Journal of Microbiology

Volume 41(3); September 2003

Regulation of Class II Bacteriocin Production by Cell-Cell Signaling: Luis E. N. Quadri; J. Microbiol. 2003;41(3):175-182.

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Abstract: Production of ribosomally synthesized antimicrobial peptides usually referred to as bacteriocins is an inducible trait in several gram positive bacteria, particularly in those belonging to the group of lactic acid bacteria. In many of these organisms, production of bacteriocins is inducible and induction requires secretion and extracellular accumulation of peptides that act as chemical messengers and trigger bacteriocin production. These inducer peptides are often referred to as autoinducers and are believed to permit a quorum sensing-based regulation of bacteriocin production. Notably, the peptides acting as autoinducers are dedicated peptides with or without antimicrobial activity or the bacteriocins themselves. The autoinducer-dependent induction of bacteriocin production requires histidine protein kinases and response regulator proteins of two-component signal transduction systems. The current working model for the regulation of class II bacteriocin production in lactic acid bacteria and the most relevant direct and indirect pieces of evidence supporting the model are discussed in this minireview.

Culture and Identification of Bacteria from Marine Biofilms: Yoo Kyung Lee , Kae-Kyung Kwon , Kyeung Hee Cho , Hyo Won Kim , Jae Hyun Park , Hong Kum Lee; J. Microbiol. 2003;41(3):183-188.

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Abstract: We isolated and cultured bacteria that inhabited marine biofilms, and identified them by phylogenetic analysis using 16S rDNA sequences. In the marine environment, biofilms cover most subtidal and intertidal solid surfaces such as rocks, ships, loops, marine animals, and algae. The bacteria in most biofilms are embedded in extracellular polymeric substances that comprise mainly of exopolysaccharides. The exopolysaccharides are excreted from multiple bacterial species; therefore, biofilms are a good source for screening exopolysaccharide-producing bacteria. Thirty-one strains were cultured, and a total of 17 unique strains were identified. Phylogenetic analysis using 16S rDNA sequences indicated that the 17 strains belonged to [alpha]-Proteobacteria (Ochrobactrum anthropi, Paracoccus carotinifaciens); [gamma]-Proteobacteria (Pseudoalteromonas agarovorans, P. piscicida, Pseudomonas aeruginosa, Shewanella baltica, Vibrio parahaemolyticus, V. pomeroyi); CFB group bacteria (Cytophaga latercula, Tenacibaculum mesophilum); high GC, Gram-positive bacteria (Arthrobacter nicotianae, Brevibacterium casei, B. epidermidis, Tsukamurella inchonensis); and low GC, Gram-positive bacteria (Bacillus macroides, Staphylococcus haemolyticus, S. warneri).

Influence of Transition-Metal Cofactors on the Reductive Dechlorination of Polychlorinated Biphenyls (PCBs): O-Seob Kwon , Young-Jin Kim § , Kyung-Je Cho , Jin Ae Lee , Young Eui Kim , In Young Hwang , Jae Hyun Kwon; J. Microbiol. 2003;41(3):189-195.

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Abstract: To enhance the reductive dechlorination of polychlorinated biphenyls (PCBs) under anaerobic conditions, we examined the adjunctive effects of cobalt (Co) and nickel (Ni), which are the central metals of transition-metal cofactors of coenzyme F_430 and vitamin B_12, respectively, on the dechlorination of Aroclor 1248. After 32 weeks of incubation, the average numbers of chlorines per biphenyl in culture vials supplemented with 0.2, 0.5, and 1.0 mM of Co reduced from 3.88 to 3.39, 2.92, and 3.28, respectively. However, the numbers of chlorine after supplementing with Ni decreased from 3.88 to 3.43, regardless of the Ni concentrations. The observed congener distribution patterns of all vials with different conditions were similar to the pattern produced by the dechlorination process of H' after 21 weeks of incubation, and these patterns were unchanged up to week 32, except for vials supplemented with 0.5 and 1.0 mM of Co. In vials containing 0.5 mM of Co, meta-rich congeners, such as 25/ 25-, 24/25-, and 25/23-chlorobiphenyls (CBPs), which were found as accumulated products of dechlorination in other conditions, were further dechlorinated, and 25/2-, 24/2-, and 2/2-CBPs were concomitantly increased after 32 weeks of incubation. In this case, the congener distribution was similar to the dechlorination pattern of process M. From these results, we suggested that the enrichment of cultures with Co might stimulate the growth of specific populations of meta-dechlorinators, and that populations might promote a change in the dechlorination process from H' to M, which is known to be less effective on the dechlorination of the more highly chlorinated congeners of PCBs.

