Journal of Microbiology

Volume 34(4); December 1996

Phylogenetic analysis of trichaptum based on the RFLP of PCR amplified DNAs: Ko, Kwan Soo , Jung, Hack Sung; J. Microbiol. 1996;34(4):295-299.

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Abstract: To infer phylogenetic relationships between species of Trichaptum (Polyporaceae), RFLP analyses of PCR-amplified DNAs were accomplished. Regions coding for ITSs of nuclear SSU rRNA genes and for mitochondrial SSU rRNA genes from thirteen strains of four Trichaptum species (T. abietinum, T. biforme, T. fusco-violaceum, and T. laricinum) were amplified and digested with eight restriction enzymes. All the fragmentation patterns were characterized and coded as 0/1 for the absence/presence of fragments. A phylogenetic tree based on the combined data sets was constructed using the Dollo parsimony method. While every two strains of T. abietinum, T. biforme, T. fusco-violaceum, and T. laricinum formed an independent group, the other strains of T. abietimum and T. fusco-violaceum made mixed groupings among compared strains. It is inferred that T. abietinum and T. fusco-violaceum have more variations, possibly geographic or physiological ones, than other species in the genus.

Image analysis of bacterial cell size by diurnal changes in lake Soyang, Korea: Choi, Seung Ik , Ahn, Tae Seok , Kato, Kenji; J. Microbiol. 1996;34(4):300-304.

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Abstract: To define the effects of zooplankton and phytoplankton to bacteria, bacterial numbers, frequency of dividing cells (FDC) and size distribution were performed with image analysis in the surface layer of Lake Soyang. In August 1992, when Anabaena was blooming, the bacterial number increased at daytime. Bacterial numbers and FDC value had a negative correlation (r = 0.83, P < 0.01). Bacterial size spectrums were dynamically changed during the day and night, especially the small bacteria less than 0.5 ㎛^3. Meanwhile, in October, after the bloom, the bacterial number was only one third of that in August, even though the FDC was higher than that in August. The bacterial numbers of small size class dropped at 13.00. But the size spectrums were relatively constant during the night time. These results suggest that the bacterial growth was tightly coupled with phytoplankton during Anabaena bloom. And after the bloom, the bacterial number was controlled grazing activity of zooplankton at daytime.

Isolation and characterization of Bacillus sp. KD1014 producing carboxymethyl-cellulase: Lee, Kyung Dong , Kim, Jung Ho , Kim Hoon; J. Microbiol. 1996;34(4):305-310.

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Abstract: A microorganism producing carboxymethyl-cellulase (CMCase) was isoalted from 300 soil and compost samples. The isolated was identified as Bacillus sp. by Biolog^TM test and fatty acid analysis, and named as Bacillus sp. KD1014. The isolate could degrade, in addition to CMC, various kinds of polysaccharides such as levan, xylan, starch, and filter paper but hardly degrade microcrystaline Avicel. The optimum growth and CMCase production of the isolate was observed between 16-and 25 hr-culture at 45℃ and pH 5.0. The maximum CMCase activity was observed at pH 4.5 and 60℃. The CMCase was found to bind to Avicel. The CMCase was internally cleaved as growth continued. When crude supernatant was used for activity staining, three major bands were detected on a native gel, however, only on major band was detected on a denaturating gel after removal of the detergent.

Identification of a cellular protein interacting with murine retrovirus Gag polyproteins: Choi , Won Ja; J. Microbiol. 1996;34(4):311-315.

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Abstract: The retroviral Gag polyprotein directs the assembly of virion particles and plays an important role in some events after entry into a host cell. The Gag polyprotein of a virus mixture is responsible for inducing murine acquired immunodeficiency syndrome (MAIDS) when injected into susceptible strains of mice. In order to identify the host cellular proteins which interact with the MAIDS virus Gag proteins and possibly mediate the function of the Gag proteins, mouse T-cell leukemic cDNA expression library was screened using the yeast GAL4 two hybrid system. Of 11 individual positive clones, the clone Y1 was selected for the study of protein-protein interaction. Its DNA sequence revealed that it was an exact match to the murine SH3 domain-containing protein SH3P8. It is expressed as 2.4 kbp transcripts in testis at higher levels and in various tissues tested at lower levels. Glutathione S-transferase-Y1 fusion protein binds tightly to Pr60^def-gag as well as Pr 65^eco-gag.

