Journal of Microbiology

Volume 43(4); August 2005

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

Enhancing the Intrinsic Bioremediation of PAH-Contaminated Anoxic Estuarine Sediments with Biostimulating Agents: Quang-Dung Bach , Sang-Jin Kim , Sung-Chan Choi , Young-Sook Oh; J. Microbiol. 2005;43(4):319-324.; DOI: https://doi.org/2259 [pii]

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Abstract: Estuarine sediments are frequently polluted with hydrocarbons from fuel spills and industrial wastes. Polycyclic aromatic hydrocarbons (PAHs) are components of these contaminants that tend to accumulate in the sediment due to their low aqueous solubility, low volatility, and high affinity for particulate matter. The toxic, recalcitrant, mutagenic, and carcinogenic nature of these compounds may require aggressive treatment to remediate polluted sites effectively. In petroleum-contaminated sediments near a petrochemical industry in Gwangyang Bay, Korea, in situ PAH concentrations ranged from 10 to 2,900 ug/kg dry sediment. To enhance the biodegradation rate of PAHs under anaerobic conditions, sediment samples were amended with biostimulating agents alone or in combination: nitrogen and phosphorus in the form of slow-release fertilizer (SRF), lactate, yeast extract (YE), and Tween 80. When added to the sediment individually, all tested agents enhanced the degradation of PAHs, including naphthalene, acenaphthene, anthracene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, and benzo[a]pyrene. Moreover, the combination of SRF, Tween 80, and lactate increased the PAH degradation rate 1.2-8.2 times above that of untreated sediment (0.01-10 ug PAH/kg dry sediment/day). Our results indicated that in situ contaminant PAHs in anoxic sediment, including high molecular weight PAHs, were degraded biologically and that the addition of stimulators increased the biodegradation potential of the intrinsic microbial populations. Our results will contribute to the development of new strategies for in situ treatment of PAH-contaminated anoxic sediments.

Isolation and Characterization of a Rhodococcus Species Strain Able to Grow on ortho- and para-Xylene: Jung Yeon Jang , Dockyu Kim , Hyun Won Bae , Ki Young Choi , Jong-Chan Chae , Gerben J. Zylstra , Young Min Kim , Eungbin Kim; J. Microbiol. 2005;43(4):325-330.; DOI: https://doi.org/2258 [pii]

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Abstract: Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1 <br>

Development of Strain-specific PCR Primers Based on a DNA Probe Fu12 for the Identification of Fusobacterium nucleatum subsp. nucleatum ATCC 25586^T: Hwa-Sook Kim , Soo Keun Song , So Young Yoo , Dong Chun Jin , Hwan Seon Shin , Chae Kwang Lim , Myung-Soo Kim , Jin-Soo Kim , Son-Jin Choe , Joong-Ki Kook; J. Microbiol. 2005;43(4):331-336.; DOI: https://doi.org/2257 [pii]

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Abstract: The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586^T (F. nucleatum ATCC 25586^T), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC 25586^T. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC 25586^T. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC 25586^T, especially with regard to the determination of the authenticity of the strain.

The Effect of Transformation on the Virulence of Streptococcus pneumoniae: Xue-Mei Zhang , Yi-Bing Yin , Dan Zhu , Bao-De Chen , Jin-Yong Luo , Yi-Ping Deng , Ming-Fang Liu , Shu-Hui Chen , Jiang-Ping Meng , Kai Lan , Yuan-Shuai Huang , Ge-Fei Kang; J. Microbiol. 2005;43(4):337-344.; DOI: https://doi.org/2256 [pii]

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Abstract: Although pneumococcus is one of the most frequently encountered opportunistic pathogen in the world, the mechanisms responsible for its infectiveness have not yet been fully understood. In this paper, we have attempted to characterize the effects of pneumococcal transformation on the pathogenesis of the organism. We constructed three transformation-deficient pneumococcal strains, which were designated as Nos. 1d, 2d, and 22d. The construction of these altered strains was achieved via the insertion of the inactivated gene, comE, to strains 1, 2 and 22. We then conducted a comparison between the virulence of the transformation-deficient strains and that of the wild-type strains, via an evaluation of the ability of each strain to adhere to endothelial cells, and also assessed psaA mRNA expression, and the survival of hosts after bacterial challenge. Compared to what was observed with the wild-type strains, our results indicated that the ability of all of the transformation-deficient strains to adhere to the ECV304 cells had been significantly reduced (p < 0.05), the expression of psaA mRNA was reduced significantly (p < 0.05) in strains 2d and 22d, and the median survival time of mice infected with strains 1d and 2d was increased significantly after intraperitoneal bacterial challenge (p < 0.05). The results of our study also clearly indicated that transformation exerts significant effects on the virulence characteristics of S. pneumoniae, although the degree to which this effect is noted appears to depend primarily on the genetic background of the bacteria.

