• Journal of Life Science
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• v.15 no.3 s.70
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• pp.415-422
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• 2005

library of the mutants was prepared by transposon mutagenesis of the Mycobacterium bovis BCG. We screened this library for the resistance to an anti-tuberculosis antibiotic, PA-824. Most of the mutants resistant to the PA-824 were not able to synthesize the coenzyme $F_{420}$ which is normally produced by the wild type M. bovis BCG strains. HPLC analysis of the cellular extract showed that one of those mutants which lost the ability to synthesize $F_{420}$ still produced F0. The insertion site of the transposon in this mutant was determined by an inverse PCR and the transposon was found to be inserted in the Rv2435c open reading frame (ORF). Rv2435c ORF is predicted to encode an 80.3 kDa protein. Rv2435c protein appears to be bound to the cytoplasmic membrane, its N-terminal present in the periplasm and C-terminal in the cytoplasm. The C-terminal portion of this protein is highly homologous with the adenylyl cyclases of both prokaryotes and eukaryotes. There are 15 ORFs which have homology with the class III AC proteins in the genome of the M. tuberculosis and M. bovis. Two of those, Rv1625c and Rv2435c, are highly homologous with the mammalian ACs. We cloned the cytoplasmic domain of the Rv2435c ORF and expressed it with six histidine residues attached on its C-terminal in Escherichia coli to find out if this protein is a genuine AC. Production of that protein in E. coli was proved by purifying the histidine-tagged protein by using the Ni-NTA resin. This protein, however, failed to complement the cya mutation in E. coli, indicating that this protein lacks the AC activity. All of the further attempts to convert this protein to a functional AC by a mutagenesis with UV or hydroxylamine, or construction of several different fusion proteins with Rv1625c failed. It is, therefore, possible that Rv2435c protein might affect the conversion of F0 to $F_{420}$ not by synthesizing cAMP but by some other way.

• Microbiology and Biotechnology Letters
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• v.37 no.1
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• pp.17-23
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• 2009

The gene encoding Thermus thermophilus HJ6 DNA polymerase (Tod) was cloned and sequenced. The open reading frame (ORF) of the Tod gene was composed of 2,505 nucleotides and encoded a protein (843 amino acids) with a predicted molecular weight of 93,795 Da. The deduced amino acid sequence of Tod showed 98% and 86% identities to the Thermus thermophilus HB8 DNA pol and Thermus aquaticus DNA pol, respectively, The Tod gene was expressed under the control of the bacteriophage $\lambda$ promoters PR and PL on the expression vector pJLA503 in Escherichia coli strain BL21 (DE3) codon plus. The expressed enzyme was purified by heat treatment, $HiTrap^{TM}$ Q column, and $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 column chromatographies. The optimal temperature and pH for DNA polymerase activity were found to be $75{\sim}80^{\circ}C$ and 9.0, respectively. The optimal concentrations of $Mg^{2+}$ and $Mn^{2+}$ were 2.5 mM and 1 mM, respectively. The enzyme activity was activated by divalent cations, and was inhibited by monovalent cations. The result of the PCR experiment with Tod DNA polymerase indicates that this enzyme might be useful in DNA amplification and PCR-based applications.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.246-251
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• 2006

A lot of mutants which cannot initiate sexual development were screened and several loci including nsdA, nsdB, nsdC, and nsdD were identified in homothallic ascomycetes Aspergillus nidulans. The NSD206, which has nsdC6 allele, showed typical phenotype of NSD (Never in sexual development) mutants. The nsdC gene was cloned by transforming NSDP697 ($nsdC^-$, $pryG^-$) with AMA1-NotI genomic library. The transforming library DNA recovered from several transformants showing wild phenotype carried about 10 kb genomic DNA insert. The DNA sequence of nsdC was analysed using GPS (Genome priming system). The nsdC gene has an open reading frame (ORF) of 1,929 bp encoding a putative polypeptide of 643 amino acids. The NsdC carries $C_2H_2C_2H_2C_2HC$ type zinc finger DNA binding domains in the middle of the polypeptide. A coiled-coil domain at its C terminus were also found. In nsdC6 allele, a single T insertion was occurred between 407-408 bp leading to the frameshift mutation and early termination of translation producing the truncated protein which has only 139 amino acids.