Effect of initial pH and L-arginine on the composition of fatty acids of streptomyces viridochromogenes: Oh, Choong Hun , Jung, Sang Oun , Pyee, Jae Ho , Kim, Jae Heon; J. Microbiol. 1996;34(4):316-319.

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Abstract: Mycelia of Streptomyces viridochromogenes grown under different pH were analysed for the fatty acid composition. The low relative proportion of 12-methyltetradecanoic acid and the high relative proportion of palmitic acid were characteristic for the young culture under slight acidic pH that caused delay of the aerial mycelium formation. The addition of L-arginine to the culture medium enabled an arginine auxotroph with bald phenotype to have the fatty acid composition similar to that of the wild type and to develop aerial mycelium. The ratio of 12-methyltetradecanoic acid to palmitic acid might be used as a parameter to explain the optimum growth in the respect of membrane fluidity.

Fate of genetically engineered 2,4-D-Degrading microorganisms in natural soils and waters: Hong, Seok Myeong , Lee, Yin Won , Kim, Chi Kyung , Ka, Jong Ok; J. Microbiol. 1996;34(4):320-326.

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Abstract: To analyze the effects of host versus plasmid on survival of 2,4-degrading bacteria in environmental samples, strains Pseudomonas cepacia/pJP4, Alcaligenes JMP228/pJP4, P. cepacia/p712, and Alcaligenes JMP228/p712 were separately inoculated into samples of field soil, paddy soil, lake water, and river water, and then the changes of their populations were measured. The strains used contained a 2,4-D degradative plasmid, either pJP4 conferring fast-growing property to the host or p712 conferring slow-growing property, and were resistant to antibiotics such that the inoculated strains could be enumerated against the indigenous microbial populations. In sterile environmental samples, these strains were stably maintained at the levels used for inoculation, except in sterile paddy soil where Alcaligenes JMP228 strains died drapidly. In natural soil samples for four strains declined steadily with time, but in natural water samples their populations fell rapidly at the early phase and then remained almost constant. When the environmental samples were treated with 2, 4-D, P. cepacia/pJP4 and P. cepacia/p712 maintained significant numbers, while Alcaligenes JMP228/pJP4 and Alcaligenes JMP228/p712 declined significantly in most of the samples. The results indicated that the survivability of genetically modified microorganisms could vary depending on the environments and that their abundance in the environments under 2,4-D selection was markedly influenced by the nature of the 2,4-D degradative plasmid as well as type of the host strain.

Identification and characterization of pH-regulated genes in saccharomyces cerevisiae: Hong, Sung Ki , Choi, Eui Yul; J. Microbiol. 1996;34(4):327-333.

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Abstract: Yeast, like many other microbes, encounters large variations in ambient pH in their natural environments. Microorganisms capable of growing over a wide pH range require a versatile, efficient pH homeostatic mechanism protecting intracellular processes against extremes of pH. In several organisms, fusions to the bacterial lacZ gene have been extremely useful for the identification of genes expressed at different time during the life cycle or under different growth conditions. In this study, using the lacZ gene screening system, we surveyed a large number of yeast strains with lacZ insertion to identify genes regulated by pH. A yeast genomic library was constructed and inserted with lacZ by a shuttle mutagenesis procedure. The yeast transformants were individually picked up with a toothpick, replica-plated, and grown in alkaline pH medium. Among the 35,000 colonies screened, 10 candidate strains were identified initially by the β-gal assay. We finally confirmed two yeast strains carrying the genes whose expression are strictly dependent on pH of growth medium. One of the fusions showing a 10-fodl induction in expression level in response to alkali pH was selected and further characterized. The pH-regulated gene was cloned by inverse PCR and a partial sequence of the gene was determined. Identification and characterization of the gene is currently under investigation.

Cloning and expression of pseudomonas cepacia catB gene in pseudomonas putida: Song, Seung Yeon , Jung, Young Hee , Lee, Myeong Sok , Lee, Ki Sung , Kim, Young Soo , Kim, Chi Kyung , Choi, Sang Ho , Min, Kyung Hee; J. Microbiol. 1996;34(4):334-340.

