Journal of Microbiology

Volume 44(6); December 2006

Journal Article

S5 Lipase : An Organic Solvent Tolerant Enzyme: Raja Noor Zaliha Raja Abdul Rahman , Syarul Nataqain Baharum , Abu Bakar Salleh , Mahiran Basri; J. Microbiol. 2006;44(6):583-590.; DOI: https://doi.org/2470 [pii]

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Abstract: In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.

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

Evaluation of the Diversity of Cyclodextrin-Producing Paenibacillus graminis Strains Isolated from Roots and Rhizospheres of Different Plants by Molecular Methods: Renata Estebanez Vollu , Rafael Fogel , Silvia Cristina Cunha dos Santos , Fabio Faria da Mota , Lucy Seldin; J. Microbiol. 2006;44(6):591-599.; DOI: https://doi.org/2469 [pii]

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Abstract: To address the diversity of cyclodextrin-producing P. graminis strains isolated from wheat roots and rhizospheres of maize and sorghum sown in Australia, Brazil, and France, restriction fragment length polymorphism analysis of part of genes encoding RNA polymerase (rpoB-RFLP) and DNA gyrase subunit B (gyrB-RFLP) was used to produce genetic fingerprints. A phylogenetic tree based on rpoB gene sequences was also constructed. The isolates originated from Brazil could be separated from those from Australia and France, when data from the rpoB-based phylogenetic tree or gyrB-RFLP were considered. These analyses also allowed the separation of all P. graminis strains studied here into four clusters; one group formed by the strains GJK201 and RSA19T, second group formed by the strains MC22.02 and MC04.21, third group formed by the strains TOD61, TOD 221, TOD302, and TOD111, and forth group formed by all strains isolated from plants sown in Cerrado soil, Brazil. As this last group was formed by strains isolated from sorghum and maize sown in the same soil (Cerrado) in Brazil, our results suggest that the diversity of these P. graminis strains is more affected by the soil type than the plant from where they have been isolated.

Phylogenetic Diversity of Acidophilic Sporoactinobacteria Isolated from Various Soils: Sung-Heun Cho , Ji-Hye Han , Chi Nam Seong , Seung Bum Kim; J. Microbiol. 2006;44(6):600-606.; DOI: https://doi.org/2468 [pii]

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Abstract: Spore forming actinobacteria (sporoactinobacteria) isolated from soils with an acidic pH in Pinus thunbergii forests and coal mine waste were subjected to taxonomic characterization. For the isolation of acidophilic actinobacteria, acidified starch casein agar (pH adjusted to 4-5) was used. The numbers of actinobacteria growing in acidic media were between 3.2 × 104 and 8.0 × 106 CFU/g soil. Forty three acidophilic actinobacterial strains were isolated and their 16S rDNA sequences were determined. The isolates were divided into eight distinctive phylogenetic clusters within the variation encompassed by the family Streptomycetaceae. Four clusters among them were assigned to the genus Streptacidiphilus, whereas the remaining four were assigned to Streptomyces. The clusters belonging to either Streptomyces or Streptacidiphilus did not form monophyletic clade. The growth pH profiles indicated that the representative isolates grew best between pH 5 and 6. It is evident from this study that acidity has played a critical role in the differentiation of the family Streptomycetaceae, and also that different mechanisms might have resulted in the evolution of two groups, Streptacidiphilus (strict acidophiles) and neutrotolerant acidophilic Streptomyces. The effect of geographic separation was clearly seen among the Streptacidiphilus isolates, which may be a key factor in speciation of the genus.

Molecular Characterization of Marine Cyanobacteria from the Indian Subcontinent Deduced from Sequence Analysis of the Phycocyanin Operon (cpcB-IGS-cpcA) and 16S-23S ITS Region: Jagadeesan Premanandh , Balakrishnan Priya , Ivanka Teneva , Balik Dzhambazov , Dharmar Prabaharan , Lakshmanan Uma; J. Microbiol. 2006;44(6):607-616.; DOI: https://doi.org/2467 [pii]

