Journal of Microbiology

Volume 39(4); December 2001

Thermostability Mechanisms in Enzymes from Thermotoga neapolitana and Pyrococcus furiosus: Claire Vieille , Dinlaka Sriprapundh , Alexei Savchenko , Suil Kang , Harini Krishnamurthy , J. Gregory Zeikus; J. Microbiol. 2001;39(4):245-249.

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Abstract: review

Molecular Microbiology of the Oil Field Sulfur Cycle: Gerrit Voordouw; J. Microbiol. 2001;39(4):250-253.

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Abstract: review

Microbial Symbiosis in Marine Sponges: Yoo Kyung Lee , Jung-Hyun Lee , Hong Kum Lee; J. Microbiol. 2001;39(4):254-264.

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Abstract: Sponges are host organisms for various symbiotic microorganisms such as archaea, bacteria, cyanobacteria and microalgae. Sponges are also sources of a wide variety of useful natural products like cytotoxins, antifouling agents, antibiotics, and anti-inflammatory and antiviral compounds. Symbiotic microorganisms in sponges can be sources of various natural products, because metabolites previously ascribed to sponges have recently been demonstrated to be biosynthesized by symbionts. If a symbiotic microorganism from which some natural products are derived can be cultured, the microorganism could be used in a mass production of the bioactive compounds. We summarize recent research on isolation and cultivation of sponge-symbiotic microorganisms and the symbiotic relationship.

Variation of the Intergenic Spacer (IGS) Region of Ribosomal DNA among Fusarium oxysporum formae speciales: Hyun-Jung Kim , Yong-Keel Choi , Byung-Re Min; J. Microbiol. 2001;39(4):265-272.

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Abstract: Variation within the intergenic spacer (IGS) of the ribosomal DNA gene for twenty-two strains of F. oxysporum and its formae speciales was examined by PCR, coupled with RFLP analysis. The length of the amplified IGS region was about 2.6 kb in all strains except F. oxysporum f. sp. cucumerinum from Korea and F. oxysporum f. sp. niveum. Those two strains were 2.5 kb long. Restriction digestion of IGS-RFLP regions by EcoRI, NruI, HincII, SalI, SmaI, BglII, HindIII, XhoI, and KpnI gave rise to nine IGS haplotypes among all strains. Cluster analysis based on the presence or absence of comigrating restriction fragments show the two groups based on 44% genetic similarity. These results demonstrated that analysis of IGS showed some difference within and between F. oxysporum formae speciales.

Growth Properties of the Iron-reducing Bacteria, Shewanella putrefaciens IR-1 and MR-1 Coupling to Reduction of Fe(III) to Fe(II): Doo Hyun Park , Byung Hong Kim; J. Microbiol. 2001;39(4):273-278.

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Abstract: Shewanella putrefaciens IR-1 and MR-1 were cultivated by using various combinations of electron donor-acceptor, lactate-Fe(III), lactate-nitrate, pyruvate-Fe(III), pyruvate-nitrate, H_2 -acetate-Fe(III) and H_2 -acetate-nitrate. Both strains grew fermentatively on pyruvate but not on lactate without an electron acceptor. In culture with Fe(III), both strains grew on pyruvate and lactate but not on H_2 -acetate-CO_2 . In cultivation with nitrate, both strains grew on pyruvate, lactate and on H_2 -acetate-CO_2 . The growth yields of IR-1 on pyruvate, pyruvate-Fe(III) and lactate-Fe(III) were about 3.4, 3.5, and 3.6 (g cell/M substrate), respectively, but the yields on lactate-nitrate, pyruvate-nitrate and H_2 -acetate-CO_2 ?trate were about 6.8, 5.9, and 9.4 (g cell/M substrate), respectively. From the growth properties of both strains on media with Fe(III) as an electron acceptor, the bacterial growth was confirmed not to be increased by addition of Fe(III) as an electron acceptor to the growth medium, which indicates a possibility that the dissimilatory reduction of Fe(III) to Fe(II) may not be coupled to free energy production.

Characteristics of Urease from Vibrio parahaemolyticus Possessing tdh and trh Genes Isolated in Korea: Young Hee Kim , Jong Sook Kim; J. Microbiol. 2001;39(4):279-285.