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Abstract: The enzyme, cis,cis-muconate lactonizing enzyme has been proposed to play a key role in the β-ketoadipate pathway of benzoate degradation. A 3.2-kb EcoRI fragment termed as pRSU2, isolated from a Pseudomonas cepacia genomic library was able to complement the catB defective mutant. Several relevant restriction enzyme sites were determined within the cloned fragment. In Pseudomonas putida SUC2 carrying pRSU2, the enzyme activity was relatively higher than those of the induced or partially induced state of wild type P. putida PRS2000. It was probably due to higher expression of P. cepacia catB in P. putida PRS2000. It was probably due to higher expression of P. cepacia catB in P. putida. One possible interpretation of these results is that the catB promoter in P. cepacia is recognized within P. putida, resulting in the almost same expression level.

Construction of genetically engineered miroorganisms for overexpression of xylE gene encoding catechol 2, 3-dioxygenase and the functional stability of the recombinant plasmid pSW3a containing xylE in aquatic environment: Han, Hyo Yung , Kim, Chi Kyung , Park, yong Keun , Ka, Jong Ok , Lee, Byeong Jae , Min, Kyung Hee; J. Microbiol. 1996;34(4):341-348.

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Abstract: The regulation of xylE gene expression was examined by using vector promoter and construction of genetically engineered microorganisms (GEMs) for application in microcosm. When the xylE gene wsa subcloned into pBluscript SK(+) under the control of lac promoter (pTY1) in E. coli, and the expression was induced by IPTG, the enzyme activity of catechol 2, 3-dioxygenase was increased 4.7 times more than that of the crude extracts from transformants harboring pTY1. We suggest that the xylE gene has its own promoter at the upstream portion, because it was able to be expressed even in the absence of IPTG. A recombinant plasmid, pSW3a harboring the xylE gene under the T7 promotor, showed the activity of 14.5 units/mg protein, higher than that of parental strain, E. coli PYT1. The xylE gene in recombinant plasmid pSW3a was used as reporter gene for the application in microcosm ecosystem, since it was used for detection of xylE-positive clones by catechol spray on the agar plates. The pSW3a in E. coli was introduced into Pseudomonas putida to construct GEM strain, and examined for the expression and functional stability in microcosms.

Cloning and expression in E. coli of the HOPDA hydrolase gene from pseudomonas sp. P 20: Lim, Jong Chul , Chae, Jong Chan , Kim, Hyong Bai , Kim, Chi Kyung; J. Microbiol. 1996;34(4):349-354.

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Abstract: Pseudomonas sp. P20 is a natural isolate which is capable of degrading biphenyl and 4-chlorobiphenyl. From a clone of pCK1022 harboring pcbCD genes of Pseudomonas sp P20, a pcbD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA) hydrolase was subcloned in Escherichia coli XL-1-Blue by using pBluescript SK(+) vector. The 2.8-kb HindII fragment harboring the pcbD gene cloned in pCK 1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 carrying pCK0124 degraded HOPDA to benzoate and 2-hydroxypenta-2, 4-dienoate by HOPDA hydrolase encoded by pcbD gene as effectively as E coli CK 1022 HARBORING pcbCD genes.

Molecular cloning of the arginine biosynthetic genes from corynebacterium glutamicum: Chun, Jae Yeon , Jung, Sam Il , Ko, Soon Young , Park, Mee Young , Kim, Soo Young , Lee, Heung Shickc , Cheon, Choong Ill , Min, Kyung Hee , Lee, Myeong Sok; J. Microbiol. 1996;34(4):355-362.

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Abstract: Complementation cloning of the argC, E, B D, F, and G genes in Corynebacterium glutamicum was done by transforming the genomic DNA library into the corresponding arginine auxotrophs fo Escherichia coli. Recombinant plasmids containing 6.7 kb and 4.8kb fragments complementing the E. coli arg B mutant were also able to complement the E. coli argC, E, A, D, and F mutants, indicating the clustered organization of the arginine biosynthetic genes within the cloned DNA fragments. The insert DNA fragments in the recombinant plasmids, named pRB1 AND pRB2, were physically mapped with several restriction enzymes. By further subcloning the entire DNA fragment containing the functions and by complementation analysis, we located the arg genes in the order of ACEBDF on the restriction map. We also determined the DNA nucleotide sequence of the fragment and report here the sequence of the argB gene. When compared to that with the nutant strain, higherh enzyme activity of N-acetylglutamate kinase was detacted in the extract of the mutant carrying the plasmid containing the putative argB gene, indicating that the plasmid contains a functional argB gene. Deduced amino acid sequence of the argB gene shows45%, 38%, and 25% identity to that from Bacillus strearothermophilus, Bacillus substilus, and E. coli respectively. Our long term goal is genetically engineering C. glutamicum which produces more arginine than a wild type strain does.