• KSBB Journal
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• v.21 no.2
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• pp.144-150
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• 2006

The exploitation of metagenome, the access to the natural extant of enormous potential resources, is the way for elucidating the functions of organism in environmental communities, for genomic analyses of uncultured microorganism, and also for the recovery of entirely novel natural products from microbial communities. The major breakthrough in metagenomics is opened by the construction of libraries with total DNAs directly isolated from environmental samples and screening of these libraries by activity and sequence-based approaches. Screening with activity-based approach is presumed as a plausible route for finding new catabolic genes under designed conditions without any prior sequence information. The main limitation of these approaches, however, is the very low positive hits in a single round of screening because transcription, translation and appropriate folding are not always possible in E. coli, a typical surrogate host. Thus, to obtain information about these obstacles, we studied the genetic organization of individual URF's(unidentified open reading frame from metagenome sequenced and deposited in GenBank), especially on the expression factors such as codon usage, promoter region and ribosome binding site(rbs), based on DNA sequence analyses using bioinformatics tools. And then we also investigated the above-mentioned properties for 4100 ORFs(Open Reading Frames) of E. coli K-12 generally used as a host cell for the screening of noble genes from metagenome. Finally, we analyzed the differences between the properties of URFs of metagenome and ORFs of E. coli. Information derived from these comparative metagenomic analyses can provide some specific features or environmental blueprint available to screen a novel biocatalyst efficiently.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.221-229
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• 2002

Japanese encephalitis virus (JEV) persistence was established and maintained in Vero cell culture for over 1 year. Eleven clones of plaque morphology mutant JEV, with large and small plaque sizes, were obtained from the cell culture supernatant. Genomic RNA replication efficiency of the mutants in naive Vero cell appeared to correspond to their different plaque sizes. No significant changes in envelop protein ORF or in non-coding regions at both ends of the RNA genome suggested that there could be an unidentified factor(s) playing role in JEV attenuation. Unlike to the replication of wild-type JEV, the mutants did not induce severe degree of cytopathic effect in Vero cells upon infection. While obvious decrease of Bcl-2 and its mRNA expression and sharp increase of p53 in naive Vero cells infected with either wild-type JEV or the large plaque-forming mutant, those changes were not observed with the small plaque-forming one. Together with these observation, internucleosomal DNA fragmentation and chromosomal DNA profile in the Vero cells infected with the mutants suggest that an overall changes in cytopathic effect in the plaque morphology mutants-infected cells should be primarily due to the reduced genomic RNA replication and the compromised degree of p53-independent apoptosis by the virus infection at least in part.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.507-513
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• 1992

Serratia marecescens RH1 isolated from soil samples produced large amount of extracellular proteases. One of the genes encoding an extracellular protease form S. marcescens RH1 was cloned in Escherichia coli by shot gun cloning method. The cloned protease, SSP, was stably expressed by its own promoter and excreted into the extracellular medium from E. coli host (ORF) of 3.135 nucleotides corresponding to 1.045 amino acids (112 kDa). The nucleotide and deduced amino acid sequence of SSP showed high overall homology (88%) to one of the S. marcescens protease (27), but low homology to other serine protease families. The optimal pH and temperature of the enzyme were pH 9.0 and 45.deg.C respectively. The activity of protease was inhibited by phenylmethylsulfonyl fluoride (PMSF), which suggests that the enzyme is a serine protease.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.700-706
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• 2000

Iron- and zinc-containing superoxide dismutase (FeZnSOD) and nickel-containing superoxide dismutase (NiSOD) are cytoplamic enzymes in Streptomyces griseus. The sodF gene coding for FeZnSOD was cloned from genomic Southern hybridization analysis with a 0.5-kb DNA probe, which was PCR-amplified with facing primers corresponding to the N-terminal amino acid of the purified FeZnSOD of S. griseus and a C-terminal region which is conserved among bacterial FeSODs and MnSODs. The sodF open reading frame (ORF) was comprised of 213 amino acid (22,430 Da), and the deduced sequence of the protein was highly homologous (86% identity) to that of FeZnSOD of Streptomyces coelicolor. The FeZnSOD expression of exponentially growing S. griseus cell was approximately doubled as the cell growth reached the early stationary phase. The growth-associated expression of FeZnSOD was mainly controlled at the transcriptional level, and the regulation was exerted through the 110 bp regulatory DNA upstream from the ATG initiation codon of the sodF gene.

