Journal of Microbiology

Volume 41(2); June 2003

In situ Delivery of Therapeutic Proteins by Recombinant Lactococcus lactis: Lothar Steidler , Sabine Neirynck; J. Microbiol. 2003;41(2):63-72.

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Abstract: Chronic inflammatory bowel disease (IBD) such as Crohn’s disease or ulcerative colitis, affects around 2 in every 1000 individuals in western countries and its incidence, particularly amongst children, is increasing. IBD shows extreme morbidity with impact on all aspects of quality of life. If left untreated, IBD can lead to death. Conventional treatment of IBD involves powerful immunosuppressive chemotherapies and surgical intervention. Long-term anti-inflammatory medication is required and so patients are often subject to a spectrum of unpleasant side effects. Interleukin-10 (IL-10) is a cytokine that acts to suppress inflammation. When however administered by injection, the high levels of IL-10 that are distributed throughout the body also lead to side effects. Lactococcus lactis can be genetically engineered to secrete biologically active cytokines. When applied to the mucosa, these L. lactis can actively deliver such cytokines. By use of this principle we developed a new therapeutic approach for IBD. Administration of L. lactis that secretes murine IL-10 cures and prevents IBD in mice. The use of the engineered L. lactis gets around the problem of delivering IL-10, allowing dramatic reduction of the effective dose. A sincere concern exists about the possible dangers of uncontrolled, deliberate release of genetically modified microorganisms, such as could occur following application in healthcare. We engaged in the establishment of adequate means for biological growth control of engineered L. lactis by targeted

Characterization of the Proteolytic Activity of Bacteria Isolated from a Rotating Biological Contactor: In Jae Park , Jerng Chang Yoon , Seong Joo Park , Eung Ho Kim , Yeon-Jae Cho , Kwang-Soo Shin; J. Microbiol. 2003;41(2):73-77.

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Abstract: Four proteolytic bacteria were isolated and identified from a rotating biological contactor based on Bacillus. The four isolates, Ni 26, 36, 39 and 49 were identified as B. vallismortis, B. subtilis, Aeromonas hydrophila and B. amyloliquefaciens, respectively, based on their biochemical properties and 16S rDNA sequence analyses. The optimal proteolytic activity was observed in the temperature and pH ranges of 40-70ºC and 8.0-8.5, respectively. The proteolytic activities of all the isolates were partially inhibited by phenylmethylsulfonylfluoride (PMSF), and the isolates Ni 26, Ni 39 and Ni 49 were inhibited by the metalloprotease inhibitor, 1,10-phenanthroline. Zymographic analyses of the culture supernatants revealed the presence of at least two proteases in all isolates.

Characteristics of Na^+ -dependent Serine Transport in Haemophilus Influenzae Rd: Young-Mog Kim , In-Koo Rhee , Mi-Yeon Park , Dong-Suck Chang , Tomofusa Tsuchiya; J. Microbiol. 2003;41(2):78-82.

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Abstract: We identified two proteins in Haemophilus influenzae Rd that exhibited high similarity to two major serine transporters of Escherichia coli (SstT and SdaC). Then, we investigated serine transport in H. influenzae Rd and detected Na + -stimulated L-serine transport activity. The optimum NaCl concentration for this stimulation was about 20 mM. The uptake of Na^+ by H. influenzae Rd was found to be elicited by L-serine influx, which supports the idea that L-serine is transported by a mechanism of Na^+ / serine symport. No uptake of H + elicited by L-serine influx was detected. Na^+ /serine symport activity was not inhibited by other amino acids such as L-threonine or D-serine. Two distinct Km values were obtained from the kinetic analysis of serine transport. Thus, two serine transport pathways may exist in H. influenzae Rd, and it appears that both systems are stimulated by Na^+ .

Tsukamurella sunchonensis sp. nov., a Bacterium Associated with Foam in Activated Sludge: Chi Nam Seong , Young Sook Kim , Keun Shik Baik , Sang Ki Choi , Min Bae Kim; J. Microbiol. 2003;41(2):83-88.

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Abstract: aeration basin of an activated sludge process, was clarified by phenotypic, chemotaxonomic and phylogenetic analyses. The strain possesses wall chemotype IV, MK-9(H_0 ), as the major menaquinone, and contains saturated, monounsaturated and 10-methyl branched fatty acids. The G+C content of its DNA is 68.1 mol%. Phenotypic data and DNA relatedness to known species indicate that the strain SCNU5^T represents a new species within the genus Tsukamurella, for which we propose the name Tsukamurella sunchonensis sp. nov. The type strain of T. sunchonensis is SCNU5^T (=KCTC 9827^T ).