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Abstract: Molecular characterization of ten marine cyanobacterial isolates belonging to the order Oscillatoriales was carried out using the phycocyanin locus (cpcBA-IGS) and the 16S-23S internally transcribed spacer region. DNA sequences from the phycocyanin operon discriminated ten genotypes, which corresponded to seven morphotypes identified by traditional microscopic analysis. The cpcB coding region revealed 17% nucleotide variation, while cpcA exhibited 29% variation across the studied species. Phylogenetic analyses support the conclusion that the Phormidium and Leptolyngbya genera are not monophyletic. The nucleotide variations were heterogeneously distributed with no or minimal informative nucleotides. Our results suggest that the discriminatory power of the phycocyanin region varies across the cyanobacterial species and strains. The DNA sequence analysis of the 16S-23S internally transcribed spacer region also supports the polyphyletic nature of the studied oscillatorian cyanobacteria. This study demonstrated that morphologically very similar strains might differ genotypically. Thus, molecular approaches comprising different gene regions in combination with morphological criteria may provide better taxonomical resolution of the order Oscillatoriales.

Increase of Yeast Survival under Oxidative Stress by the Expression of the Laccase Gene from Coprinellus congregatus: Dongsik Kim , Eunju Kwak , Hyoung T. Choi; J. Microbiol. 2006;44(6):617-621.; DOI: https://doi.org/2466 [pii]

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Abstract: Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress (H2O2) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.

A Fibrinolytic Enzyme from the Medicinal Mushroom Cordyceps militaris: Jae-Sung Kim , Kumar Sapkota , Se-Eun Park , Bong-Suk Choi , Seung Kim , Nguyen Thi Hiep , Chun-Sung Kim , Han-Seok Choi , Myung-Kon Kim , Hong-Sung Chun , Yeal Park , Sung-Jun Kim; J. Microbiol. 2006;44(6):622-631.; DOI: https://doi.org/2465 [pii]

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Abstract: In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9%. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and 37°C, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin α-chain followed by the γ-γ chains. It also hydrolyzed the β-chain, but more slowly. The Aα, Bβ, and γ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by Cu2+ and Co2+, but enhanced by the additions of Ca2+ and Mg2+ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it’s a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

Characterization of β-Ketoadipate Pathway from Multi-Drug Resistance Bacterium, Acinetobacter baumannii DU202 by Proteomic Approach: Sonn-Ho Park , Jae-Woo Kim , Sung-Ho Yun , Sun Hee Leem , Hyung-Yeel Kahng , Seung Il Kim; J. Microbiol. 2006;44(6):632-640.; DOI: https://doi.org/2464 [pii]

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Abstract: In this study, the biodegradative activities of monocyclic aromatic compounds were determined from the multi-drug resistant (MDR) Acinetobacter baumannii, which were studied in the form of clinical isolates from a hospital in Korea. These bacteria were capable of biodegrading monocyclic aromatic compounds, such as benzoate and p-hydroxybenzoate. In order to determine which pathways are available for biodegradation in these stains, we conducted proteome analyses of benzoate and p-hydroxybenzoate-cultured A. baumannii DU202, using 2-DE/MS analysis. As genome DB of A. baumannii was not yet available, MS/MS analysis or de novo sequencing methods were employed in the identification of induced proteins. Benzoate branch enzymes [catechol 1,2-dioxygenase (CatA) and benzoate dioxygenase α subunit (BenA)] of the β-ketoadipate pathway were identified under benzoate culture condition and p-hydroxybenzoate branch enzymes [protocatechuate 3,4-dioxygenase α subunit (PcaG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB)] of the β-ketoadipate pathway were identified under p-hydroxybenzoate culture condition, respectively, thereby suggesting that strain DU202 utilized the β-ketoadipate pathway for the biodegradation of monocyclic aromatic compounds. The sequence analysis of two purified dioxygenases (CatA and PcaGH) indicated that CatA is closely associated with the CatA of Acinetobacter radiresistance, but PcaGH is only moderately associated with the PcaGH of Acinetobacter sp. ADP1. Interestingly, the fused form of PcaD and PcaC, carboxymuconolactone decarboxylase (PcaCD), was detected on benzoate-cultured A. baumannii DU202. These results indicate that A. baumannii DU202 exploits a different β-ketoadipate pathway from other Acinetobacter species.

