Journal of Microbiology

Volume 45(6); December 2007

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

Bacterial Diversity at Different Depths in Lead-Zinc Mine Tailings as Revealed by 16S rRNA Gene Libraries: Han-Bo Zhang , Wen Shi , Ming-Xia Yang , Tao Sha , Zhi-Wei Zhao; J. Microbiol. 2007;45(6):479-484.; DOI: https://doi.org/2648 [pii]

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Abstract: Bacterial communities at 10 cm, 100 cm, and 200 cm depths in a 100-year-old lead-zinc tailing heap were evaluated by constructing 16S rRNA gene libraries. In total, 98 operational taxonomic units (OTUs) were identified from 193 clones at a 3% sequence difference level. The OTU number and species richness decreased with the depth. Species composition was significantly different between the three libraries. Fifty-seven percent of the examined clones were Acidobacteria and 27% belonged to Proteobacteria. Other sequences included Chloroflexi, Firmicutes, Chlamydiae, Actinobacteria, Gemmatimonadetes, Nitrospira, and three unclassified OTUs. Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were mainly distributed in the rhizosphere of naturally colonizing plants; however, Deltaproteobacteria, Acidobacteria, and Chloroflexi tended to inhabit the deeper tailings (below the 100 cm-depth).

Biological Pretreatment of Softwood Pinus densiflora by Three White Rot Fungi: Jae-Won Lee , Ki-Seob Gwak , Jun-Yeong Park , Don-Ha Choi , Mi Kwon , In-Gyu Choi; J. Microbiol. 2007;45(6):485-491.; DOI: https://doi.org/2647 [pii]

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Abstract: The effects of biological pretreatment on the Japanese red pine Pinus densiflora, was evaluated after exposure to three white rot fungi Ceriporia lacerata, Stereum hirsutum, and Polyporus brumalis. Change in chemical composition, structural modification, and their susceptibility to enzymatic saccharification in the degraded wood were analyzed. Of the three white rot fungi tested, S. hirsutum selectively degraded the lignin of this sortwood rather than the holocellulose component. After eight weeks of pretreatment with S. hirsutum, total weight loss was 10.7%, while lignin loss was the highest at 14.52% among the tested samples. However, holocellulose loss was lower at 7.81% compared to those of C. lacerata and P. brumalis. Extracelluar enzymes from S. hirsutum showed higher activity of ligninase and lower activity of cellulase than those from other white rot fungi. Thus, total weight loss and changes in chemical composition of the Japanese red pine was well correlated with the enzyme activities related with lignin- and cellulose degradation in these fungi. Based on the data obtained from analysis of physical characterization of degraded wood by X-ray Diffractometry (XRD) and pore size distribution, S. hirsutum was considered as an effective potential fungus for biological pretreatment. In particular, the increase of available pore size of over 120 nm in pretreated wood powder with S. hirsutum made enzymes accessible for further enzymatic saccharification. When Japanese red pine chips treated with S. hirsutum were enzymatically saccharified using commercial enzymes (Cellulclast 1.5 L and Novozyme 188), sugar yield was greatly increased (21.01%) compared to non-pretreated control samples, indicating that white rot fungus S. hirsutum provides an effective process in increasing sugar yield from woody biomass.

Effect of gacS and gacA Mutations on Colony Architecture, Surface Motility, Biofilm Formation and Chemical Toxicity in Pseudomonas sp. KL28: Kyung Soon Choi , Yaligara Veeraragouda , Kyoung Mi Cho , Soo O Lee , Geuk Rae Jo , Kyungyun Cho , Kyoung Lee; J. Microbiol. 2007;45(6):492-498.; DOI: https://doi.org/2646 [pii]

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Abstract: GacS and GacA proteins form a two component signal transduction system in bacteria. Here, Tn5 transposon gacS and gacA (Gac) mutants of Pseudomonas sp. KL28, an alkylphenol degrader, were isolated by selecting for smooth colonies of strain KL28. The mutants exhibited reduced ability to migrate on a solid surface. This surface motility does not require the action of flagella unlike the well-studied swarming motility of other Pseudomonas sp. The Gac mutants also showed reduced levels of biofilm and pellicle formation in liquid culture. In addition, compared to the wild type KL28 strain, these mutants were more resistant to high concentrations of m-cresol but were more sensitive to H2O2, which are characteristics that they share with an rpoS mutant. These results indicate that the Gac regulatory cascade in strain KL28 positively controls wrinkling morphology, biofilm formation, surface translocation and H2O2 resistance, which are important traits for its capacity to survive in particular niches.

