Journal of Microbiology

Volume 45(2); April 2007

Review

Biosynthesis, Modification, and Biodegradation of Bacterial Medium-Chain-Length Polyhydroxyalkanoates: Do Young Kim , Hyung Woo Kim , Moon Gyu Chung , Young Ha Rhee; J. Microbiol. 2007;45(2):87-97.; DOI: https://doi.org/2528 [pii]

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Abstract: Medium-chain-length polyhydroxyalkanoates (MCL-PHAs), which have constituents with a typical chain length of C6-C14, are polyesters that are synthesized and accumulated in a wide variety of Gram-negative bacteria, mainly pseudomonads. These biopolyesters are promising materials for various applications because they have useful mechanical properties and are biodegradable and biocompatible. The versatile metabolic capacity of some Pseudomonas spp. enables them to synthesize MCL-PHAs that contain various functional substituents; these MCL-PHAs are of great interest because these functional groups can improve the physical properties of the polymers, allowing the creation of tailor-made products. Moreover, some functional substituents can be modified by chemical reactions to obtain more useful groups that can extend the potential applications of MCL-PHAs as environmentally friendly polymers and functional biomaterials for use in biomedical fields. Although MCL-PHAs are water-insoluble, hydrophobic polymers, they can be degraded by microorganisms that produce extracellular MCL-PHA depolymerase. MCL-PHA-degraders are relatively uncommon in natural environments and, to date, only a limited number of MCL-PHA depolymerases have been investigated at the molecular level. All known MCL-PHA depolymerases share a highly significant similarity in amino acid sequences, as well as several enzymatic characteristics. This paper reviews recent advances in our knowledge of MCL-PHAs, with particular emphasis on the findings by our research group.

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

Alternative Alert System for Cyanobacterial Bloom, Using Phycocyanin as a Level Determinant: Chi-Yong Ahn , Seung-Hyun Joung , Sook-Kyoung Yoon , Hee-Mock Oh; J. Microbiol. 2007;45(2):98-104.; DOI: https://doi.org/2527 [pii]

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Abstract: Chlorophyll a concentration and cyanobacterial cell density are regularly employed as dual criteria for determinations of the alert level for cyanobacterial bloom. However, chlorophyll a is not confined only to the cyanobacteria, but is found universally in eukaryotic algae. Furthermore, the determination of cyanobacterial cell counts is notoriously difficult, and is unduly dependent on individual variation and trained skill. A cyanobacteria-specific parameter other than the cell count or chlorophyll a concentration is, accordingly, required in order to improve the present cyanobacterial bloom alert system. Phycocyanin has been shown to exhibit a strong correlation with a variety of bloom-related factors. This may allow for the current alert system criteria to be replaced by a three-stage alert system based on phycocyanin concentrations of 0.1, 30, and 700 μg/L. This would also be advantageous in that it would become far more simple to conduct measurements without the need for expensive equipment, thereby enabling the monitoring of entire lakes more precisely and frequently. Thus, an alert system with superior predictive ability based on highthroughput phycocyanin measurements appears feasible.

Phylogenetic Diversity of Bacteria in an Earth-Cave in Guizhou Province, Southwest of China: JunPei Zhou , YingQi Gu , ChangSong Zou , MingHe Mo; J. Microbiol. 2007;45(2):105-112.; DOI: https://doi.org/2526 [pii]

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Abstract: The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.

Dominance of Endospore-forming Bacteria on a Rotating Activated Bacillus Contactor Biofilm for Advanced Wastewater Treatment: Seong Joo Park , Jerng Chang Yoon , Kwang-Soo Shin , Eung Ho Kim , Soobin Yim , Yeon-Je Cho , Gi Moon Sung , Dong-Geun Lee , Seung Bum Kim , Dong-Uk Lee , Sung-Hoon Woo , Ben Koopman; J. Microbiol. 2007;45(2):113-121.; DOI: https://doi.org/2525 [pii]

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Abstract: The bacterial diversity inherent to the biofilm community structure of a modified rotating biological contactor wastewater treatment process, referred to as the Rotating Activated Bacillus Contactor (RABC) process, was characterized in this study, via both culture-dependent and culture-independent methods. On the basis of culture-dependent methods, Bacillus sp. were found to exist in large numbers on the biofilm (6.5% of the heterotrophic bacteria) and the microbial composition of the biofilms was quite simple. Only three phyla were identified-namely, the Proteobacteria, the Actinobacteria (High G+C Gram-positive bacteria), and the Firmicutes (Low G+C Gram-positive bacteria). The culture-independent partial 16S rDNA sequence analysis revealed a considerably more diverse microbial composition within the biofilms. A total of eight phyla were recovered in this case, three of which were major groups: the Firmicutes (43.9%), the Proteobacteria (28.6%), and the Bacteroidetes (17.6%). The remaining five phyla were minor groups: the Planctomycetes (4.4%), the Chlorobi (2.2%), the Actinobacteria (1.1%), the Nitrospirae (1.1%), and the Verrucomicrobia (1.1%). The two most abundant genera detected were the endospore-forming bacteria (31.8%), Clostridium and Bacillus, both of which are members of the Firmicutes phylum. This finding indicates that these endospore-forming bacteria successfully colonized and dominated the RABC process biofilms. Many of the colonies or clones recovered from the biofilms evidenced significantly high homology in the 16S rDNA sequences of bacteria stored in databases associated with advanced wastewater treatment capabilities, including nitrification and denitrification, phosphorus accumulation, the removal of volatile odors, and the removal of chlorohydrocarbons or heavy metals. The microbial community structures observed in the biofilms were found to correlate nicely with the enhanced performance of advanced wastewater treatment protocols.