Structural Analysis of the fcbABC Gene Cluster Responsible for Hydrolytic Dechlorination of 4-Chlorobenzoate from pJS1 Plasmid of Comamonas sp. P08: Jeong-Soon Lee , Kyoung Lee , Jong-Ok Ka , Jong-Chan Chae , Chi-Kyung Kim; J. Microbiol. 2003;41(2):89-94.

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Abstract: Bacterial strain No. P08 isolated from wastewater at the Cheongju industrial complex was found to be capable of degrading 4-chlorobenzoate under aerobic condition. P08 was identified as Comamonas sp. from its cellular fatty acid composition and 16S rDNA sequence. The fcb genes, responsible for the hydrolytic dechlorination of 4-chlorobenzoate, were cloned from the plasmid pJS1 of Comamonas sp. P08. The fcb gene cluster of comamonas sp. P08 was organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC. This organization of the fcb genes was very similar to that of the fcb genes carried on the chromosomal DNA of Pseudomonas sp. DJ-12. However, it differed from the fcbA-fcbB-fcbC ordering of Arthrobacter sp. SU. The nucleotide sequences of the fcbABC genes of strain P08 showed 98% and 53% identities to those of Pseudomonas sp. DJ-12 and Arthrobacter sp. SU, respectively. This suggests that the fcb genes might have been derived from Pseudomonas sp. DJ-12 to form plasmid pJS1 in Comamonas sp. P08, or that the fcb genes in strain DJ-12 were transposed from Comamonas sp. P08 plasmid.

Cloning, Expression in Escherichia coli, and Enzymatic Properties of a Lipase from Pseudomonas sp. SW-3: Sun-Young An , Sang-Wan Kim , Yong-Lark Choi , Young-Su Cho , Woo-Hong Joo , oung-Choon Lee; J. Microbiol. 2003;41(2):95-101.

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Abstract: The lipase gene (lipA) and its activator gene (lipB) of Pseudomonas sp. SW-3 were cloned and sequenced. The lipB was found to be present immediately downstream of lipA. The deduced amino acid sequences of lipA and lipB showed a high level of homology to those of other lipases belonging to the family I.1 of bacterial lipases. When lipA was expressed in Escherichia coli using T7 promoter, an active lipase was produced in cells carrying both lipA and lipB, but not in cells harboring only lipA. Recombinant lipase (rPSL) overproduced in an insoluble form was solubilized in the presence of 8 M urea, purified in a ureadenatured form and refolded by removing urea in the presence of the Ca^2+ ion. RPLS had maximum activity at pH 8.0 and 50℃, was stable at pHs from 7.0 to 9.0 and below 50 ℃, and showed the highest activity toward the p-nitrophenyl ester of palmitate (C16).

Cloning and Sequence Analysis of Two Catechol-degrading Gene Clusters from a Phenol-utilizing Bacterium Pseudomonas putida SM25: Young-Hee Jung , Jong-Ok Ka , Choong-Ill Cheon , Myeong-Sok Lee , Eun-Sook Song , Soon-Young Choi , Daeho Cho , Sang-Ho Choi , Kwon-Soo Ha , Young Mok Park , Jong-Soon Choi , Kyung-Hee Min; J. Microbiol. 2003;41(2):102-108.

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Abstract: A 6.1 kb Sph I fragment from the genomic DNA of Pseudomonas putida SM 25 was cloned into the vector pUC19. The open reading frame of catB was found to consist of 1,122 nucleotides. The sequence alignment of the catB gene products from different kinds of bacteria revealed an overall identity ranging from 40 to 98%. The catC gene contained an open reading frame of 96 codons, from which a protein with a molecular mass of about 10.6 kDa was predicted. The amino acids in the proposed activesite region of CatC were found to be almost conserved, including the charged residues. Since the catBC genes in P. putida SM25 were tightly linked, they could be regulated under coordinate transcription, and transcribed from a single promoter located upstream of the catB gene, as in P. putida RB1.

Regulation of the sufABCDSE Operon by Fur: Joon-Hee Lee , Won-Sik Yeo , Jung-Hye Roe; J. Microbiol. 2003;41(2):109-114.

