• BMB Reports
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• v.38 no.1
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• pp.97-103
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• 2005

Under the condition of expression of $\lambda$ P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the $\lambda$ P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the $\lambda$ P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of $\lambda$ P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.

• Animal cells and systems
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• v.2 no.3
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• pp.351-353
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• 1998

A 245 bp segment of E. coli chromosomal replication origin, oriC, contains 11 repeats of the GATC sequence in which adenine is methylated by Dam methylase. Newly replicated oriC is hemimethylated. The parental strand of the newly replicated oriC is methylated, but the nascent strand is not yet methylated until methylated by Dam methylase. The hemimethylated oriC plays an important role in the regulation of chromosomal replication. Activity in the seqA mutant was identified to bind preferentially to hemimethylated DNA, but not to fully-methylated DNA. This activity may participate in the sequestration of initiation of chromosomal replication.

• Korean Journal of Microbiology
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• v.32 no.3
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• pp.186-191
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• 1994

The origin of the leading strand replication (ori) and of lagging strand replication (M-O) of R-plasmid pSBK203 was identified and its base sequence was determined. About 50 bp of ori sequence residues overlapped with the structural gene of rep. Sequence comparison reveals that pSBK-ori shares obvious identities with those of pT181 family and consists of two regions, one is conserved and the other is variable region. Of two palindrome sequence located one after another in upstream region of rep gene, palA' instead of palA which shares sequence homology with diverse family of plasmids such as pOX6, pC194, and pE194 seems to act as a signal for conversion of primarily replicated ssDNA to dsDNA (minus origin (M-O)).

• Journal of Microbiology
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• v.33 no.3
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• pp.191-195
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• 1995

IciA protein has been shown as an inhibitor for the initiation of E. coli chromosomal DNA replication at oriC. IciA protein binds the AT-rich region in oriC and then blocks the initiation of chromosomal DNA replication. Two binding sites for IciA protein were identified in dnaA gene, encoding the initiator for the E. coli chromosomal replication, promoter region by gel-shift assay and DNase I footprinting, One, named as IciA site I, is located upstream of the dnaA promoter 1P. The other, named as IciA site II, is located downstream of the dnaA promoter 2P. The sequence comparison of the regions protected from the DNase I cleavage did not result in a clear consensus sequence for the binding of IciA protein, suggesting that IciA protein may be a member of multimeric complex dsDNA binding proteins. This study provided information about the binding mode of IciA protein. Even though the IciA site II and IciA binding site in oriC seem to be composed of two IciA binding units, one binding unit is likely enough to cause the binding of IciA protein to the IciA site I. The binding of IciA protein to the dna4 promoter implies that IciA protein may involve not only the control of the initiation of chromosomal DNA replication but also the control of the dna4 gene expression.

• Korean Journal of Microbiology
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• v.41 no.2
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• pp.140-145
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• 2005

Streptomyces natalensis ATCC27448 produces natamycin, a commercially important macrolide antifungal antibiotic. For molecular genetic study of S. natalensis, we have developed a system for introducing DNA into S. natalensis via conjugal transfer from Escherichia coli. An effective transformation procedure for S. natalensis was established based on transconjugation from E, coli ET12567/pUZ8002 using a ${\Phi}C31$-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 10 mM $MgCl_2$ using $6.25\times10^8$ of E.coli donor cells without heat treatment of spores. In addition, southern blot analysis of exconjugants and the sequence of plasmids containing DNA flanking the insertion sites from the chromosome revealed that S. natalensis contains a single ${\Phi}C31$ attB site and at least a secondary or pseudo attB site. Similar to the case of various Streptomyces species, a single ${\Phi}C31$ attB site of S. natalensis is present within an ORF encoding a pirin-homolog, but a pseudo-attB site is present within a distinct site (GenBank accession no. $YP\_117731$) and also its sequence deviates from the consensus sequences of attB sequence.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.535-538
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• 2000