Isolation and Identification of Newly Isolated Antagonistic Streptomyces sp. Strain AP19-2 Producing Chromomycins: Xue-Chang Wu , Wei-Feng Chen , Chao-Dong Qian , Ou Li , Ping Li , Yan-Ping Wen; J. Microbiol. 2007;45(6):499-504.; DOI: https://doi.org/2645 [pii]

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Abstract: A new antagonistic strain of actinomycete, designated AP19-2, was isolated from the feces of giant pandas inhabiting the Foping National Nature Reserve in China. Cultural characteristic studies strongly suggested that this strain is a member of the genus Streptomyces. The nucleotide sequence of the 16S rRNA gene of strain AP19-2 evidenced profound similarity (97-99%) with other Streptomyces strains. Two pure active molecules were isolated from a fermentation broth of Streptomyces sp. strain AP19-2 via extraction, concentration, silica gel G column chromatography, and HPLC. The chemical structures of the two related compounds (referred to as chromomycin A2 and chromomycin A3) were established on the basis of their Infrared spectra (IR), High Resolution Electrospray Ionization Mass Spectrometry (HR-ESI-MS), and Nuclear Magnetic Resonance (NMR) data, and by comparison with published data.

Dasania marina gen. nov., sp. nov., of the Order Pseudomonadales, Isolated from Arctic Marine Sediment: Yoo Kyung Lee , Soon Gyu Hong , Hyun Hee Cho , Kyeung Hee Cho , Hong Kum Lee; J. Microbiol. 2007;45(6):505-509.; DOI: https://doi.org/2644 [pii]

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Abstract: An obligately aerobic bacterium, strain KOPRI 20902T, was isolated from a marine sediment in Ny-Ålesund, Spitsbergen Islands, Norway. Cells were irregular rods and motile with polar monotrichous flagellum. The optimum growth temperature was 17-22°C. Cells grew best in pH 7.0-10.0 and 3-4% sea salts (corresponding to 2.3-3.1% NaCl). The novel strain required Ca2+ or Mg2+ in addition to NaCl for growth. Sequence analysis of 16S rRNA gene revealed that the Arctic isolate is distantly related with established species (<92.4% sequence similarity) and formed a monophyletic group with Cellvibrio, which formed a distinct phylogenetic lineage in the order Pseudomonadales. Predominant cellular fatty acids [C16:1 ω7c/15:0 iso 2OH (45.3%), C16:0 (18.4%), ECL 11.799 (11.2%), C10:0 3OH (10.4%)]; DNA G+C content (37.0 mol%); nitrate reduction to nitrogen; absence of aesculin hydrolysis, N-acetyl-β-glucosaminidase and esterase; no assimilation of arabinose, galactose, glucose, lactose, maltose, and trehalose differentiated the strain from the genus Cellvibrio. Based on the phylogenetic and phenotypic characteristics, Dasania marina gen. nov., sp. nov. is proposed in the order Pseudomonadales. Strain KOPRI 20902T (=KCTC 12566T=JCM 13441T) is the type strain of Dasania marina.

Cryoprotective Properties of Exopolysaccharide (P-21653) Produced by the Antarctic Bacterium, Pseudoalteromonas arctica KOPRI 21653: Sung Jin Kim , Joung Han Yim; J. Microbiol. 2007;45(6):510-514.; DOI: https://doi.org/2643 [pii]

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Abstract: Twenty-five bacterial strains that secrete mucous materials were isolated from sediment obtained from King George Island, Antarctica. Seven of these strains proved capable of producing cryoprotective exopolysaccharides. The strain KOPRI 21653 was selected for the further study of an anti-ice-nucleating polysaccharide (ANP), which originated from a polar region. KOPRI 21653 was identified as Pseudoalteromonas arctica as the result of 16S rRNA analysis. The exopolysaccharide, P-21653, was purified completely from the KOPRI 21653 cell culture via column chromatography and protease treatment. The principal sugar components of P-21653 were determined to be galactose and glucose, at a ratio of 1:1.5, via GC-MS analysis. The cryoprotective activity of P-21653 was characterized via an E. coli viability test. In the presence of 0.1% (w/v) P-21653, the survival ratio of E. coli cells was as high as 82.6% over three repeated freeze-thaw cycles. The survival ratio decreased drastically to 71.5 and 48.1%, respectively, in five and seven repeated cycle conditions; however, the survival ratios were greater over three (96.6-92.1%) to seven (100.5-91.6%) freeze-thaw cycles in the presence of 0.5 and 1.0% (w/v) P-21653. In addition, at much lower concentrations (0.1-1.0%), P-21653 resulted in survival ratios (83.1-98.4%) similar to those of two commercially available cryoprotectants (VEG plus X-1000, 92.9% and VM3, 95.3%), which were utilized at the recommended concentrations (90%). The biochemical characteristics of exopolysaccharide P-21653 reflect that this compound may be developed as a useful cryoprotectant for use in medical applications and in the food industry.

