Journal of Microbiology

Volume 43(5); October 2005

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

Diversity of Denitrifying Bacteria Isolated from Daejeon Sewage Treatment Plant: Young-Woon Lim , Soon-Ae Lee , Seung Bum Kim , Hae-Young Yong , Seon-Hee Yeon , Yong-Keun Park , Dong-Woo Jeong , Jin-Sook Park; J. Microbiol. 2005;43(5):383-390.; DOI: https://doi.org/2286 [pii]

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Abstract: The diversity of the denitrifying bacterial populations in Daejeon Sewage Treatment Plant was examined using a culture-dependent approach. Of the three hundred and seventy six bacterial colonies selected randomly from agar plates, thirty-nine strains that showed denitrifying activity were selected and subjected to further analysis. According to the morphological and biochemical properties, the thirty nine isolates were divided into seven groups. This grouping was supported by an unweighted pair group method, using an arithmetic mean (UPGMA) analysis with fatty acid profiles. Restriction pattern analysis of 16S rDNA with four endonucleases (AluI, BstUI, MspI and RsaI) again revealed seven distinct groups, consistent with those defined from the morphological and biochemical properties and fatty acid profiles. Through the phylogenetic analysis using the 16S rDNA partial sequences, the main denitrifying microbial populations were found to be members of the phylum, Proteobacteria; in particular, classes Gammaproteobacteria (Aeromonas, Klebsiella and Enterobacter) and Betaproteobacteria (Acidovorax, Burkholderia and Comamonas), with Firmicutes, represented by Bacillus, also comprised a major group.

Fluoroquinolone Resistance and gyrA and parC Mutations of Escherichia coli Isolated from Chicken: Young-Ju Lee , Jae-Keun Cho , Ki-Seuk Kim , Ryun-Bin Tak , Ae-Ran Kim , Jong-Wan Kim , Suk-Kyoung Im , Byoung-Han Kim; J. Microbiol. 2005;43(5):391-397.; DOI: https://doi.org/2285 [pii]

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Abstract: Escherichia coli is a common inhabitant of the intestinal tracts of animals and humans. The intestines of animals also represent an ideal environment for the selection and transfer of antimicrobial resistance genes. The aim of this study was to investigate the resistance of E. coli isolated from chicken fecal samples to fluoroquinolones and to analyze the characterization of mutations in its gyrA and parC gene related resistance. One hundred and twenty-eight E. coil isolates showed a high resistance to ciprofloxacin (CIP; 60.2%), enrofloxacin (ENO; 73.4%) and norfloxacin (NOR; 60.2%). Missense mutation in gyrA was only found in the amino acid codons of Ser-83 or Asp-87. A high percentage of isolates (60.2%) showed mutations at both amino acid codons. Missense mutation in parC was found in the amino acid codon of Ser-80 or Glu-84, and seven isolates showed mutations at both amino acid codons. Isolates with a single mutation in gyrA showed minimal inhibitory concentrations (MIC) for CIP (≤0.5 to 0.75 ug/ml), ENO (1 to 4 ug/ml) and NOR (0.75 to 4 ug/ml). These MIC were level compared to isolates with two mutations, one in gyrA and one in parC, and three mutations, one in gyrA and two in parC (CIP, ≤0.5 to 3 ug/ml; ENO, 2 to 32< ug/ml; NOR, 1.5 to 6 ug/ml). However, the isolates with two mutation in gyrA regardless of whether there was a mutation in parC showed high MIC for the three fluoroquinolones (CIP, 0.75 to 32 ≤ug/ml; ENO, 3 to 32 ≤ug/ml; NOR, 3 to 32 ≤ug/ml ). Interestingly, although the E. coil used in this study was isolated from normal flora of chicken, not clinical specimens, a high percentage of isolates showed resistance to fluoroquinolones and possessed mutations at gyrA and parC associated with fluoroquinolone resistance.

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

Utilization of Putrescine by Streptococcus pneumoniae During Growth in Choline-limited Medium: D. Ware , J. Watt , E. Swiatlo; J. Microbiol. 2005;43(5):398-405.; DOI: https://doi.org/2284 [pii]

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Abstract: Polyamines such as putrescine are small, ubiquitous polycationic molecules that are required for optimal growth of eukaryotic and prokaryotic cells. These molecules have diverse effects on cell physiology and their intracellular content is regulated by de novo synthesis and uptake from the environment. The studies presented here examined the structure of a putative polyamine transporter (Pot) operon in Streptococcus pneumoniae (pneumococcus) and growth of pneumococci in medium containing putrescine substituted for choline. RT-PCR experiments demonstrated that the four genes encoding the Pot system are co-transcribed with murB, a gene involved in an intermediary step of peptidoglycan synthesis. Pneumococci grown in chemically-defined media (CDM) containing putrescine without choline enter logarithmic phase growth after 36-48 hs. However, culture density at stationary phase eventually reaches that of choline-containing medium. Cells grown in CDM-putrescine formed abnormally elongated chains in which the daughter cells failed to separate and the choline-binding protein PspA was no longer cell-associated. Experiments with CDM containing radiolabeled putrescine demonstrated that pneumococci concentrate this polyamine in cell walls. These data suggest that pneumococci can replicate without choline if putrescine is available and this polyamine may substitute for aminoalcohols in the cell wall teichoic acids.

