch Signal Transduction Induces a Novel Profile of Kaposi’s Sarcoma-Associated Herpesvirus Gene Expression: Heesoon Chang; J. Microbiol. 2006;44(2):217-225.; DOI: https://doi.org/2362 [pii]

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Abstract: Kaposi’s sarcoma-associated herpesvirus (KSHV) RTA transcription factor is recruited to its responsive elements through interaction with RBP-Jк that is a downstream transcription factor of the Notch signaling pathway that is important in development and cell fate determination. This suggests that KSHV RTA mimics cellular Notch signal transduction to activate viral lytic gene expression. Here, I demonstrated that unlike other B lymphoma cells, KSHV-infected primary effusion lymphoma BCBL1 cells displayed the constitutive activation of ligand-mediated Notch signal transduction, evidenced by the Jagged ligand expression and the complete proteolytic process of Notch receptor I. In order to investigate the effect of Notch signal transduction on KSHV gene expression, human Notch intracellular (hNIC) domain that constitutively activates RBP-Jк transcription factor activity was expressed in BCBL1 cells, TRExBCBL1-hNIC, in a tetracycline inducible manner. Gene expression profiling showed that like RTA, hNIC robustly induced expression of a number of viral genes including K5 immune modulatory gene resulting in downregulation of MHC I and CD54 surface expression. Finally, the genetic analysis of KSHV genome demonstrated that the hNIC-mediated expression of K5 during viral latency consequently conferred the downregulation of MHC I and CD54 surface expression. These results indicate that cellular Notch signal transduction provides a novel expression profiling of KSHV immune deregulatory gene that consequently confers the escape of host immune surveillance during viral latency.

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

Swarming Differentiation of Vibrio vulnificus Downregulates the Expression of the vvhBA Hemolysin Gene via the LuxS Quorum-Sensing System: Moon-Young Kim , Ra-Young Park , Mi-Hwa Choi , Hui-Yu Sun , Choon-Mee Kim , Soo-Young Kim , Joon-Haeng Rhee , Sung-Heui Shin; J. Microbiol. 2006;44(2):226-232.; DOI: https://doi.org/2361 [pii]

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Abstract: Swarming has proven to be a good in vitro model for bacterial surface adherence and colonization, and the swarming differentiation of a bacterium has been shown to be coupled with changes in the expression of virulence factors associated with its invasiveness, particularly in the early stages of infection. In this study, we attempted to determine whether the expression of vvhA, which encodes for hemolysin/cytolysin (VvhA), is either upregulated or downregulated during the swarming differentiation of V. vulnificus. The insertional inactivation of vvhA itself exerted no detectable effect on the expression of V. vulnificus swarming motility. However, in our lacZ-fused vvhA transcriptional reporter assay, vvhA expression decreased in swarming V. vulnificus as compared to non-swarming or planktonic V. vulnificus. The reduced expression of vvhA in swarming V. vulnificus increased as a result of the deletional inactivation of luxS, a gene associated with quorum sensing. These results show that vvhA expression in swarming V. vulnificus is downregulated via the activity of the LuxS quorum-sensing system, suggesting that VvhA performs no essential role in the invasiveness of V. vulnificus via the adherence to and colonization on the body surfaces required in the early stages of the infection. However, VvhA may play a significant role in the pathophysiological deterioration occurring after swarming V. vulnificus is differentiated into planktonic V. vulnificus.

Enhanced Production of Exopolysaccharides by Fed-batch Culture of Ganoderma resinaceum DG-6556: Hyun Mi Kim , Soon-Young Paik , Kyung Soo Ra , Kwang Bon Koo , Jong Won Yun , Jang Won Choi; J. Microbiol. 2006;44(2):233-242.; DOI: https://doi.org/2360 [pii]

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Abstract: The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl2 at an initial pH 6.0 and temperature 31°C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.

Presence of an Inducible Semicarbazide-Sensitive Amine Oxidase in Mycobacterium sp. Strain JC1 DSM 3803 Grown on Benzylamine: Young-Tae Ro , Hyun-Il Lee , Young-Min Kim; J. Microbiol. 2006;44(2):243-247.; DOI: https://doi.org/2359 [pii]

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Abstract: Mycobacterium sp. strain JC1 was capable of growth on benzylamine as a sole source of carbon and energy. The primary deamination of benzylamine was mediated by an inducible amine oxidase, which can also oxidize tyramine, histamine, and dopamine. Inhibitor study identified this enzyme as a copper-containing amine oxidase sensitive to semicarbazide.

Validation Study

Differentiation of Lymphocystis Disease Virus Genotype by Multiplex PCR: Shin-Ichi Kitamura , Sung-Ju Jung , Myung-Joo Oh; J. Microbiol. 2006;44(2):248-253.; DOI: https://doi.org/2358 [pii]

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Abstract: Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease. The viruses have been divided into three genotypes (genotype I for LCDV-1, II for Japanese flounder isolates, and III for rockfish isolates) on the basis of major capsid protein (MCP) gene sequences. In this study, we developed a multiplex PCR primer set in order to distinguish these genotypes. We also analyzed the MCP gene of a new LCDV isolate from the sea bass (SB98Yosu). Comparison of sequence identities between SB98Yosu and eight Japanese flounder isolates, revealed identity of more than 90.1% at nucleotide level and 96.5% at deduced amino acid level, respectively. Phylogenetic analyses based on the MCP gene showed that SB98Yosu belongs to genotype II, along with Japanese flounder isolates. Multiplex PCR based on the MCP gene allowed us to identify these genotypes in a simple and rapid manner, even in a sample that contained two genotypes, in this case genotypes II and III.

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