• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.103-110
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• 2003

The regulation of pyrimidine nucleotide synthesis has been proved to be controlled by a regulatory protein PyrR-mediated attenuation in the Gram-positive bacteria. After several bacterial genome sequencing projects, we have discovered the PyrR orthologues in the databases for Haemophilus influenzae and Synechocystis and sp. PCC6803 genome sequences. To investigate whether these PyrR orthologue proteins regulate pyrimidine nucleotide synthesis as well as the cases of Bacillus, the PyrR regions of each strains were amplified by PCR and cloned with pUC19 or T-vector in Escherichia coli and with a shuttle vector pHPS9 for E. coli and B. subtilis. For the regulation test of the PyrR orthologues, the aspartate-transcarbamylase (ATCase) assay was carried out. From the results of the ATCase assay, it was confirmed that Synechocystis sp. PCC6803 could not restore by pyrimidines to a B. subtilis, PyrR but H. influenzae PyrR could. For Purification of PyrR orthologue proteins, PyrR orthologue genes were cloned into the expression vector (pET14b). Over-expressed product of PyrR orthologue genes was purified and analyzed by the SDS-PACE. The purified PyrR orthologue proteins from H. influenzae and Synechocystis sp. PCC6803 turned out to be molecular mass of 18 kDa and 21 kDa, respectively. The result of uracil phosphoribosyl transferase (UPRTase) assay with purified PyrR orthologue proteins showed that H. influenzae PyrR protein only has UPRTase activity. In addition, we could predict several regulatory mechanisms that PyrR orthologue proteins regulate pyrimidine de novo synthesis in bacteria, through phylogenetic analysis for PyrR orthologue protein sequences.

• Molecules and Cells
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• v.25 no.1
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• pp.138-141
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• 2008

HP0892 (SwissProt/TrEMBL ID O25552) is a 90-residue conserved hypothetical protein from Helicobacter pylori strain 26695, with a calculated pI of 9.38 and a molecular mass of 10.41 kDa. It belongs to the Plasmid stabilization system protein family (PF05016) in the Pfam database. Proteins with sequence similarity to HP0892 exist in Vibrio choierae, Enterococcus faecalis, Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli O157. Here we report the sequence-specific backbone resonance assignments of HP0892 using multidimentional heteronuclear NMR spectroscopy. About 97.0% (422/435) of the HN, N, CO, $C{\beta}$, $C{\alpha}$ resonances of 90 residues of HP0892 were assigned. On the basis of the resonance assignments, three helical regions and four strand regions were identified using the CSI program. This study is a prerequisite for calculating the solution structure of HP0892, and will be useful for studying its interaction with other molecules.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2000.11a
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• pp.83-85
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• 2000

A lot of microbial genome sequencing projects is being done in many genome centers around the world, since the first genome, Haemophilus influenzae, was sequenced in 1995. The deluge of microbial genome sequence data demands new and highly automatic data flow system in order for genome researchers to manage and analyze their own bulky sequence data from low-level to high-level. In such an aspect, we developed the automatic data management system for microbial genome projects, which consists mainly of local database, analysis programs, and user-friendly interface. We designed and implemented the local database for large-scale sequencing projects, which makes systematic and consistent data management and retrieval possible and is tightly coupled with analysis programs and web-based user interface, That is, parsing and storage of the results of analysis programs in local database is possible and user can retrieve the data in any level of data process by means of web-based graphical user interface. Contig assembly, homology search, and ORF prediction, which are essential in genome projects, make analysis programs in our system. All but Contig assembly program are open as public domain. These programs are connected with each other by means of a lot of utility programs. As a result, this system will maximize the efficiency in cost and time in genome research.