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Abstract: Vibrio parahaemolyticus is a halophilic bacterium associated with seafood gastroenteritis. An unusual strain of Kanagawa-positive urease producing Vibrio parahaemolyticus O1 : K1 was isolated from the environment and identified. A polymerase chain reaction assay revealed that this strain harbored both the tdh and trh genes. The urease from this strain was studied. Maximum urease production was induced in LB medium containing 0.2% urea, 0.5% glucose, 2% NaCl and pH 5.5 with 6 h of cultivation at 37 C under aeration. Purification of urease was achieved by the process of whole cell lysate, 65% ammonium sulphate precipitation, DEAE-cellulose ion exchange column chromatography, Sepharose CL-6B gel filtration and oxirane activated Sepharose 6B-urea affinity chromatography with 203 fold purification and 2.2% yield. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of the subunits with a molecular weight of 85 kDa, 59 kDa, 41 kDa and the molecular weight for the native enzyme by nondenaturing PAGE and gel filtration chromatography was 255 kDa. The purified urease was stable at pH 7.5 and the optimal pH in HEPES buffer was 8.0. The enzyme was stable at 60 C for 2 h with a residual activity of 32%. The addition of 10 uM of NiCl_2 maintained stability for 30 min. The Km value of the purified enzyme was 35.6 mM in urea substrate. The TD_50 (median toxic dose) of the purified urease was 2.5 ug/ml on human leukemia cells.

Characterization of a Putative F-box Motif in Ibd1p/Bfa1p, a Spindle Checkpoint Regulator of Budding Yeast Saccharomyces cerevisiae: Kyum-Jung Lee , Hyung-Seo Hwang , Kiwon Song; J. Microbiol. 2001;39(4):286-292.

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Abstract: During mitosis, the proper segregation of duplicated chromosomes is coordinated by a spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the misorientation of the mitotic spindle. Ibd1p/Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for mitotic exit and its disruption abolishes mitotic arrest when proper organization of the mitotic spindle is inhibited. Ibd1p/Bfa1p localizes to the spindle pole body, a microtubule-organizing center in yeast, and its overexpression arrests the cell cycle in 80% of cells with an enlarged bud at mitosis and in 20% of cells with multiple buds. In this study, we found that the C-terminus of Ibd1p/Bfa1p physically interacts with Skp1p, a key component of SCF (Skp1/cullin/F-box) complex for ubiquitin-mediated proteolysis of cell cycle regulators as well as an evolutionally conserved kinetochore protein for cell cycle progression. A putative F-box motif was found in the C-terminus of Ibd1p/Bfa1p and its function was investigated by making mutants of conserved residues in the motif. These Ibd1p/Bfa1p mutants of a putative F-box interacted with Skp1p in vitro by two-hybrid assays as wild type Ibd1p/Bfa1p. Also, these Ibd1p/Bfa1p mutants displayed the overexpression phenotypes of wild type Ibd1p, when over-expressed under inducible promoters. These results suggest that a putative F-box motif of Ibd1p/Bfa1p is not essential for the interaction with Skp1p and its function in mitotic exit and cytokinesis.

Production of the Nucleocapsid Protein of Newcastle Disease Virus in Escherichia coli and its Assembly into Ring- and Nucleocapsid-like Particles: Chiew Ling Kho , Wen Siang Tan , Khatijah Yusoff; J. Microbiol. 2001;39(4):293-299.

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Abstract: The nucleocapsid (NP) protein of Newcastle disease virus (NDV) and its derivative (NP_cfus ) containing the myc region and six histidine residues fused to its C-terminus were expressed abundantly in Escherichia coli. The proteins were purified by sucrose gradient centrifugation. Both the NP and NP_cfus proteins self-assembled into ring-like particles with a diameter of 24 +- 2 nm around a central hole of 7 +- 1 nm. Some of these ring-like particles stacked together to form nucleocapsid-like structures which are heterogeneous in length with a diameter of 20 +- 2 nm and a central hollow of 5 +- 1 nm. Only a very small amount of the monomers in the particles was linked by inter-molecular disulfide bonds. Fusion of the C-terminal end to 29 amino acids inclusive of the myc epitope and His-tag did not impair ring assembly but inhibited the formation of the long herringbone structures. Immunogold labeling of the particles with the anti-myc antibody showed that the C-terminus of the NP_cfus protein is exposed on the surface of these ring-like particles.

Efficient Generation of BLCL Expressing Foreign Antigen as Antigen-presenting Cells with Recombinant Retroviruses: Hyun-Il Cho , Soon-Young Paik , Il-Hoan Oh , Kyu-Jung Ahn , Dong-Wook Kim , Jin-Han Kang , Wan-Shik Shin , Chun-Choo Kim , Hoon Han; J. Microbiol. 2001;39(4):300-304.