Streptomyces lavendulae FRI-5 produces the ${\gamma}$-butyrolactone autoregulator IM-2, which is required for nucleoside antibiotic producetion. We have developed a system for introducing DNA into S. lavendule FRI-5 via conjugal transfer from Esherichia cole. Conditions were established for conjugation of the oriT-and attP-containing plasmid pSET152 from E. coli ET12567 (pUZ8002) to FRI-5. Conjugation resulted in integration of the plasmid at the chromosomal C31 attB site. The frequency of intergeneric conjugation varied with the medium used. The highest frequency ($1.6\times10-5$ per recipient) was obtained on ISP medium 2 containing 10mM MgCl2. Southern blot and phenotypic analyses of exconjugants revealed that S. lavendulae FRI-5 contains a unique C31 attB site, and that integration of heterologous DNA into the attB site did not interfere with morphological differentiation or IM-2-dependent signal transduction, including the production of a blue pigment. This system will now enable detailed genetic analysis of the regulation of antibiotic production in S. lavendulae FRI-5.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.516.2-516
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• 1986

DNA subfragments, sopA, sopB, and sopC supporting stable maintenance of an oriC plasmid, were derived from mini-F plasmid DNA (EcoRI restriction fragment, f5) after digestion with restriction endonucleases, and cloned in vector plasmid pBR322. The recombinant plasmid obtained were introduced into E. coli KY7231 and E. coli CSR603, and proteins specified by the mini-F fragments were analysed by SDS-polyacrylamide gel electrophoresis. Two proteins encoded by the F fragments were detected, having molecular weights of 41,000 and 37.000. The sopA protein (41K) encoded by a plasmid pXX288 was observed in the cytoplasm, whereas the sopB protein (37K) encoded by a plasmid pXX157 was in the membrane fraction. There was no novel protein band detected in the cell with a plasmid pXX300, which contained sopC fragment. Gene products of a plasmid pXX167, which is comprised of sopA, sopB, and sopC, were not detectable. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins were overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of the sopA and sepB proteins were 6.6 and 7.0, respectively.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.256-260
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• 1993

DNA subfragments, sopA, sopB and sopC which help to maintain the stability of an ori C plasmid, were derived from a mini-F plasmid DNA (EcoRI restriction fragment f5) after digestion with restriction endonuclease, and cloned in the vector plasmid pBR322. The recombinant plasmids obtained were introduced into E. coli KY7231 and E. coli CSR603 strains, and proteins specified by the mini-F fragments were analysed by SDS-PAGE. Two proteins encoded by the F fragments were detected, and their molecular weights were 41,000 and 37,000 daltons. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins had been overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of sopA and sopB proteins were 6.6 and 7.0, respectively.

• KSBB Journal
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• v.25 no.2
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• pp.167-172
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• 2010

Streptomyces scabiei producing phytotoxin called thaxtomin, which cause scab disease on economically important crops such as potato. For molecular genetics study of S. scabiei an effective transformation method was established based on conjugal transfer from Escherichia coli ET12567 (pUZ8002) using a phiC31-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 50 mM $MgCl_2$. In addition, the sequence and location of the chromosomal integration attB site of S. scabiei was identified for the first time in the strains producing thaxtomin by the southern blot analysis of exconjugants and the sequencing of plasmid containing DNA flanking the insertion sites from exconjugant chromosome. Similar to the case of Streptomyces species, a single phiC31 attB site of S. scabiei is present within an ORF encoding a pirin-homolog.

• Journal of Microbiology and Biotechnology
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• v.13 no.3
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• pp.457-462
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• 2003

The complete nucleotide sequence of a plasmid, pMG1, isolated from Bifidobacterium longum MG1 has been determined. This plasmid, composed of 3,862 base pairs with 65.1% of G+C content. harbors two major open reading frames (ORF) encoding putative proteins of 29 kDa (ORF I) and 71 kDa (ORF II). ORF I showed relatively high amino acid sequence homology with replication proteins of other plasmids from Gr Im-positive and -negative bacteria. Upstream of ORF I, four sets of tandem repeat sequences resembling the iteron structure of related plasmids were found. S1 endonuclease treatment and Southern blot analysis revealed that pMG1 accumulates single-stranded DNA (ssDNA) intermediate, which indicate i the rolling circle replication (RCR) mechanism of this plasmid. Homology search indicated that ORF II encodes plasmid mobilization protein, and the presence of highly conserved oriT sequence in the upstream of this gene supported this assumption. RT-PCR showed that only ORF I is expressed in vivo. Based on these results, pMG 1 was exploited to construct a shuttle vector, pBES2. It was successfully transformed into Bifidobacterium and maintained stably.