• International Journal of Oral Biology
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• v.39 no.4
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• pp.215-220
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• 2014

Toothbrushes play an essential role in oral hygiene. However, they can be significant in microbial transmission and can increase the risk of infection, since they can serve as a reservoir for microorganisms in healthy, oral-diseased and medically ill adults. This study was conducted to evaluate toothbrush contamination in six toothbrushes donated from four people. Two participants each supplied two toothbrushes - one used in the bathroom and one used in the workplace. The other two people each donated two toothbrushes used in the workplace. Polymerase chain reaction was used to construct a 16S rRNA clone library. Sequences of cloned DNA were compared with those from the reference organisms provided by GenBank. A total 120 clones, representing 20 clones for each toothbrush, were analyzed. They are composed of six pylum, 46 genera and 79 species. The most dominant species were Streptococcus oralis, Streptococcus parasanguinis and Haemophilus parainfluenzae. Enterobacter and Escherichia were recovered from toothbrushes used domestically. Toothbrushes used in the workplace did not contain Enterobacteria.

• Journal of Microbiology
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• v.43 no.2
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• pp.209-212
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• 2005

The purpose of this study was to develop species-specific PCR primers for use in the identification and detection of Actinobacillus actinomycetemcomitans. These primers target variable regions of the 168 ribosomal RNA coding gene (rDNA). We assessed the specificity of the primers against 9 A. actinomycetemcomitans strains and 11 strains (3 species) of the Haemophilus genus. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC$ 33384^$T Our obtained data revealed that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 4 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these PCR primers are incredibly sensitive, and should prove suitable for application in epidemiological studies, as well as the diagnosis and monitoring of periodontal pathogens after treatment for periodontitis.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.836-843
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• 2004

Nontypeable H. influenzae (NTHi), a Gram-negative obligate human pathogen, causes pneumonia, chronic bronchitis, and otitis media, and the respiratory epithelium is the first line of defense that copes with the pathogen. In an effort to identify transcriptional responses of human respiratory epithelial cells to infection with NTHi, we examined its differential gene expression using high density cDNA microarrays. BEAS-2B human bronchial epithelial cells were exposed to NTHi for 3 hand 24 h, and the alteration of mRNA expression was analyzed using microarrays consisting of 8,170 human cDNA clones. The results indicated that approximately 2.6% of the genes present on the microarrays increased in expression over 2-fold and 3.8% of the genes decreased during the 24-h infection period. Upregulated genes included cytokines (granulocyte-macrophage colony stimulating factor 2, granulocyte chemotactic protein 2, IL-6, IL-10, IL-8), transcription factors (Kruppel-like factor 7, CCAAT/enhancer binding protein $\beta$, E2F-1, NF-$\kappa$B, cell surface molecules (CD74, ICAM-1, ICAM-2, HLA class I), as well as those involved in signal transduction and cellular transport. Selected genes were further confirmed by reverse-transcription-PCR. These data expand our knowledge of host cellular responses during NTHi infection and should provide a molecular basis for the study of host-NTHi interaction.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.72-80
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• 2000

Lipopolysaccharides (LPS) of Pasteurella multocida (P multocida) and several Gram-negative bacterial pathogens were analyzed by methanolysis, trifluoroacetylation and gas chromatography (GC) on a fused-silica capillary column. The GC analysis indicated that LPS prepared from a strain of P multocida by phenol-water (PW) or trichloroacetic acid (TCA) extraction were quite different in chemical composition. However, LPS prepared from Salmonella enteritidis by the two extraction methods were very similar. PW-LPS extracts from different Pasteurella strains of a serotype had essentially identical GC patterns. Endotoxic LPS extracted from 16 different serotypes of P multocida by PW or by phenol-chloroform-petroleum ether procedures yielded chromatograms indicating similar composition of the fatty acid moieties but minor differences in carbohydrate content. When the chemical composition of endotoxic LPS extracted from several Gram-negative bacteria (P multocida, Pasteurella hacmolytica, Haemophilus somnus, Actinobacillus ligniersii, Brucella abortus, Treponema hyodysenteriae, Escherichia coli, Bacteriodes fragilis, Salmonella abortus equi and Salmonella enteritidis) were examined, each bacteria showed a unique GC pattern. The carbohydrate constituents in LPS of various Gram-negative bacteria were quite variable not only in the O-specific polysaccharides but also in the core polysaccharides. The LPS of closely related bacteria shared more fatty acid constituents with each other than with unrelated bacteria.