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

Complete Sequence of a Gene Encoding KAR3-Related Kinesin-like Protein in Candida albicans: Min-Kyoung Kim , Young Mi Lee , Wankee Kim , Wonja Choi; J. Microbiol. 2005;43(5):406-410.; DOI: https://doi.org/2283 [pii]

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Abstract: In contrast to Saccharomyces cerevisiae, little is known about the kinesin-like protein (KLP) in Candida albicans. The motor domain of kinesin, or KLP, contains a subregion, which is well conserved from yeast to humans. A similarity search, with the murine ubiquitous kinesin heavy chain region as a query, revealed 6 contigs that contain putative KLPs in the genome of C. albicans. Of these, the length of an open reading (ORF) of 375 amino acids, temporarily designated CaKAR3, was noticeably short compared with the closely related S. cerevisiae KAR3 (ScKAR3) of 729 amino acids. This finding prompted us to isolate a [lambda] genomic clone containing the complete CaKAR3 ORF, and here the complete sequence of CaKAR3 is reported. CaKAR3 is a C-terminus motor protein, of 687 amino acids, encoded by a non-disrupting gene. When compared with ScKAR3, the amino terminal region of 112 amino acids was unique, with the middle part of the 306 amino acids exhibiting 25% identity and 44% similarity, while the remaining C-terminal motor domain exhibited 64% identity and 78% similarity, and have been submitted to GeneBank under the accession number AY182242.

Journal Article

Computational Detection of Prokaryotic Core Promoters in Genomic Sequences: Ki-Bong Kim , Jeong Seop Sim; J. Microbiol. 2005;43(5):411-416.; DOI: https://doi.org/2282 [pii]

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Abstract: The high-throughput sequencing of microbial genomes has resulted in the relatively rapid accumulation of an enormous amount of genomic sequence data. In this context, the problem posed by the detection of promoters in genomic DNA sequences via computational methods has attracted considerable research attention in recent years. This paper addresses the development of a predictive model, known as the dependence decomposition weight matrix model (DDWMM), which was designed to detect the core promoter region, including the -10 region and the transcription start sites (TSSs), in prokaryotic genomic DNA sequences. This is an issue of some importance with regard to genome annotation efforts. Our predictive model captures the most significant dependencies between positions (allowing for non-adjacent as well as adjacent dependencies) via the maximal dependence decomposition (MDD) procedure, which iteratively decomposes data sets into subsets, based on the significant dependence between positions in the promoter region to be modeled. Such dependencies may be intimately related to biological and structural concerns, since promoter elements are present in a variety of combinations, which are separated by various distances. In this respect, the DDWMM may prove to be appropriate with regard to the detection of core promoter regions and TSSs in long microbial genomic contigs. In order to demonstrate the effectiveness of our predictive model, we applied 10-fold cross-validation experiments on the 607 experimentally-verified promoter sequences, which evidenced good performance in terms of sensitivity.

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

Transformation and Mutagenesis of the Nematode-trapping Fungus Monacrosporium sphaeroides by Restriction Enzyme-mediated Integration (REMI): Xu Jin , Ming-He Mo , Zhou Wei , Xiao-Wei Huang , Ke-Qin Zhang; J. Microbiol. 2005;43(5):417-423.; DOI: https://doi.org/2281 [pii]

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Abstract: In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants g-1 per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. 90% of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.

Finding the Sources of Korean Salmonella enterica Serovar Enteritidis PT4 Isolates by Pulsed-field Gel Electrophoresis: Yong-Ku Woo; J. Microbiol. 2005;43(5):424-429.; DOI: https://doi.org/2280 [pii]

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Abstract: In previous studies, it has been reported that both S. enteritidis, the most common serotype, and S. enteritidis Phage Type 4 (SEPT 4) isolates were identified as the most prevalent PT in domestic poultry and also in humans in Korea until 2002. The aim of this study was to analyze the genetic diversity and epidemiological properties of both PT isolates, and also to trace the source of SEPT 4 isolates from domestic poultry and humans by Pulsed-field gel electrophoresis (PFGE). In order to understand the molecular epidemiologic properties of SEPT 4 isolates, which have very similar phenotypic properties to our preliminary investigations (serotyping, phage typing, large plasmids and antibiograms), PFGE analysis with XbaI enzyme was performed on the representative SEPT 4 isolates. Thirty-six SEPT 4 isolates were analyzed and differentiated with 10 pulsed-field profiles (PFP) expressing very high discriminative ability (SID: 0.921). In PFP, SEPT 4 isolates from human patients showed a perfect genetic match with those from broiler chickens and meats. Therefore, this study was able to successfully trace the major source of SEPT 4 isolates and also to determine the usefulness of the PFGE method for genetic analysis of epidemic strains.