Analysis of Vaginal Lactic Acid Producing Bacteria in Healthy Women: Hyeran Nam , Kyunghee Whang , Yeonhee Lee; J. Microbiol. 2007;45(6):515-520.; DOI: https://doi.org/2642 [pii]

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Abstract: Vaginal lactic acid-producing bacteria of 80 pre-menopausal women were studied by isolation on Blood and DeMan-Rogosa-Sharpe agar, PCR with group-specific primers for Lactobacillus-denaturing gradient gel electrophoresis (DGGE), and PCR with specific primers for V3 region in 16S rRNA-temporal temperature gel electrophoresis (TTGE). Conventional isolation method on media detected only one lactobacillus (Lactobacillus brevis) while TTGE detected only Lactobacillus sp. DGGE detected seven Lactobacillus species; L. coleohominis, L. crispatus, L. iners, L. reuteri, L. rhamnosus, L. vaginalis, and Leuconostoc lactis. L. acidophilus and L. gasseri, which are prevalent in Western women, were not detected in Korean women. Furthermore, L. rhamnosus, Leuc. lactis, L. coleohominis, and Weissella cibaria, which were not previously reported in the vaginal microbiota of Korean women, were detected. The five most prevalent LABs in vaginal microbiota in Korean women were L. iners, Enterococcus faecalis, L. crispatus, Leuc. lactis, and W. cibaria.

Degradation of Malic Acid by Issatchenkia orientalis KMBL 5774, an Acidophilic Yeast Strain Isolated from Korean Grape Wine Pomace: Sung-Hee Seo , Chang-Ho Rhee , Heui-Dong Park; J. Microbiol. 2007;45(6):521-527.; DOI: https://doi.org/2641 [pii]

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Abstract: Several yeast strains degrading malic acid as a sole carbon and energy source were isolated from Korean wine pomace after enrichment culture in the presence of malic acid. Among them, the strain designated as KMBL 5774 showed the highest malic acid degrading ability. It was identified as Issatchenkia orientalis based on its morphological and physiological characteristics as well as the nucleotide sequences of the internal transcribed spacer (ITS) I-5.8S rDNA-ITS II region. Phylogenetic analysis of the ITS I-5.8S rDNAITS II sequences showed that the KMBL 5774 is the closest to I. orientalis zhuan 192. Identity of the sequences of the KMBL 5774 was 99.5% with those of I. orientalis zhuan 192. The optimal pH of the media for the growth and malic acid degradation by the yeast was between 2.0 and 3.0, suggesting that the strain is an acidophile. Under the optimized conditions, the yeast could degrade 95.5% of the malic acid after 24 h of incubation at 30°C in YNB media containing 2% malic acid as a sole carbon and energy source.

Affinity Maturation of an Anti-Hepatitis B Virus PreS1 Humanized Antibody by Phage Display: Gi-Hyeok Yang , Sun Ok Yoon , Myung Hee Jang , Hyo Jeong Hong; J. Microbiol. 2007;45(6):528-533.; DOI: https://doi.org/2640 [pii]

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Abstract: In a previous study we generated an anti-Hepatitis B Virus (HBV) preS1 humanized antibody (HzKR127) that showed in vivo HBV-neutralizing activity in chimpanzees. However, the antigen-binding affinity of the humanized antibody may not be sufficient for clinical use and thus affinity maturation is required for better therapeutic efficacy. In this study, phage display technique was employed to increase the affinity of HzKR127. All six amino acid residues (Glu95-Tyr96-Asp97-Glu98-Ala99-Tyr100) in the heavy (H) chain complementarydetermining region 3 (HCDR3) of HzKR127 were randomized and phage-displayed single chain Fv (scFv) library was constructed. After three rounds of panning, 12 different clones exhibiting higher antigen-binding activity than the wild type ScFv were selected and their antigen-binding specificity for the preS1 confirmed. Subsequently, five ScFv clones were converted to whole IgG and subjected to affinity determination. The results showed that two clones (B3 and A19) exhibited an approximately 6 fold higher affinities than that of HzKR127. The affinity-matured humanized antibodies may be useful in anti-HBV immunotherapy.