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Abstract: A promoter that is inducible by paraquat and menadione, the superoxide generators, independently of soxRS has been found in front of the sufABCDSE operon in Escherichia coli. Based on the observation that SufA is a holomog of IscA that functions in the assembly of iron sulfur cluster and the sufA promoter (sufAp) contains a putative Fur-binding consensus, we investigated whether this gene is regulated by Fur, a ferric uptake regulator. When examined in several sufAp-lacZ chromosomal fusion strains, sufAp was induced by EDTA, an iron chelator and a well-known Fur-inducer. The basal level of sufA expression increased dramatically in fur mutant, suggesting repression of sufAp by Fur. The derepression in fur mutant and EDTA-induction of sufA expression required nucleotides up to -61, where a putative Fur box is located. Purified Fur protein bound to the DNA fragment containing the putative Fur box between -35 and -10 promoter elements. The regulation by Fur and menadione induction of sufAp acted independently. The rpoS mutation increased sufA induction by menadione, suggesting that the stationary sigma factor RpoS acts negatively on sufA induction.

Synthetic Lethal Mutations with spmex67 of Schizosaccharomyces pombe in the Mediation of mRNA Export: Jin Ho Yoon; J. Microbiol. 2003;41(2):115-120.

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Abstract: Mex67p/Tap are evolutionally conserved mRNA export factors. To identify mutations in genes that are functionally linked to mex67 with respect to mRNA export, we used a synthetic lethal genetic screen in Schizosaccharomyces pombe. Three synthetic lethal mutants were isolated and mutations in these mutants defined separate complementation groups. These mutants exhibited the accumulation of poly(A)^+ RNA in the nucleus, with a decrease in the cytoplasm under synthetically lethal conditions, suggesting that the mutations cause an mRNA nuclear export defect. In addition, the S. pombe genes that were found to be involved in mRNA export did not suppress the synthetic lethality of these mutants. These results indicate that the isolated mutants contain mutations in new genes, which are involved in mRNA export from the nucleus.

Isolation and Characterization of Bud6p, an Actin Interacting Protein, from Yarrowia lipolytica: Yunkyoung Song , Seon Ah Cheon , So-Yeon Lee , Ji-Sook Hwang , Jeong-Yoon Kim; J. Microbiol. 2003;41(2):121-128.

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Abstract: The identification of genes involved in true hypha formation is important in the study of mechanisms underlying the morphogenetic switch in yeast. We isolated a gene responsible for the morphogenetic switch in Yarrowia lipolytica, which forms true hyphae in response to serum or N-acetylglucosamine. The isolated gene, encoding 847 amino acids, had sequence identities of 27% and 25% with the Bud6 (Aip3) proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. Disruption of this gene, designated YlBUD6, in haploid and diploid strains significantly reduced the ability of Y. lipolytica to switch from the yeast form to the hyphal form in hypha-inducing media. It was also found that YlBud6 mutants were rounder than the wild type when grown in the yeast form. These results indicate that the YlBud6 protein is necessary for hyphal growth and cell polarity in both haploid and diploid Y. lipolytica cells.

High Prevalence of the China 1 Strain of Epstein-Barr Virus in Korea as Determined by Sequence Polymorphisms in the Carboxy-Terminal Tail of LMP1: Sung-Gyu Cho , Won-Keun Lee; J. Microbiol. 2003;41(2):129-136.

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Abstract: The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) exhibits considerable sequence heterogeneity among EBV isolates. Seven distinct EBV strains have been defined based on sequence polymorphisms in the LMP1 gene, which are designated China 1, China 2, China 3, Alaskan, Mediterranean, NC, and the B95-8 strains. In this study, we analyzed a 30-bp deletion and sequence variations in the carboxy-terminal region of the LMP1 gene in 12 EBV isolates from spontaneous lymphoblastoid cell lines derived from individuals with non-EBV associated cancers in Korea. Eleven of the 12 isolates showed a 30-bp deletion spanning LMP1 amino acids 342 to 353, suggesting a high prevalence of the LMP1 30-bp deletion variant among EBV isolates in Korea. In addition, all 12 isolates had a 15-bp common deletion in the 33-bp repeat region and multiple base-pair changes relative to the prototype B95-8 EBV strain along with variations in the number of the 33-bp repeats. The bp changes at positions 168746, 168694, 168687, 168395, 168357, 168355, 168631, 168320, 168308, 168295, and 168225 were highly conserved among the isolates. Comparative analysis of sequence change patterns in the LMP1 carboxy-terminal coding region identified nine 30-bp deletion variants as China 1, two deletion variants as a possible interstrain between the Alaskan and China 1 strains, and a single undeleted variant as a possible variant of the Alaskan strain. These results suggest the predominance of the China 1 EBV strain in the Korean population.