A New Function of Skp1 in the Mitotic Exit of Budding Yeast Saccharomyces cerevisiae: Namil Kim , Hayoung Yoon , Eunhwa Lee , Kiwon Song; J. Microbiol. 2006;44(6):641-648.; DOI: https://doi.org/2463 [pii]

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Abstract: We previously reported that Skp1, a component of the Skp1-Cullin-F-box protein (SCF) complex essential for the timely degradation of cell cycle proteins by ubiquitination, physically interacts with Bfa1, which is a key negative regulator of the mitotic exit network (MEN) in response to diverse checkpoint-activating stresses in budding yeast. In this study, we initially investigated whether the interaction of Skp1 and Bfa1 is involved in the regulation of the Bfa1 protein level during the cell cycle, especially by mediating its degradation. However, the profile of the Bfa1 protein did not change during the cell cycle in skp1-11, which is a SKP1 mutant allele in which the function of Skp1 as a part of SCF is completely impaired, thus indicating that Skp1 does not affect the degradation of Bfa1. On the other hand, we found that the skp1-12 mutant allele, previously reported to block G2-M transition, showed defects in mitotic exit and cytokinesis. The skp1-12 mutant allele also revealed a specific genetic interaction with Δbfa1. Bfa1 interacted with Skp1 via its 184 C-terminal residues (Bfa1-D8) that are responsible for its function in mitotic exit. In addition, the interaction between Bfa1 and the Skp1-12 mutant protein was stronger than that of Bfa1 and the wild type Skp1. We suggest a novel function of Skp1 in mitotic exit and cytokinesis, independent of its function as a part of the SCF complex. The interaction of Skp1 and Bfa1 may contribute to the function of Skp1 in the mitotic exit.

Cloning and Analysis of a Type II Polyketide Synthase Gene Cluster from Streptomyces toxytricini NRRL 15,443: Anna Yoo , Atanas V. Demirev , Ji Seon Lee , Sang Dal Kim , Doo Hyun Nam; J. Microbiol. 2006;44(6):649-654.; DOI: https://doi.org/2462 [pii]

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Abstract: A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), β-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.

Molecular Phylogenetic Analysis of HIV-1 vif Gene from Korean Isolates: Chan Seung Park , Mi Sook Kim , Sung Duk Lee , Sung Soo Kim , Keon Myung Lee , Chan-Hee Lee; J. Microbiol. 2006;44(6):655-659.; DOI: https://doi.org/2461 [pii]

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Abstract: Phylogenetic studies of nef, pol, and env gene sequences of HIV-1 isolated from Koreans suggested the presence of a Korean clade in which Korean sequences are clustered to the exclusion of foreign sequences. We attempted to identify and characterize the Korean clade using all vif gene sequences isolated from Koreans registered in the NCBI GenBank database (n = 233). Most (77%) of the Korean isolates belonged to the Korean clade as a large subcluster in subtype B, designated the Korean clade subtype B (KCB). KCB sequences were relatively homogenous compared to Korean subtype B sequences that did not belong to the KCB (non-Korean clade subtype B; NKCB). Comparison of amino acid frequencies of KCB and NKCB sequences revealed several positions where the amino acid frequencies were significantly different. These amino acid residues were critical in separating KCB from NKCB or from foreign sequences, since substitution of these amino acids in KCB with the NKCB amino acids relocated the KCB sequences to NKCB, and vice versa. Further analyses of KCB will help us to understand the origin and evolutionary history of KCB.

Journal Article

Claritromycin Resistance and Helicobacter pylori Genotypes in Italy: Vincenzo De Francesco , Marcella Margiotta , Cesare Hassan , Nicola Della Valle , Osvaldo Burattini , Roberto D’Angelo , Giuseppe Stoppino , Ugo Cea , Floriana Giorgio , Rosa Monno , Sergio Morini , Carmine Panella , Enzo Ierardi; J. Microbiol. 2006;44(6):660-664.; DOI: https://doi.org/2460 [pii]