Partial Purification of Factors for Differential Transcription of the rrnD Promoters for Ribosomal RNA Synthesis in Streptomyces coelicolor: Mi-Young Hahn , Jung-Hye Roe; J. Microbiol. 2007;45(6):534-540.; DOI: https://doi.org/2612 [pii]

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Abstract: The Streptomyces coelicolor A3(2) genome contains six operons (rrnA to F) for ribosomal RNA synthesis. Transcription from rrnD occurs from four promoters (p1 to p4). We found that transcripts from the p1 and p3 promoters were most abundant in vivo in the early exponential phase. However, at later phases of exponential and stationary growth, transcripts from the p1 promoter decreased drastically, with the p3 and p4 transcripts constituting the major forms. Partially purified RNA polymerase supported transcription from the p3 and p4 promoters, whereas pure reconstituted RNA polymerase with core enzyme (E) and the major vegetative sigma factor sigmaHrdB (E.sigmaHrdB) did not. In order to assess any potential requirement for additional factor(s) that allow transcription from the p3 and p4 promoters, we fractionated a partially purified RNA polymerase preparation by denaturing gel filtration chromatography. We found that transcription from the p3 and p4 promoters required factor(s) of about 30-35 kDa in addition to RNAP holoenzyme (E.sigmaHrdB). Therefore, transcription from the p3 and p4 promoters, which contain a consensus -10 region but no -35 for sigmaHrdB recognition, are likely to be regulated by transcription factor(s) that modulate RNA polymerase holoenzyme activity in S. coelicolor.

Analysis of Expressed Sequence Tags from the Red Alga Griffithsia okiensis: Hyoungseok Lee , Hong Kum Lee , Gynheung An , Yoo Kyung Lee; J. Microbiol. 2007;45(6):541-546.; DOI: https://doi.org/2611 [pii]

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Abstract: Red algae are distributed globally, and the group contains several commercially important species. Griffithsia okiensis is one of the most extensively studied red algal species. In this study, we conducted expressed sequence tag (ESTs) analysis and synonymous codon usage analysis using cultured G. okiensis samples. A total of 1,104 cDNA clones were sequenced using a cDNA library made from samples collected from Dolsan Island, on the southern coast of Korea. The clustering analysis of these sequences allowed for the identification of 1,048 unigene clusters consisting of 36 consensus and 1,012 singleton sequences. BLASTX searches generated 532 significant hits (E-value <10-4) and via further Gene Ontology analysis, we constructed a functional classification of 434 unigenes. Our codon usage analysis showed that unigene clusters with more than three ESTs had higher GC contents (76.5%) at the third position of the codons than the singletons. Also, the majority of the optimal codons of G. okiensis and Chondrus crispus belonging to Bangiophycidae were C-ending, whereas those of Porphyra yezoensis belonging to Florideophycidae were G-ending. An orthologous gene search for the P. yezoensis EST database resulted in the identification of 39 unigenes commonly expressed in two rhodophytes, which have putative functions for structural proteins, protein degradation, signal transduction, stress response, and physiological processes. Although experiments have been conducted on a limited scale, this study provides a material basis for the development of microarrays useful for gene expression studies, as well as useful information for the comparative genomic analysis of red algae.

Generation and Characterization of a Monoclonal Antibody with Specificity for Mycoplasma arginini: Yeon Sung Son , Hyo Jeong Hong; J. Microbiol. 2007;45(6):547-552.; DOI: https://doi.org/2610 [pii]

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Abstract: Previously, we generated monoclonal antibodies (MAbs) that bound to the surface of human embryonic stem cells (hESCs) in an attempt to discover new hESC-specific surface markers. In this study, MAb 47-235 (IgG1, κ) was selected for further characterization. The MAb bound to the surface of undifferentiated hESCs but did not bind to mouse ESCs or mouse embryonic fibroblast cells in flow cytometric analysis. The antibody immunoprecipitated a 47 kDa protein from the lysates of cell surface-biotinylated hESCs. Identification of the protein by quadrupole time of flight tandem mass spectrometry revealed that 47-235 binds to Ag 243-5 protein of Mycoplasma arginini. BM-Cyclin treatment of the hESCs that reacted with 47-235 resulted in loss of mycoplasma DNA and the reactivity to 47-235. Nevertheless, the hESCs that were reactive to 47-235 maintained self-renewal and pluripotency and thus could be differentiated into three embryonic germ layers.