Isolation and Characterization of Bacillus Strains for Biological Control: Han-Soo Kim , Jiyong Park , Sung-Won Choi , Kee-Hyun Choi , Gung Pyo Lee , Soo Jung Ban , Chang Hoo Lee , Chung Sun Kim; J. Microbiol. 2003;41(3):196-201.

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Abstract: The object of this study was to characterize Bacillus strains GB-017 and GB-0356, which produce antifungal substances, especially for plant pathogens. In addition, this study was undertaken to characterize the culture conditions required for the production of antifungal substances and to document some of the properties of the antifungal substance produced by these soil-isolated strains. Strains GB-0365 and GB-017 were found to be bacillus-shaped, gram-positive and motile, and to inhibit Botrytis cineria, Fusarium sp., Pythium sp., and Rhizoctonia solani. Antagonistic activity was maintained up to pH 9.0, and the antifungal activity was stable to heat at 80oC for 1 h. Antifungal substances were separated and purified using ion exchange and adsorption columns including WK-10(H^+) (pH 7.0), HP-20 column (pH 3.0) and IPA (pH 3.0). and IPA. Its UV absorption spectrum showed major peaks at 231 and 259 nm, corresponding to polyene and lactone. A fast atom bombardment mass spectrum (FAB MS) showed a highest peak at 441 m/z and major peaks at 192, 205, and 370 m/z.

Cloning of the Gene for Na+/Serine-Threonine Symporter (sstT) from Haemophilus influenzae Rd and Characteristics of the Transporter: Young-Mog Kim; J. Microbiol. 2003;41(3):202-206.

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Abstract: A protein, exhibiting a high similarity to the major serine transporter of Escherichia coli, SstT, was found in Haemophilus influenzae Rd. A Na+-stimulated serine transport activity was also detected in the cells. The gene (sstT) for the Na^+/serine symporter from the chromosome of H. influenzae was cloned, and the properties of the transporter investigated. The serine transport activity was stimulated by Na^+. The uptake of Na^+ was elicited by the addition of serine or threonine into the cells, supporting the idea that these amino acids are transported by a mechanism of Na^+/substrate symport. No uptake of H^+ was elicited by the influx of serine. The serine transport via the SstT of H. influenzae was inhibited by excess threonine, which was used as another substrate. The K_m and the V_max values for the serine transport were 2.5 mM and 14 nmol/min/mg protein, respectively.

Purification and Characterization of Extracellular Temperature-Stable Serine Protease from Aeromonas hydrophila: Soo-Jin Cho , Jong-Ho Park , Seong Joo Park , Jong-Soon Lim , Eung Ho Kim , Yeon-Jae Cho , Kwang-Soo Shin; J. Microbiol. 2003;41(3):207-211.

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Abstract: Extracellular protease, from Aeromonas hydrophila Ni 39, was purified 16.7-fold to electrophoretic homogeneity with an overall yield of 19.9%, through a purification procedure of acetone precipitation, and Q Sepharose and Sephacryl S-200 chromatographies. The isoelectric point of the enzyme was 6.0 and the molecular mass, as determined by Sephacryl S-200 HR chromatography, was found to be about 102 kDa. SDS/PAGE revealed that the enzyme consisted of two subunits, with molecular masses of 65.9 kDa. Under standard assay conditions, the apparent K_m value of the enzyme toward casein was 0.32 mg/ml. About 90% of the proteolytic activity remained after heating at 60 ℃ for 30 min. The highest rate of azocasein hydrolysis for the enzyme was reached at 60℃, and the optimum pH of the enzyme was 9.0. The enzyme was inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), by about 87.9%, but not by E64, EDTA, pepstatin or 1,10-phenanthroline. The enzyme activity was inhibited slightly by Ca_2^+, Mg_2^+ and Zn_2^+ ions.

Expression and Activity of Citrus Phytoene Synthase and [beta]-Carotene Hydroxylase in Escherichia coli: In-Jung Kim , Kyong-Cheol Ko , Tae-Sik Nam , Yu-Wang Kim , Won-Il Chung , Chan-Shick Kim; J. Microbiol. 2003;41(3):212-218.

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Abstract: Citrus phytoene synthase (CitPsy) and [beta]-carotene hydroxylase (CitChx), which are involved in caroteinoid biosynthesis, are distantly related to the corresponding bacterial enzymes from the point of view of amino acid sequence similarity. We investigated these enzyme activities using Pantoea ananatis carotenoid biosynthetic genes and Escherichia coli as a host cell. The genes were cloned into two vector systems controlled by the T7 promoter. SDS-polyacrylamide gel electrophoresis showed that CitPsy and CitChx proteins are normally expressed in E. coli in both soluble and insoluble forms. In vivo complementation using the Pantoea ananatis enzymes and HPLC analysis showed that [beta]-carotene and zeaxanthin were produced in recombinant E. coli, which indicated that the citrus enzymes were functionally expressed in E. coli and assembled into a functional multi-enzyme complex with Pantoea ananatis enzymes. These observed activities well matched the results of other researchers on tomato phytoene synthase and Arabidopsis and pepper [beta]-carotene hydroxylases. Thus, our results suggest that plant carotenoid biosynthetic enzymes can generally complement the bacterial enzymes and could be a means of carotenoid production by molecular breeding and fermentation in bacterial and plant systems.