Molecular Survey of Latent Pseudorabies Virus Infection in Nervous Tissues of Slaughtered Pigs by Nested and Real-time PCR: Hyun A Yoon , Seong Kug Eo , Abi George Aleyas , Seong Ok Park , John Hwa Lee , Joon Seok Chae , Jeong Gon Cho , Hee Jong Song; J. Microbiol. 2005;43(5):430-436.; DOI: https://doi.org/2279 [pii]

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Abstract: In this study, the prevalence and quantity of a latent pseudorabies virus (PrV) infection in the nervous tissues of randomly selected pigs was determined via nested and real-time PCR. The nervous tissues, including the trigeminal ganglion (TG), olfactory bulb (OB), and brain stem (BS), were collected from the heads of 40 randomly selected pigs. The majority of the nervous tissues from the selected pigs evidenced a positively amplified band on nested PCR. In particular, nested PCR targeted to the PrV glycoprotein B (gB) gene yielded positive results in all of the BS samples. Nested PCR for either the gE or gG gene produced positive bands in a less number of nervous tissues (57.5% and 42.5%, respectively). Real-time PCR revealed that the examined tissues harbored large copy numbers of latent PrV DNA, ranging between 100.1 and 107.2 (1-1.58x107) copies per 1 g of genomic DNA. Real-time PCR targeted to the PrV gE gene exhibited an accumulated fluorescence of reporter dye at levels above threshold, thereby indicating a higher prevalence than was observed on the nested PCR (100% for BS, 92% for OB, and 85% for TG). These results indicate that a large number of farm-grown pigs are latently infected with a field PrV strain with a variety of copy numbers. This result is similar to what was found in association with the human herpes virus.

Journal Article

Immunization with Major Outer Membrane Protein of Vibrio vulnificus Elicits Protective Antibodies in a Murine Model: Cho-Rok Jung , Min-Jung Park , Moon-Soo Heo; J. Microbiol. 2005;43(5):437-442.; DOI: https://doi.org/2278 [pii]

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Abstract: Sera from rabbits were infected with Vibrio vulnificus containing an antibody against major outer membrane protein (MOMP). MOMP of V. vulnificus ATCC 27562 were isolated and purified by Sarkosyl and TritonX-100 dual treatment. Molecular size of MOMP was identified as 36-kDa on 13% SDS-PAGE. The sequence of the first 26 amino acid residues from the N-terminal end of the protein is AELYNQDGTSLDMGGRAEARLSMKDG , which is a perfect match with OmpU of V. vulnificus CMCP6 and YJ016. MOMP specific IgM and IgG were investigated in groups of mice. The group of mice immunized with MOMP and Alum showed higher levels of IgG2b than the group immunized with only MOMP. Vaccination with MOMP resulted in protective antibodies in the mouse infection experiment.

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

Conserved Virulence Factors of Pseudomonas aeruginosa are Required for Killing Bacillus subtilis: Shin-Young Park , Yun-Jeong Heo , Young-Seok Choi , Eric Deziel , You-Hee Cho; J. Microbiol. 2005;43(5):443-450.; DOI: https://doi.org/2277 [pii]

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Abstract: The multi-host pathogen, Pseudomonas aeruginosa, possesses an extraordinary versatility which makes it capable of surviving the adverse conditions provided by environmental, host, and, presumably, competing microbial factors in its natural habitats. Here, we investigated the P. aeruginosa-Bacillus subtilis interaction in laboratory conditions and found that some P. aeruginosa strains can outcompete B. subtilis in mixed planktonic cultures. This is accompanied by the loss of B. subtilis viability. The bactericidal activity of P. aeruginosa is measured on B. subtilis plate cultures. The bactericidal activity is attenuated in pqsA, mvfR, lasR, pilB, gacA, dsbA, rpoS, and phnAB mutants. These results suggest that P. aeruginosa utilizes a subset of conserved virulence pathways in order to survive the conditions provided by its bacterial neighbors.