A Highly Pathogenic Strain of Bacillus thuringiensis serovar kurstaki in Lepidopteran Pests: Hatice Kati , Kazim Sezen , Remziye Nalcacioglu , Zihni Demirbag; J. Microbiol. 2007;45(6):553-557.; DOI: https://doi.org/2609 [pii]

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Abstract: In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.

Initial Characterization of yliH in Salmonella typhimurium: Kyung-Hwa Park , Miryung Song , Hyon E. Choy; J. Microbiol. 2007;45(6):558-565.; DOI: https://doi.org/2608 [pii]

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Abstract: Using microarray analysis, we determined those Salmonella genes induced at the entry of stationary phase, and subsequently discovered that uncharacterized yliH was induced most dramatically. We set out to establish the molecular mechanism underlying the stationary phase induction of yliH under the standard culture condition, LB with vigorous aeration, by analyzing its promoter activity in various mutant backgrounds, lacking stationary phase σ, RpoS-, or stringent signal molecules ppGpp, ΔrelA ΔspoT. It was found that the stationary phase induction of yliHp was partially dependent on rpoS but entirely dependent on ppGpp. DNA sequence analysis revealed that the Salmonella yliH gene is composed of 381 base-pair nucleotides, with overall amino acid sequence revealing 76.38% amino acid identity and 88.98% similarity with Escherichia coli yliH, although no motif from data base was noted for its possible role. Recently however, it has been reported that yliH in E. coli was implicated in biofilm formation and motility by repressing these activities (Domka et al., 2006). We have constructed a mutant Salmonella deleting yliH gene by allele replacement and examined its phenotype, and found that the yliH in Salmonella more or less affects motility and adherence by enhancing these activities. The effect on biofilm formation in Salmonella was uncertain. Moreover, addition of cloned yliH of E. coli into Salmonella did not reduce motility or adherence. Taken together, it appears that the pathways implicating yliH for biofilm formation and motility in E. coli and in Salmonella are somewhat different.

Mouse Strain-Dependent Osteoclastogenesis in Response to Lipopolysaccharide: Ho Gil Choi , Jin Moon Kim , Bong-Ju Kim , Yun-Jung Yoo , Jeong-Heon Cha; J. Microbiol. 2007;45(6):566-571.; DOI: https://doi.org/2607 [pii]

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Abstract: Bacterial lipopolysaccharide (LPS) is a potent stimulator of bone resorption in periodontitis. Co-culture systems of mouse calvaria-derived osteoblasts and bone marrow-derived preosteoclasts were used as an in vitro osteoclast differentiation. This study revealed that co-cultures using ddY or ICR mouse strain responded differently to LPS while responded equally to 1α,25(OH)2D3. Thus, the different response to LPS indicates dissimilarity of two mouse stains in their capacity for generating osteoclasts while the two mouse strains share the similarity in response to 1α,25(OH)2D3. To identify which cells between osteoblasts and preosteoclasts in the co-culture are responsible for the dissimilarity, the reciprocal co-cultures were performed between ddY and ICR mouse strains. The treatment of 1,25(OH)2D3 to ddY/ICR (osteoblasts from ddY/preosteoclasts from ICR) and ICR/ddY reciprocal co-cultures also showed the similarity. In case of LPS treatment, the results of ddY/ICR were similar to ddY/ddY and the results of the other reciprocal co-culture, ICR/ddY combination, were consistent with those of ICR/ICR. It suggests that the dissimilarity between the two mouse strains may resident in osteoblasts but not in preosteoclasts. Therefore, the osteoblast is responsible for mouse strain-dependent osteoclastogenesis in response to LPS. Although mouse models will continue to provide insights into molecular mechanisms of osteoclastogenesis, caution should be exercised when using different mouse strains, especially ddY and ICR strains as models for osteoclast differentiation.

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