Factors Indicating Culture Status During Cultivation of Spirulina (Arthrospira) platensis: Choong-Jae Kim , Yun-Ho Jung , Hee-Mock Oh; J. Microbiol. 2007;45(2):122-127.; DOI: https://doi.org/2524 [pii]

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Abstract: Factors indicating culture status of two Spirulina platensis strains were monitored in a batch mode cultivation for 36 days. Changing mode in all factors showed a common turning point, indicating shift of cell or culture status. Mean biomass productivity was highly sustained until day 22, chlorophyll a concentration peaked on day 22, pH value was >12 on day 22, coil number was abruptly shortened on day 22, and floating activity was sustained at greater than 79% after day 22, indicating that day 22 is a criterion reflecting phase-transfer in cell physiology in a batch culture system. Many of these changes may have been caused by increased pH, suggesting that pH control is essential for mass production of S. platensis. Fluctuations in floating activity were likely induced by the number of cellular gas vacuoles. Consequently, coil number per trichome and floating activity of S. platensis could readily act as simple indicators for determination of culture status or harvesting time of cells.

Increased Carotenoid Production in Xanthophyllomyces dendrorhous G276 Using Plant Extracts: Soo-Ki Kim , Jun-Hyeong Lee , Chi-Ho Lee , Yoh-Chang Yoon; J. Microbiol. 2007;45(2):128-132.; DOI: https://doi.org/2523 [pii]

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Abstract: The red yeast Xanthophyllomyces dendrorhous (previously named Phaffia rhodozyma) produces astaxanthin pigment among many carotenoids. The mutant X. dendrorhous G276 was isolated by chemical mutagenesis. The mutant produced about 2.0 mg of carotenoid per g of yeast cell dry weight and 8.0 mg/L of carotenoid after 5 days batch culture with YM media; in comparison, the parent strain produced 0.66 mg/g of yeast cell dry weight and a carotenoid concentration of 4.5 mg/L. We characterized the utilization of carbon sources by the mutant strain and screened various edible plant extracts to enhance the carotenoid production. The addition of Perilla frutescens (final concentration, 5%) or Allium fistulosum extracts (final concentration, 1%) enhanced the pigment production to about 32 mg/L. In a batch fermentor, addition of Perilla frutescens extract reduced the cultivation time by two days compared to control (no extract), which usually required five-day incubation to fully produce astaxanthin. The results suggest that plant extracts such as Perilla frutescens can effectively enhance astaxanthin production.

Propagation of Bombyx mori Nucleopolyhedrovirus in Nonpermissive Insect Cell Lines: Soo-Dong Woo , Jong Yul Roh , Jae Young Choi , Byung Rae Jin; J. Microbiol. 2007;45(2):133-138.; DOI: https://doi.org/2522 [pii]

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Abstract: This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via β-galactosidase expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs.

Retracted Publication

Transcriptional Analysis of the DNA Polymerase Gene of Bombyx mori Parvo-like Virus (China Isolate): Yong-Jie Wang , Ke-Ping Chen , Qin Yao , Xu Han; J. Microbiol. 2007;45(2):139-145.; DOI: https://doi.org/2521 [pii]

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Abstract: The Bombyx mori parvo-like virus (China isolate) DNA polymerase (BmDNV-3 dnapol) gene has been tentatively identified based on the presence of conserved motifs. In the present study, we perform a transcriptional analysis of the BmDNV-3 dnapol gene using the total RNA isolated from BmDNV-3 infected silkworm at different times. Northern blot analysis with a BmDNV-3 dnapol-specific riboprobe showed a major transcript of 3.3 kb. 5′-RACE revealed that the major transcription start point was located 20 nucleotides downstream of the TATA box. In a temporal expression analysis using differential RT-PCR, BmDNV-3 dnapol transcript was detected at low levels at 6 h.p.i., increased from 6 to 36 h.p.i., and remained fairly constant thereafter. Analysis of the predicted DNA polymerase sequence using neighborjoining and protein parsimony algorithms indicated that the predicted 1115-residue polypeptide contained five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3′ to 5′ exonuclease activity. The molecular phylogenetic analysis of this gene supported the placement of Bombyx mori parvo-like virus in a separate virus family.