Electrochemical Reduction of Xylose to Xylitol by Whole Cells or Crude Enzyme of Candida peltata: Sun Mi Park , Byung In Sang , Dae Won Park , Doo Hyun Park; J. Microbiol. 2005;43(5):451-455.; DOI: https://doi.org/2276 [pii]

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Abstract: In this study, whole cells and a crude enzyme of Candida peltata were applied to an electrochemical bioreactor, in order to induce an increment of the reduction of xylose to xylitol. Neutral red was utilized as an electron mediator in the whole cell reactor, and a graphite-Mn(IV) electrode was used as a catalyst in the enzyme reactor in order to induce the electrochemical reduction of NAD+ to NADH. The efficiency with which xylose was converted to xylitol in the electrochemical bioreactor was five times higher than that in the conventional bioreactor, when whole cells were employed as a biocatalyst. Meanwhile, the xylose to xylitol reduction efficiency in the enzyme reactor using the graphite-Mn (IV) electrode and NAD+ was twice as high as that observed in the conventional bioreactor which utilized NADH as a reducing power. In order to use the graphite-Mn(IV) electrode as a catalyst for the reduction of NAD+ to NADH, a bioelectrocatalyst was engineered, namely, oxidoreductase (e.g. xylose reductase). NAD+ can function in this biotransformation procedure without any electron mediator or a second oxidoreductase for NAD+/NADH recycling

Microbial Conversion of Major Ginsenoside Rb1 to Pharmaceutically Active Minor Ginsenoside Rd: Myung Kyum Kim , Jun Won Lee , Ki Young Lee , Deok-Chun Yang; J. Microbiol. 2005;43(5):456-462.; DOI: https://doi.org/2275 [pii]

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Abstract: More than seventy strains of aerobic bacteria showing [beta]-glucosidase activity were isolated from a ginseng field, using a newly designed Esculin-R2A agar, and identified by their 16S rRNA gene sequences. Of these microorganisms, twelve strains could convert the major ginsenoside, Rb1, to the pharmaceutically active minor ginsenoside Rd. Three strains, Burkholderia pyrrocinia GP16, Bacillus megaterium GP27 and Sphingomonas echinoides GP50, were phylogenetically studied, and observed to be most potent at converting ginsenoside Rb1 almost completely within 48 h, as shown by TLC and HPLC analyses.

Investigations on Bacteria as a Potential Biological Control Agent of Summer Chafer, Amphimallon solstitiale L. (Coleoptera: Scarabaeidae): Kazlm Sezen , Ismail Demir , Hatice Katl , Zihni Demirbag; J. Microbiol. 2005;43(5):463-468.; DOI: https://doi.org/2274 [pii]

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Abstract: Studying the bacteria of hazardous insects allows the opportunity to find potentially better biological control agents. Therefore, in this study, bacteria from summer chafer (Amphimallon solstitiale L., Coleoptera: Scarabaeidae) we isolated and identified the insecticidal effects of bacteria isolated from A. solstitiale and Melolontha melolontha L. (common cockchafer, Coleoptera: Scarabaeidae) and the mixtures of these bacterial isolates were investigated on A. solstitiale larvae. Crystals from Bacillus sp. isolated from M. melolontha were also purified, and tested against the second and third-stage larvae of A. solstitiale. The bacterial isolates of A. solstitiale were identified as Pseudomonas sp., Pseudomonas sp., Bacillus cereus and Micrococcus luteus, based on their morphology, spore formation, nutritional features, and physiological and biochemical characteristics. The insecticidal effects of the bacterial isolates determined on the larvae of A. solstitiale were 90% with B. cereus isolated from A. solstitiale, and 75% with B. cereus, B. sphaericus and B. thuringiensis isolated from M. melolontha within ten days. The highest insecticidal effects of the mixed infections on the larvae of A. solstitiale were 100% both with B. cereus+B. sphaericus and with B. cereus+B. thuringiensis. In the crystal protein bioassays, the highest insecticidal effect was 65% with crystals of B. thuringiensis and B. sphaericus isolated from M. melolontha within seven days. Finally, our results showed that the mixed infections could be utilized as microbial control agents, as they have a 100% insecticidal effect on the larvae of A. solstitiale.

Journal Article

Isolation of Cryptococcus neoformans var. grubii (serotype A) from Pigeon Droppings in Seoul, Korea: Hee Youn Chee , Kyung Bok Lee; J. Microbiol. 2005;43(5):469-472.; DOI: https://doi.org/2273 [pii]

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Abstract: Seventy-two pigeon dropping samples were collected from 26 different localities in Seoul and investigated for the occurrence of Cryptococcus neoformans. Seventeen samples from 8 different localities were found to be positive for C. neoformans. All isolates were obtained from withered pigeon droppings. Identification and serotyping of the isolates were determined by means of serological testing and DNA fingerprinting. All isolates belonged to C. neoformans var. grubbi (serotype A).

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