Partial Characterization of the Pathogenic Factors Related to Chlamydia trachomatis Invasion of the McCoy Cell Membrane: Myeong-Gu Yeo , Young-Ju Kim , Yeal Park; J. Microbiol. 2003;41(2):137-143.

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Abstract: The present study was performed to identify pathogenic factors of Chlamydia trachomatis, which invade the host cell membrane. We prepared monoclonal antibody against C. trachomatis and searched for pathogenic factors using this antibody, and subsequently identified the surface components of the elementary body of C. trachomatis, i.e., major outer membrane protein (MOMP), lipopolysaccharide (LPS), and two other surface exposure proteins. These proteins are believed to be important in the pathogenesis of host cell chlamydial infection. Additionally, to identify factors related to the host cell and C. trachomatis, we prepared C. trachomatis infected and non-infected McCoy cell extracts, and reacted these with anti-chlamydial LPS monoclonal antibody. We found that anti-chlamydial LPS monoclonal antibody reacted with a 116 kDa proteinaceous McCoy cell membrane component.

Isolation of Listeria monocytogenes by Immunomagnetic Separation and Atomic Force Microscopy: Birce Mercanoglu , S. Aykut Aytac , M. Ali Ergun , Erdal Tan; J. Microbiol. 2003;41(2):144-147.

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Abstract: Listeria monocytogenes is a pathogen of major concern to the food industry and the potential cause of severe infections such as listeriosis. Early detection of this foodborne pathogen is important in order to eliminate its potential hazards. So, immunomagnetic separation (IMS) has been suggested as a means of reducing the total analysis time and for improving the sensitivity of detection. Atomic force microscopy (AFM) has been used for measuring the topographic properties of sample surfaces at nanometer scale. In this study, we used AFM to confirm both the sensitivity and the specificity of IMS. Regarding AFM analysis, the length and the width of the bacteria, which were in agreement with literature values, were found to be 2.993 mm and 0.837 mm, respectively. As a result, AFM helped us both characterize and measure the bacterial and bead structures.

Investigation of Waterborne Parasites in Drinking Water Sources of Ankara, Turkey: Bilal Bakir , Mehmet Tanyuksel , Fatma Saylam , Sultan Tanriverdi , R. Engin Araz , Ali Kasim Hacim , Metin Hasde; J. Microbiol. 2003;41(2):148-151.

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Abstract: Waterborne parasite infections are considered a re-emerging threat. Most studies on the epidemiology of human cryptosporidiosis, giardiasis, and amebiasis have been carried out in developed countries, and there is little data on the occurrence of these infections in other areas. The objective of this study was to investigate the presence of waterborne parasites such as Cryptosporidium parvum, Giardia lam-blia, and Entamoeba histolytica in various water samples in Ankara, Turkey. A total of 85 samples were examined, 43 from the municipal water supply, 34 from wells, 6 from the Ankara River, and 2 from two untreated dams; by conventional microscopy, immunologically and by polymerase chain reaction (PCR). Oocysts of C. parvum and cysts of G. lamblia were detected by using an indirect fluorescence (antigen) assay, whereas an enzyme linked immunosorbent assay was used to detect the cysts of E. his-tolytica and E. dispar. In addition, PCR was used for E. histolytica, E. dispar, C. parvum and G. lamblia detection. G. lamblia was found in 2 of the 34 well water samples, and parasites were found in 3 of the 6 Ankara River samples. The 1^st contained E. histolytica cysts and Strongyloides stercoralis larvae, the 2^nd E. histolytica cysts, and Trichuris trichiura eggs, and the 3^rd C. parvum oocysts only. No parasite was observed in the municipal water samples and untreated dam water samples. These results extend our knowledge on waterborne parasites, such occurrence information on waterborne pathogens assists the management and treatment of municipal water.

The Phylotype of Thermus from the Rehai Geothermal Area, Tengchong, China: Chunlei Guo , Tao Wang , Wei Zhu , Donghua Zhang , Xiaolong Cui , Lihua Xu , Qian Peng; J. Microbiol. 2003;41(2):152-156.

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Abstract: Through enrichment on two nutrient agars, 57 Thermus isolates were recovered from 15 hot spring samples taken from the Rehai geothermal area, Tengchong, China. Unique growth characteristics were observed when the strains were transferred from YIM14 medium to Thermus medium. Phylogenetic analysis showed that the 16S rDNA sequences of the isolates and clones from the Rehai geothermal area formed a monophyletic group on the phylogenetic tree. A secondary structure comparison showed that their 16S rRNAs have unique secondary structure characteristics.

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