Molecular Analysis of Colonized Bacteria in a Human Newborn Infant Gut: Hee-Kyung Park , Sung-Sub Shim , Su-Yung Kim , Jae-Hong Park , Su-Eun Park , Hak-Jung Kim , Byeong-Chul Kang , Cheol-Min Kim; J. Microbiol. 2005;43(4):345-353.; DOI: https://doi.org/2255 [pii]

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Abstract: The complex ecosystem of intestinal microflora is estimated to harbor approximately 400 different microbial species, mostly bacteria. However, studies on bacterial colonization have mostly been based on culturing methods, which only detect a small fraction of the whole microbiotic ecosystem of the gut. To clarify the initial acquisition and subsequent colonization of bacteria in an infant within the few days after birth, phylogenetic analysis was performed using 16S rDNA sequences from the DNA isolated from feces on the 1st, 3rd, and 6th day. 16S rDNA libraries were constructed with the amplicons of PCR conditions at 30 cycles and 50^oC annealing temperature. Nine independent libraries were produced by the application of three sets of primers (set A, set B, and set C) combined with three fecal samples for day 1, day 3, and day 6 of life. Approximately 220 clones (76.7%) of all 325 isolated clones were characterized as known species, while other 105 clones (32.3%) were characterized as unknown species. The library clone with set A universal primers amplifying 350 bp displayed increased diversity by days. Thus, set A primers were better suited for this type of molecular ecological analysis. On the first day of the life of the infant, Enterobacter, Lactococcus lactis, Leuconostoc citreum, and Streptococcus mitis were present. The largest taxonomic group was L. lactis. On the third day of the life of the infant, Enterobacter, Enterococcus faecalis, Escherichia coli, S. mitis, and Streptococcus salivarius were present. On the sixth day of the life of the infant, Citrobacter, Clostridium difficile, Enterobacter sp., Enterobacter cloacae, and E. coli were present. The largest taxonomic group was E. coli. These results showed that microbiotic diversity changes very rapidly in the few days after birth, and the acquisition of unculturable bacteria expanded rapidly after the third day.

Journal Article

Expression of Escherichia coli Heat-labile Enterotoxin B Subunit (LTB) in Saccharomyces cerevisiae: Mohammad Ahangarzadeh Rezaee , Abbas Rezaee , Seyed Mohammad Moazzeni , Ali Hatef Salmanian , Yoko Yasuda , Kunio Tochikubo , Shahin Najar Pirayeh , Mohsen Arzanlou; J. Microbiol. 2005;43(4):354-360.; DOI: https://doi.org/2254 [pii]

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Abstract: Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

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

Establishment of a Micro-Particle Bombardment Transformation System for Dunaliella salina: Congping Tan , Song Qin , Qun Zhang , Peng Jiang , Fangqing Zhao; J. Microbiol. 2005;43(4):361-365.; DOI: https://doi.org/2253 [pii]

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Abstract: In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. salina with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells'' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 ug/ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

Cells in the Respiratory and Intestinal Tracts of Chickens Have Different Proportions of both Human and Avian Influenza Virus Receptors: Jin A Kim , Si Yun Ryu , Sang Heui Seo; J. Microbiol. 2005;43(4):366-369.; DOI: https://doi.org/2252 [pii]

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Abstract: Avian influenza viruses play a crucial role in the creation of human pandemic viruses. In this study, we have demonstrated that both human and avian influenza receptors exist in cells in the respiratory and intestinal tracts of chickens. We have also determined that primarily cultured chicken lung cells can support the replication of both avian and human influenza viruses.

Journal Articles

Optimization of Bacteriocin ST311LD Production by Enterococcus faecium ST311LD, Isolated from Spoiled Black Olives: Svetoslav D. Todorov , Leon M.T. Dicks; J. Microbiol. 2005;43(4):370-374.; DOI: https://doi.org/2251 [pii]

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Abstract: Bacteriocin ST311LD is approximately 2.3 kDa in size. Low levels of bacteriocin activity were recorded in BHI and M17 broth (800 AU/ml) and in 10% (w/v) soy milk (3,200 AU/ml). No bacteriocin production was recorded in 10% (w/v) molasses, despite good growth. Optimal levels (12,800 AU/ml) were detected in MRS broth which had been supplemented with tryptone (20.0 g/l), saccharose (5.0 or 10.0 g/l) or vitamin C (1 ppm). Increased potassium levels did not result in higher levels of activity, and glycerol (1.0 g/l) inhibited the production of bacteriocin ST311LD.

Regulation of Branched-Chain, and Sulfur-Containing Amino Acid Metabolism by Glutathione during Ultradian Metabolic Oscillation of Saccharomyces cerevisiae: Ho-Yong Sohn , Eun-Joo Kum , Gi-Seok Kwon , Ingnyol Jin , Hiroshi Kuriyama; J. Microbiol. 2005;43(4):375-380.; DOI: https://doi.org/2250 [pii]

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Abstract: Autonomous ultradian metabolic oscillation (T~=50 min) was detected in an aerobic chemostat culture of Saccharomyces cerevisiae. A pulse injection of GSH (a reduced form of glutathione) into the culture induced a perturbation in metabolic oscillation, with respiratory inhibition caused by H_2S burst production. As the production of H_2S in the culture was controlled by different amino acids, we attempted to characterize the effects of GSH on amino acid metabolism, particularly with regard to branched chain and sulfur-containing amino acids. During stable metabolic oscillation, concentrations of intracellular glutamate, aspartate, threonine, valine, leucine, isoleucine, and cysteine were observed to oscillate with the same periods of dissolved O_2 oscillation, although the oscillation amplitudes and maximal phases were shown to differ. The methionine concentration was stably maintained at 0.05 mM. When GSH (100 uM) was injected into the culture, cellular levels of branched chain amino acids increased dramatically with continuous H_2S production, whereas the cysteine and methionine concentrations were noticeably reduced. These results indicate that GSH-dependent perturbation occurs as the result of the promotion of branched chain amino acid synthesis and an attenuation of cysteine and methionine synthesis, both of which activate the generation of H_2S. In a low sulfate medium containing 2.5 mM sulfate, the GSH injections did not result in perturbations of dissolved O_2, NAD(P)H redox oscillations without burst H_2S production. This suggests that GSH-dependent perturbation is intimately linked with the metabolism of branched-chain amino acids and H_2S generation, rather than with direct GSH-GSSG redox control.

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