Resistance of Saccharomyces cerevisiae to Fungicide Chlorothalonil: Jae-Ho Shin , Young-Mog Kim , Jong-Woo Park , Jang-Eok Kim , In-Koo Rhee; J. Microbiol. 2003;41(3):219-223.

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Abstract: The toxicity of chlorothalonil on the growth of yeasts was investigated using several yeast strains. An alcohol tolerant yeast, Saccharomyces cerevisiae F38-1, was the most chlorothalonil-tolerant. The glutathione content and the glutathione S-transferase activity were related to the chlorothalonil-tolerant phenotype. Several thiol compounds affect the dissipation of chlorothalonil. However, there was no significant difference on the effects of chlorothalonil dissipation among the thiol compounds tested. The growth of yeast cells was arrested by chlorothalonil. It took about 13 h to dissipate 1 mg/l of chlorothalonil, and the growth was restored as the chlorothalonil content decreased. The glutathione content and glutathione S-transferase are suggested to be among the most important factors of yeast resistance to chlorothalonil.

Ultrastructure of the Cell Wall of a Null Pigmentation Mutant, npgA1, in Aspergillus nidulans: Yun Shin Chung , § , Jung-Mi Kim , Dong-Min Han , Keon-Sang Chae , Kwang-Yeop Jahng; J. Microbiol. 2003;41(3):224-231.

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Abstract: The null pigmentation mutant (npgA1) of Aspergillus nidulans was previously characterized by its production of no pigment at any stage of its life cycle, its reduction in hyphal branching, and its delay in the asexual spore development. The chemical composition of the cell wall was also altered in npgA1 mutants that became more sensitive to Novozyme 234_TM, which is possibly due to a structural defect in the cell wall. To investigate the effects of the cell wall structure on these pleiomorphic phenomena, we examined the ultrastructure of the cell wall in the npgA1 mutant (WX17). Scanning electron micrographs (SEM) showed that after being cultured for six days, the outermost layer of the conidial wall of WX17 peeled off. Although this phenotype suggested that the cell wall structure in WX17 may be modified, examination using TEM of the fine structure of cross-sectioned hyphal wall of WX17 did not show any differences from that of FGSC4. However, staining for carbohydrates of wall layers showed that the electron-translucent layer of the cell wall was missing in WX17. In addition, the outermost layer H1 of the hyphal wall was also absent in WX17. The ultrastructural observation and cytochemical analysis of cell walls suggested that the pigmentation defect in WX17 may be attributed to the lack of a layer in the cell wall.

Phylogenetic Analysis of the HIV-1 nef Gene from Korean Isolates: Dong-Hun Lee , Yeup Yoon , Chan-Hee Lee; J. Microbiol. 2003;41(3):232-238.

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Abstract: Previous phylogenetic studies on human immunodeficiency virus type 1 (HIV-1) isolated from Korean patients suggest that the major subtype of Korean isolate is subtype B. In this subtype, some of the Korean isolates seem to be clustered exclusively of foreign isolates. Presence of this so-called “Korean clade” among Korean isolates is unique but needs verification since the number of Korean isolates used in previous studies was limited. This study aimed to identify the presence of the “Korean clade” by molecular phylogenetic analysis using all the Korean nef gene sequences registered in the NCBI GenBank (N=243) together with 32 reference strains and 77 foreign isolates. Extensive analysis of the nef gene nucleotide sequences by neighbor-joining method revealed the following. Most (83.1%) of the Korean isolates belonged to subtype B, and 81.2% of subtype B were clustered together and excluded foreign isolates (bootstrap value=91.9%). Within Korean subtype B cluster, no characteristic subcluster formation was evident since the bootstrap values for the subcluster were very low. Due to limited information, the phylogenetic analysis failed to identify the epidemiological linkage among specific groups such as homosexuals and hemophiliacs within the Korean subtype B cluster. Detailed analysis and epidemiological information are needed to clarify the origin and significance of the Korean subtype B cluster.

Mutational Analysis of an Essential RNA Stem-loop Structure in a Minimal RNA Substrate Specifically Cleaved by Leishmania RNA Virus 1-4 (LRV1-4) Capsid Endoribonuclease: Youngtae Ro , Jean L. Patterson; J. Microbiol. 2003;41(3):239-247.