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

Identification and Characterization of the Vibrio vulnificus rtxA Essential for Cytotoxicity in vitro and Virulence in Mice: Jeong Hyun Lee , Myung Won Kim , Byoung Sik Kim , Seung Min Kim , Byung Cheol Lee , Tae Sung Kim , Sang Ho Choi; J. Microbiol. 2007;45(2):146-152.; DOI: https://doi.org/2520 [pii]

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Abstract: A mutant exhibiting decreased cytotoxic activity toward INT-407 intestinal epithelial cells and carrying a mutation in the rtx gene cluster that consists of rtxCA and rtxBDE operons was screened from a library of V. vulnificus mutants. The functions of the rtxA gene, assessed by constructing an isogenic mutant and evaluating its phenotypic changes, demonstrated that RtxA is essential for the virulence of V. vulnificus in mice as well as in tissue cultures.

Cloning, Expression, and Functional Characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate Synthase Gene in Escherichia coli: Yi Yi , Dairong Qiao , Linhan Bai , Hui Xu , Ya Li , Xiaolin Wang , Yi Cao; J. Microbiol. 2007;45(2):153-157.; DOI: https://doi.org/2519 [pii]

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Abstract: 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.

Cloning and Expression of the Cathepsin F-like Cysteine Protease Gene in Escherichia coli and Its Characterization: Han Seung Joo , Kwang Bon Koo , Kyung In Park , Song Hwan Bae , Jong Won Yun , Chung Soon Chang , Jang Won Choi; J. Microbiol. 2007;45(2):158-167.; DOI: https://doi.org/2518 [pii]

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Abstract: In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the 32P-labeled partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3''-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the Cys90, His226, and Asn250 residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3% to 12.5% of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and 35°C, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

Mutations in the GyrA Subunit of DNA Gyrase and the ParC Subunit of Topoisomerase IV in Clinical Strains of Fluoroquinolone-Resistant Shigella in Anhui, China: Li-Fen Hu , Jia-Bin Li , Ying Ye , Xu Li; J. Microbiol. 2007;45(2):168-170.; DOI: https://doi.org/2517 [pii]

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Abstract: In this research 26 Shigella isolates were examined by PCR and direct nucleotide sequencing for genetic alterations in the quinolone-resistance determining regions (QRDRs). We tested for the presence of qnr genes by PCR in 91 strains, but no qnr genes were found. The results did show, however, some novel mutations at codon 83 of gyrA (Ser→Ile) and codon 64 of parC (Ala64→Cys, Ala64→Asp), which were related to fluroquinolone resistance.

Revegetation of a Lakeside Barren Area by the Application of Plant Growth-promoting Rhizobacteria: Tae-Seok Ahn , Jong-Ok Ka , Geon-Hyoung Lee , Hong-Gyu Song; J. Microbiol. 2007;45(2):171-174.; DOI: https://doi.org/2516 [pii]

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Abstract: The growth stimulation of wild plants by several bacterial species showing plant growth-promoting capabilities was examined in a barren lakeside area at Lake Paro, Korea. Microbial numbers and activities in the field soil were monitored for 73 days after inoculation of the bacteria. The acridine orange direct counts for the total soil bacterial populations ranged between 2.0-2.3×109 cells/g soil and 1.4-1.8×109 cells/g soil in the inoculated and uninoculated soils, respectively. The numbers of Pseudomonas spp., which is known as a typical plant growth-promoting rhizobacteria, and the total microbial activity were higher in the inoculated soil compared to those in the uninoculated soil. The average shoot and root lengths of the wild plants grown in the inoculated soil were 17.3 cm and 12.4 cm, respectively, and longer than those of 11.4 cm and 8.5 cm in the uninoculated soil. The total dry weight of the harvested wild plants was also higher in the inoculated soil (42.0 g) compared to the uninoculated soil (35.1 g). The plant growth-promoting capabilities of the inoculated bacteria may be used for the rapid revegetation of barren or disturbed land, and as biofertilizer in agriculture.

Recombinant Expression and Purification of Functional XorII, a Restriction Endonuclease from Xanthomonas oryzae pv. oryzae: Dong Kyu Hwang , Jae-Yong Cho , Young Kee Chae; J. Microbiol. 2007;45(2):175-178.; DOI: https://doi.org/2515 [pii]

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Abstract: An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at 25°C in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.9.

Journal Article

Outer Membrane Protein H for Protective Immunity Against Pasteurella multocida: Jeongmin Lee , Young Bong Kim , Moosik Kwon; J. Microbiol. 2007;45(2):179-184.; DOI: https://doi.org/2514 [pii]

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Abstract: Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.

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