• Proceedings of the Korea Crystallographic Association Conference
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• 2003.05a
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• pp.17-17
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• 2003

tRNA (m¹ G37) methyltransferase (TrmD) catalyze s the trans for of a methyl group from S-adenosyl-L-methionine (AdoMet) to G/sup 37/ within a subset of bacterial tRNA species, which have a residue G at 36th position. The modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. We have determined the first crystal structure of TrmD from Haemophilus influenzae, as a binary complex with either AdoMet or S-adenosyl-L-homocysteine (AdoHcy), as a ternary complex with AdoHcy/phosphate, and as an apo form. The structure indicates that TrmD functions as a dimer (Figure 1). It also suggests the binding mode of G/sup 36/G/sup 37/ in the active site of TrmD and catalytic mechanism. The N-terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C-terminal domain shows a structural similarity to DNA binding domain of trp or tot repressor. We propose a plausible model for the TrmD₂-tRNA₂ complex, which provides insights into recognition of the general tRNA structure by TrmD (Figure 2).

• Korean Journal of Veterinary Service
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• v.19 no.2
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• pp.115-125
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• 1996

A total of 315 microorgainisms were isolated from 256 pneumonic lung samples of pig in abattoirs of northern Chungnam area during February to November 1994, and Identified 97 strains as Pusteurella multocida, 89 strains as Staphylococcus spp, 54 strains as Streptococcus spp, 22 strains as Mycoplasma spp, 21 strains as Escherichia coli, 18 strains as Haemophilus parsuis, 11 strains as Corymebacteroi, pyogenes, and 3 strains as Acrinobacillus spp by Gram's and Dienes stain, and biological properties test Involved API system. After that, they were examined anti biotic susceptibility for ampicillin(AM), cephalothin(CF), chloramphenicol(CP), erythromycin(EM), kanamycin(KM), gentamicin(GM), neomycin(NM), penicillin(PC), streptomycin(SM), tetracycline(TE), tiamulin(TIA), tylosin(TYL), methicillin(DP), colistin(CL) and trimet hoprim(SXT). In antibiotics susceptibility test, 293 isolates except Mycoplasma spp 22 strain were highly susceptible to DF(79.2%) and AM(76.2%), but resistant to PC(14.0%), NM(19.5%) and KM (23.2%) The multiple drug resistant patterns were noted in most isolates, whereas only 7 isolates resistant to single drugs.

• Korean Journal of Veterinary Research
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• v.44 no.3
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• pp.399-405
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• 2004

Haemophilus somnus, Mycoplasma bovis and Pasteurella multocida were responsible for respiratory diseases in bovine. Methods for identifying these bacteria had poor sensitivity and specificity. In this paper, PCR assays were applied for rapid identification of H. somnus, M. bovis, P. multocida B:2 and P. multocida capsular types. The specific PCR products were amplified from H. somnus, but not from other bacteria. Ten-fold diluted H. somnus were mixed with P. multocida and then the mixed cultures were inoculated on agar plates. After incubation, PCR was performed with harvest from agar plates and could detect as few as 3.4 CFU/ml of H. somnus. The primers MboF and MboR produced an amplification product unique to M. bovis and sensitivity of PCR was as low as 100 pg of DNA. Only serotype B:2 of P. multocida, the causal agent of haemorrhagic septicemia in bovine, was specifically amplified in PCR among the 16 reference serotypes. The multiplex capsular PCR typing for P. multocida was produced the P. multocida specific product as well as the capsular serogroup-specific product. The present PCR assays should be useful for the rapid identification of bacterial pathogens from bovine respiratory diseases.