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Abstract: Epstein-Barr Virus (EBV)-transformed lymphoblastoid B cell lines, BLCL, which expresse antigens, are potential antigen-presenting cells (APCs) for the induction of CTL in vitro. However, transfection of BLCLs with subsequent selection by antibiotics is notoriously difficult because plating efficiencies of BLCLs are reported to be 1% or less. To generate stable transfectants of BLCLs, we produced high titers of retroviruses encoding pp65 antigen of human cytomegalovirus as foreign antigens and transduced them to BLCLs. The pp65 gene was cloned into the retroviral vector pLXSN. The recombinant retroviral vector was transfected to ecotropic packaging cell line, GP&E86, and this polyclonal recombinant retrovirus was transduced to PA317 that is amphotropic packaging cell line. The titers of cloned PA317 amphotropic retroviruses ranged from 5 to 7 X 10^6 colony forming units (CFU) per ml (CFU/ml). We performed three rounds of consecutive transductions to BLCLs in order to improve the cloning efficiencies. The expression of recombinant HCMV-pp65 antigen was more than 20% after the final transduction. The third-transduced BLCLs were easily selected in optimal concentration of G418. BLCLs expressing foreign antigens could be used as target cells for CTL assay and/or as APCs for induction of in vitro CTL responses specific for viral and tumor antigens.

Overexpression of the sprD Gene Encoding Streptomyces griseus Protease D Stimulates Actinorhodin Production in Streptomyces lividans: Si-Sun Choi , Won-Jae Chi , Jae Hag Lee , Sang-Soon Kang , Byeong Chul Jeong , Soon-Kwang Hong; J. Microbiol. 2001;39(4):305-313.

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Abstract: The sprD gene encoding Streptomyces griseus protease D (SGPD), a chymotrypsin-like protease, was cloned from Streptomyces griseus IFO13350 and sequenced. Most of the amino-acid sequence deduced from the nucleotide sequence is identical to that of Streptomyces griseus IMRU3499 except that one amino acid has been deleted and Trp369 has been substituted into Cys369 in the SGPD from S. griseus IFO13350 without affecting the protease activity. The sprD gene was overexpressed in Streptomyces lividans TK24 as a heterologous host. Various media with different compositions were also used to maximize the productivity of SGPD in the heterologous host. The SGPD productivity was best when the transformant of S. lividans TK24 was cultivated in R2YE medium. The relative chymotrypsin activity of the culture broth measured with an artificial chromogenic substrate, N-succinyl-ala-ala-pro-phe-[rho]-nitroanilide, was 16 units/ml. A high level of SGPD was also produced in YEME and SAAM media but it was relatively lower than in R2YE medium, and negligible amounts of SGPD were produced in GYE, GAE and Benedict media. The growth of S. lividans reached the maximum level of cell mass at days 3 and 4 of the culture, but SGPD production started in the stationary phase of cell growth and kept increasing till the 10th day of culture in R2YE and YEME medium, but in GYE media the productivity reached maximum level at 8 days of cultivation. The introduction of the sprD gene into S. lividans TK24 triggered biosynthesis of the pigmented antibiotic, actinorhodin, which implies some protease may play a very important role in secondary-metabolite formation in Streptomyces.

A Ser/Thr Specific Protein Kinase Activates the Mouse Rantes Gene after Lipopolysaccharide Stimulation: Youn- Uck Kim , Youn-Hwoan Kim , Duek-Jun An , Hyuk-Chu Kwon; J. Microbiol. 2001;39(4):314-320.

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Abstract: Macrophages stimulated by lipopolysaccharide (LPS) from gram negative bacteria undergo activation of a group of immediate early genes including Rantes. The mouse Rantes gene promoter region contains an LPS responsive element (LRE). We detected 3 specific bands, termed B1, B2, and B3, formed by the interaction of the LRE and proteins found in LPS-stimulated RAW 264.7 cells. An additional band, B4, was determined to be an AP-1 binding protein. The B1 band appears within 1 hour of LPS stimulation. The observed binding pattern could be changed by a specific heparin column fraction of nuclear extracts from LPS-stimulated cells. We have determined that a Ser/Thr-specific protein kinase is activated by LPS stimulation, and this protein kinase enhances B1 band formation. The B1 band can be converted to band B2/B3 by adding specific heparin column fraction. Purified Ser/Thr-specific protein phosphatases PP-1 and PP-2A can stimulate the same conversion to about the same extent. Thus, the formation of the LRE sequence binding complex appears to be regulated by Ser/Thr protein kinase and one or more Ser/Thr specific phosphatases. At least four proteins are involved in the regulation of the LRE-dependent Rantes expression: two binding factors that bind directly to the target sequences, and two factors that control their binding. The future purification and characterization of these binding proteins will reveal in detail the mechanism of Rantes gene activation after LPS stimulation.