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Abstract: The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single sitespecific cleavage in the 5’ untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5’ 249-342) and several synthetic RNA transcripts encoding pointmutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5’ 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg2+ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg_2^+ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca2+ and Mn2+ ions, among the reagents tested, could change the mobility of the substrate RNA 5’ 249-342 on a gel similarly to that of Mg_2^+ ions, but only Ca_2^+ ions identically showed the stabilizing effect of Mg_2^+ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg_2^+ or Ca_2^+) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

Glutathione Content and the Activities of GlutathioneSynthesizing Enzymes in Fission Yeast are Modulated by Oxidative Stress: Yuk-Young Lee , Su-Jung Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2003;41(3):248-251.

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Abstract: Glutathione (GSH) is an important factor in determining tolerance against oxidative stress in living organisms. It is synthesized in two sequential reactions catalyzed by [gamma]-glutamylcysteine synthetase (GCS) and glutathione synthetase (GS) in the presence of ATP. In this work, the effects of three different oxidative stresses were examined on GSH content and GSH-related enzyme activities in the fission yeast Schizosaccharomyces pombe. GSH content in S. pombe was significantly enhanced by treatment with hydrogen peroxide, [beta]-naphthoflavone (BNF) and tert-butylhydroquinone (BHQ). Simultaneously, they greatly induced GCS and GS activity. However, they did not have any effects on glutathione reductase activity. These results suggest that GCS and GS activities in S. pombe are upregulated by oxidative stress.

Determination of Carbon Source Utilization of Bacillus and Pythium Species by Biolog?Microplate Assay: Se-Chul Chun , R.W. Schneider , Ill-Min Chung; J. Microbiol. 2003;41(3):252-258.

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Abstract: The carbon utilizations of Bacillus species and Pythium species were investigated by using a Biolog? microplate assay to determine if there are differences in the carbon utilizations of selected strains of these species. It may be possible to afford a competitive advantage to bacterial biological control agents by providing them with a substrate that they can readily use as a carbon source, for example, in a seed coating formulation. Microplates, identified as SFP, SFN and YT were used to identify spore-forming bacteria, nonspore-forming bacteria, and yeast, respectively. Bacterial and mycelial suspensions were adjusted to turbidities of 0.10 to 0.11 at 600 nm. One hundred microliters of each of the bacterial and mycelial suspension were inoculated into each well of each of the three types of microplates. L-arabinose, D-galactose, D-melezitose and D-melibiose of the 147 carbohydrates tested were found to be utilized only by bacteria, and not by Pythium species, by Biolog? microplate assay, and this was confirmed by traditional shake flask culture. Thus, it indicated that the Biolog? microplate assay could be readily used to search for specific carbon sources that could be utilized to increase the abilities of bacterial biological control agents to adapt to contrived environments.

Regulation of nsdD Expression in Aspergillus nidulans: Kap Hoon Han , Kyu-Yong Han , Min-Su Kim , Dong-Beom Lee , Jong-Hak Kim , Suhn-Kee Chae , Keon-Sang Chae , Dong-Min Han; J. Microbiol. 2003;41(3):259-261.

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Abstract: The nsdD gene has been predicted to encode a GATA type transcription factor with the type IVb zinc finger DNA binding domain functions in activating sexual development of A. nidulans. In several allelic mutants of nsdD producing truncated NsdD polypeptides lacking the C-terminal zinc finger, the transcription level of nsdD gene was greatly increased. Also in an over-expressed mutant, the transcription under its own promoter was reduced. These results suggest that the expression of nsdD is negatively autoregulated. When the nsdD gene was over-expressed, cleistothecia were formed in excess amounts even in the presence of 0.6M KCl that inhibited sexual development of the wild type. Northern blot analysis revealed that the expression of nsdD was repressed by 0.6M KCl. These results strongly suggest that the inhibition of sexual development by salts was carried out via the nsdD involved regulatory network.

Freezing Seawater for the Long-term Storage of Bacterial Cells for Microscopic Enumeration: Jung-Ho Hyun , Eun-Jin Yang; J. Microbiol. 2003;41(3):262-265.

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Abstract: Although enumerating bacterial cells is a fundamental step in understanding microbial ecosystems in marine environments, substantial decrease in bacterial counts with increasing sample storage time hampers the accurate estimation of bacterial biomass. We compared the variations in bacterial cell numbers caused by freezing and thawing of sample bottles or slides. Bacterial counts of seawater samples frozen only once in a sampling bottle yielded approximately 95% of the original numbers after 90 days, whereas 80% of the original count was obtained for samples prepared on slides. Only 67% and 58% of the original counts were recovered in samples repeatedly frozen and thawed in bottles or on slides, respectively. The results indicated that freezing a seawater sample in a bottle increased the consistency of the epifluorescence microscopic enumeration of bacterial cells.

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