• Korean Journal of Plant Resources
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• v.18 no.3
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• pp.374-381
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• 2005

A full-length cDNA encoding ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) has been isolated and its nucleotide sequence determined from root in ginseng plant (Panax ginseng). The rbcS cDNA of ginseng is 790 nucleotides long and has an open reading frame of 549 bp with deduced amino acid of 183 residues (pI 8.37), 20.5 kDa. The deduced amino acid sequence of rbcS matched to the previously reported rbcS protein genes and showed a high similarity with the $78\%$ identity with rbcS of Helianthus annuus (CAA68490). In the phylogenetic analysis based on the amino acid residues, the ginseng rbcS was clustered with H. annuus (CAA68490), C. morifolium (AA025119) and L. sativa (Q40250).

• Journal of Life Science
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• v.27 no.11
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• pp.1276-1289
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• 2017

This study isolated and purified the antimicrobial peptide McSSP-31 from an acidified hemocyte extract of a Mytilus coruscus. The antimicrobial peptide was purified by using a $C_{18}$ reversed-phase high-performance liquid chromatography (HPLC). The peptide was determined to be 3330.549 Da by matrix assisted-laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS). The N-terminus of a 14 amino-acid sequence was identified as P-S-P-T-R-R-S-T-S-R-S-K-S-R by Edman degradation method. The acquired sequence showed a 93% similarity with the sperm-specific protein Phi-1, which is from M. californianus. The identified open-reading frame (ORF) of peptide was 306 bp encoding 101 amino acids, which was analyzed by rapid amplification of cDNA ends (RACE), cloning and sequencing analysis. We compared the full sequence with other known proteins that reveal the sperm-specific protein Phi-1 (93.5%) of M. californianus. Synthesized antimicrobial peptide (McSSP-31) showed antibacterial activity against gram-positive bacteria including B. subtilis, S. mutans, S. aureus and gram-negative bacteria including E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and fungi, C. albicans. Also, synthesized peptide showed strong antibacterial activity against antibiotic-resistant strains, including S. aureus. The cytotoxicity of the peptide was determined by using the HUVEC human cell line. The peptide did not exhibit any significant cytotoxic effects on the normal human cell line, and it had very low hemolytic activity with flounder hemoglobin. The results demonstrated that peptide purified from the hemocyte of a M. coruscus exhibits antibacterial activity against various bacteria and has the potential to be an alternative antibiotic agent.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.88-94
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• 2008

Two sugar biosynthetic cassette plasm ids were used to direct the biosynthesis of a deoxyaminosugar. The pOTBP1 plasmid containing TDP-glucose synthase (desIII), TDP-glucose-4,6-dehydratase (desIV), and glycosyltransferase (desVII/desVIII) was constructed and transformed into S. venezuelae YJ003, a strain in which the entire gene cluster of desosamine biosynthesis is deleted. The expression plasmid pOTBP3 containing 4-aminotransferase (gerB) and 3,5-epimerase (orf9) was transformed again into S. venezuelae YJ003-OTBP1 to obtain S. venezuelae YJ003-OTBP3 for the production of 4-amino-4,6-dideoxy-L-glucose derivatives. The crude extracts obtained from S. venezuelae ATCC 15439, S. venezuelae YJ003, and S. venezuelae YJ003-OTBP3 were further analyzed by TLC, bioassay, HPLC, ESI/MS, LC/MS, and MS/MS. The results of our study clearly shows that S. venezuelae YJ003-OTBP3 constructs other new hybrid macrolide derivatives including 4-amino-4,6-dideoxy-L-glycosylated YC-17 (3, [M+ $Na^+$] m/z=464.5), methymycin (4, m/z=480.5), novamethymycin (6, m/z=496.5), and pikromycin (5, m/z=536.5) from a 12-membered ring aglycon (10-deoxymethynolide, 1) and a 14-membered ring aglycon (narbonolide, 2). These results suggest a successful engineering of a deoxysugar pathway to generate novel hybrid macrolide derivatives, including deoxyaminosugar.