Assessment of Baird-Parker Agar as Screening Test for Determination of Staphylococcus aureus in Poultry Meat: Rosa Capita , Carlos Alonso-Calleja , Benito Moreno , Maria del Camino Garcia-Fernandez; J. Microbiol. 2001;39(4):321-325.

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Abstract: Baird-Parker agar with egg yolk/tellurite emulsion (BPA) is widely accepted as a medium for the enumeration of Staphylococcus aureus in foods. However, it is not completely selective and colonies of other genera or species could be similar to those of Staphylococcus aureus. Moreover, the strains of Staphylococcus aureus that are lecithinase negative could go unnoticed. Both facts could affect the counts. The aim of this study was to determine whether the enumeration of the colonies with the typical morphology of Staphylococcus aureus on BPA is sufficient to quantify this species in poultry meat. Forty chicken carcasses were tested for Staphylococcus aureus by surface plating using BPA. Results indicate that the predictive value of the morphology of the colonies on BPA is 85.71% and 68.42% for typical and atypical colonies of Staphylococcus aureus, respectively. However, Staphylococcus aureus counts (after identification) and counts of typical colonies did not show any significant differences (P>0.05) and are significantly (P<0.001) correlated (r = 0.996). These results suggest that, for screening purposes, enumeration of Staphylococcus aureus from poultry meat does not require any identification of strains, resulting in a saving of time and money.

Phylogenetic Analysis of Mycobacterium sp. C2-3 Degrading Polycyclic Aromatic Hydrocarbons: Il-Gyu Lee , Suk-Kyun Han , You-Seak Go , Tae-Young Ahn; J. Microbiol. 2001;39(4):326-330.

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Abstract: Mycobacterium sp. C2-3 was isolated from petroleum-contaminated soil around an oil reservoir and identified by analysis of its 16S rRNA gene sequence. Strain C2-3 was able to use fluorene, phenanthrene, fluoranthene and pyrene as sole sources of carbon and energy, yet unable to degrade naphthalene. The strain was also able to use n-alkanes, such as hexadecane and heptadecane, and phenanthrene and pyrene, in particular, were degraded rapidly. The phylogenetic data suggested that the isolate C2-3 is a thermosensitive, fast-growing strain of Mycobacterium sp.

Reaction Route for Enzymatic Production of Neofructo-oligosaccharides from Sucrose Using Penicillium citrinum Cells: Jae Heung Lee , Satoru Shinohara; J. Microbiol. 2001;39(4):331-333.

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Abstract: The production of oligosaccharides using Penicillium citrium cells at high sugar concentrations was investigated at 50 C and pH 5.0. Both 1-kestose and neokestose were produced from sucrose, while both nystose and tetrasaccharide were produced from 1-kestose. However, no reaction product was obtained from neofructo-oligosaccharides such as neokestose. Based on these experimental results, a hypothetical reaction route was proposed to illustrate how neofructo-oligosaccharides are formed from 1-kestose.

Cloning and Sequence Analysis of the hpaD Gene Responsible for Homoprotocatechuate 2,3-Dioxygenase from Pseudomonas sp. DJ-12: Sang-Mahn Lee , Jong-Chan Chae , Youngsoo Kim , Chi-Kyung Kim; J. Microbiol. 2001;39(4):334-337.

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Abstract: The degradative pathway of homoprotocatechuate (HPC) is the bacterial route whereby 3,4-dihydroxyphenylacetic acid is catabolized to pyruvate and succinate by a series of sequential reactions. The HPC is catalized by homoprotocatechuate 2,3-dioxygenase (HPC-2,3O) to form 5-carboxymethyl-2-hydroxy-muco semialdehyde. In this study, the hpaD gene encoding HPC-2,3O was cloned from the chromosomal DNA of Pseudomonas sp. DJ-12 and its nucleotide sequence was analyzed. The open reding frame of hpaD gene was found to be composed of 864 nucleotide pairs and to encode a polypeptide with 287 amino acid residues. The deduced amino acid sequence of the HPC-2,3O from Pseudomonas sp. DJ-12 exhibited 60~64% homology with those of the corresponding enzymes from E. coli, Salmonella enterica, and Klebsiella pneumoniae.

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