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Abstract: The relationship between H. pylori clarithromycin resistance and genetic pattern distribution has been differently explained from different geographic areas. Therefore, we aimed to assess the clarithromycin resistance rate, to evaluate the bacterial genetic pattern, and to search for a possible association between clarithromycin resistance and cagA or vacA genes. This prospective study enrolled 62 consecutive H. pylori infected patients. The infection was established by histology and rapid urease test. Clarithromycin resistance, cagA and vacA status, including s/m subtypes, were assessed on paraffin-embedded antral biopsy specimens by TaqMan real time polymerase chain reaction (PCR). Primary clarithromycin resistance was detected in 24.1% of cases. The prevalence of cagA was 69.3%, and a single vacA mosaicism was observed in 95.1% cases. In detail, the s1m1 was observed in 23 (38.9%) patients, the s1m2 in 22 (37.2%), and the s2m2 in 14 (23.7%), whereas the s2m1 combination was never found. The prevalence of cagA and the vacA alleles distribution did not significantly differ between susceptible and resistant strains. Primary clarithromycin resistance is high in our area. The s1m1 and s1m2 are the most frequent vacA mosaicisms. There is no a relationship between clarithromycin resistance and bacterial genotypic pattern and/or cagA positivity.

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

Effect of Mycelial Extract of Clavicorona pyxidata on the Production of Amyloid β-Peptide and the Inhibition of Endogenous β-Secretase Activity in vitro: Tae-Hee Lee , Young-Il Park , Yeong-Hwan Han; J. Microbiol. 2006;44(6):665-670.; DOI: https://doi.org/2459 [pii]

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Abstract: Amyloid β-peptide (Aβ), which is a product of the proteolytic effect of β-secretase (BACE) on an amyloid precursor protein, is closely associated with Alzheimer’s disease (AD) pathogenesis. There is sufficient evidence to suggest that a BACE inhibitor may reduce Aβ levels, thus decreasing the risk of AD. In a previous study, an extract of Clavicorona pyxidata DGUM 29005 mycelia was found to inhibit the production of a soluble β-amyloid precursor protein (sβAPP), Aβ, and BACE in neuronal cell lines. We sought to determine whether this mycelial extract exerts the same effect in human rhabdomyosarcoma A-204 and rat pheochromocytoma PC-12 cells. We found that the production of Aβ decreased in a dose-dependent manner in the presence of the mycelial extract and that the concentration of Aβ never exceeded 50 μg/ml. The presence of sAPP was detected in every culture medium to which the mycelial extract had been added and its concentration remained the same, regardless of the concentration of the extract used. Endogenous β-secretase activity in A-204 and PC-12 cellular homogenates also decreased in the presence of this extract. These cells, in culture, were not susceptible to the cytotoxic activity of the mycelial extract.

Construction of an Escherichia-Pseudomonas Shuttle Vector Containing an Aminoglycoside Phosphotransferase Gene and a lacZ'''' Gene for α-Complementation: Bheong-Uk Lee , Ja-Heon Hong , Hyung-Yeel Kahng , Kye-Heon Oh; J. Microbiol. 2006;44(6):671-673.; DOI: https://doi.org/2458 [pii]

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Abstract: A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminoglycoside phosphotransferase gene (aph) from Tn903, a lacZ'''' gene for α-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DH5α and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

Diversity of Yeasts Associated with Panax ginseng: Soon Gyu Hong , Kang Hyun Lee , Jangyul Kwak , Kyung Sook Bae; J. Microbiol. 2006;44(6):674-679.; DOI: https://doi.org/2457 [pii]

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Abstract: Biodiversity of yeasts was investigated in the ginseng cultivation field. Among 34 isolates tested in this study, 26 isolates belonged to the hymenomycetous yeast group. These 26 strains were classified into 12 species including four new-species candidates that did not have clear affiliation to any established species. Seven isolates among the remaining strains were classified into three ascomycetous yeast species, and one isolate was identified as a urediniomycetous yeast species.

Isolation of Quinolone-Resistant Escherichia coli Found in Major Rivers in Korea: Dahye Jung , Min Young Lee , Jung Min Kim , Je Chul Lee , Dong Taek Cho , Yeonhee Lee; J. Microbiol. 2006;44(6):680-684.; DOI: https://doi.org/2456 [pii]

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Abstract: Twenty isolates resistant to seven quinolones were isolated from major rivers in Korea. All isolates had three mutations, Ser83→Leu and Asp87→Asn in GyrA and Ser80→Ile or Ser80→Arg in ParC and three isolates had an additional mutation Glu84→Gly or Glu84→Val in ParC. In addition, a clonal spread was not found in these isolates.

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