Emulsification of crude oil by acinetobacter sp. SH-14: Son, Hong Joo , Go, Sun Hee , Lee, Geon , Lee, Sang Joon; J. Microbiol. 1996;34(4):363-369.

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Abstract: As basic study to evaluate the treatability of oil-contaminated environment with bacteria, isolation and characterization of crude oil-degrading bacterium were carried out. A bacterial strain SH-14 capable of degrading crude oil was isolated from contaminated soils by enrichment culture technique and identified as Acinetobacter sp. by morphological, cultural and biochemical characteristics, and so named Acinetobacter sp. SH-14. The optimal medium composition and cultural conditions for the growth and emulsification of crude oil by Acinetobacter sp. SH-14 used were crude oil of 2.0%, KNO of 0.2%, K₂HPO₄of 0.05%, and MgSO₄· 7H₂O of 1.0%, along with initial pH 7.0at 30℃. Acinetobacter sp. SH-14 showed to be resistant to chloramphenicol and utilized various hydrocarbons such as dodecane, hexadecane, isooctane, cyclo-hexane etc., as a sole carbon source. Acinetobacter sp. SH-14 harbored a single plasmid. By agarose gel electrophoresis and curing experiment it was found that the genes for crude oil components degradation were encoded on the plasmid.

Characterization of bacillus thuringiensis isolates form soil in wonju area: Yoo, Kwan Hee , Kim, Soo Young , Kang, Min Ho , Cho, Myung Hwan , Lee, Hyung Hoan; J. Microbiol. 1996;34(4):370-373.

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Abstract: There strains (KW-1, KW-14, KW-15) of Bacillus thuringiensis were isolated from soil in Wonju area and characterized. The three strains produced parasporal inclusion bodies (crystals) and spores in their cells. The KW-1 strain produces spherical crystals. The crystals of strain KW-14 are bipyramidal crystal. The KW-15 strain harbors irregular crystals. Only minor biochemical characteristics of the three isolates were different and distinctive, however general characteristics were similar to the known serotypes of B. thuringiensis. Three strains were resistant to penicilin G, oxacillin and cephalothin. This strains were highly toxic to Bombyx mori larvae, but not to the Culex pipiens larvae.

Stage-specific change and regulation of endogenous protein phosphorylation in allomyces macrogynus: Park, Young Shik , Oh, Keun Hee , Lee, Soo Woong , Seong, Chang Soo , Park, I Ha , Yim, Jeong Bin; J. Microbiol. 1996;34(4):374-378.

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Abstract: In the aquatic fungus Allomyces macrogynus the effects of Ca^2+ and cAMP on the intracellular signal transduction of zeoospore germination were studied using in vitro protein phosphorylation assay system. An endogenuously phosphorylated protein (p50) having molecular weight of 50 kDa on SDS-PAGE was found in soluble fractions of both zeoospore and mycelium. In zoospore extract, the endogenous phophorylation of p50 was weak without any effectors, but was enhanced by Ca^2+ and even more by cAMP. Phosphorylation of the same protein in mycelial extract was high only in the absence of cAMP. Irrespective of the presence of Ca^2+ and cAMP, its phosphorylation was antagonistically suppressed in assay of combined zoospore and mycelial extracts. These results suggest that p50 is interconvertible in phosphorylation/dephosphorylation as a novel protein involved in germination of A. macrogynus. The antagonistic effect of cAMP to the phosphorylation of p50s from different developmental stages may be important in the regulation of cellular differentiation.

Production of a monoclonal antibody and ultrastructure of the sporozoite of cryptosporidium parvum: Choi, Young Sook , Lee, Sung Tae , Cho, Myung Hwan; J. Microbiol. 1996;34(4):379-383.

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Abstract: Cryptosporidium Parvum causes a life-threatening diarrhea in acquired immunodeficiency syndrome (AIDS) patients. The sporozoite stage of C. parvum has been known to be a target in treating cryptosporidiosis in AIDS patients as it is an extracellular stage. A sporozoite was ultrastructurally observed. It has a crescent shape with a rounded posterior end and a tapering body. The compact nucleus was located at the posterior end. A monoclonal antibody was produced, which recognized a 43 kDa of sporozoite antigens in a western blot analysis and showed the surface labeling in immunofluorescence.

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