• Microbiology and Biotechnology Letters
• /
• v.39 no.4
• /
• pp.337-344
• /
• 2011

Integrase MJ1 from the bacteriophage ${\Phi}FC1$ carries out recombination between two DNA sequences (the phage attachment site, attP and the bacterial attachment site, attB) in NIH3T3 mouse cells. In this study, the integration vector containing attP, attB and the integrase gene MJ, was constructed. The integration mediated by integrase MJ1 in Escherichia coli led to excision of LacZ. Therefore, the frequency of integration was measured by the counting of the white colony, which is detectable on X-Gal plates. The extrachromosomal integration in NIH3T3 mouse cells was monitored by the expression of the green fluorescent protein (GFP) as a reporter. To demonstrate integration mediated integrase MJ1 in NIH3T3 cells, vectors containing attP and attB were co-transfected into NIH3T3 cells. The integration was confirmed by fluorescent microscopy. The expression of GFP was induced in NIH3T3 cells expressing MJ1 without accessory factors. By contrast, the excision mediated by the MJ1 between attR and attL had no effect on the expression of GFP. These results suggest that integrase MJ1 may enable a variety of genomic modifications for research and therapeutic purposes in higher living cells.

• Korean Journal of Microbiology
• /
• v.41 no.2
• /
• pp.140-145
• /
• 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
• /
• v.17 no.2
• /
• pp.342-347
• /
• 2007

Bacteriophage ${\Phi}FC1$ integrase (MJ1) was previously shown to perform a site-specific recombination between a phage attachment site (attP) and a host attachment site (attB) in its host, Enterococcus faecalis, and also in a non-host bacterium, Escherichia coli. Here, we investigated biochemical features of MJ1 integrase. First, MJ1 integrase could perform in vitro recombination between attP and attB in the absence of additional factors. Second, MJ1 integrase interacted with att sites. Electrophoretic mobility shift assays and DNase I footprinting revealed that MJ1 integrase could efficiently bind to all the att sites and that MJ1 integrase recognized relatively short sequences (${\sim}50bp$) containing an overlapping region within attB and attP. These results demonstrate that MJ1 integrase indeed catalyzes an integrative recombination between attP and attB, the mechanism of which might be simple and unidirectional, as found in serine integrases.

• Journal of Life Science
• /
• v.20 no.1
• /
• pp.97-102
• /
• 2010

Streptomyces platensis YK-2, newly isolated from forest soil, produces transglutaminase (TGase), which catalyses an acyl transfer reaction between the primary grade amine and protein or $\gamma$-carboxyamide group of peptide bound glutamine residues. For a molecular genetic study of S. platensis, an effective transformation method was established by using a conjugal transfer of DNA from Escherichia coli to spores of actinomycetes. The highest transconjugation frequency of S. platensis was obtained on an MS medium containing 50 mM $MgCl_2$, using $5{\times}10^7\;E$. coli as a DNA donor and $1{\times}10^8$ spores without heat treatment as a host. We also identified that S. platensis contains a single attB site within an ORF encoding a pirin-homolog, and that its attB site sequence shows high homology to that of S. logisporoflavus. In addition, it was confirmed by phenotypic analyses of exconjugants that the introduction of heterologous DNA into the attB site of the S. platensis chromosome does not affect its morphological differentiation and TGase production.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.5
• /
• pp.783-788
• /
• 2003

A 27 bp $tRNA^{Gly}$ region (att1) was identified as the integration site for a 12,384 bp Pfl-derived genomic island containing 15 open reading frames (ORFs) from PA0715 to PA0729 in P. aeruginosa strain PAOl. Homologous island was observed in P. aeruginosa strain PA14, but not in P. aeruginosa strain K (PAK). We isolated the Pfl island from PA14, and determined its 10,657 bp sequences containing 14 ORFs, with significant sequence variations near the borders. In contrast to the PAO1 Pfl island, the PA14 Pfl island was integrated at the 10 bp att2 site between PA1191 and PA1192. The attl site of PA14, however, was still occupied by a third genetic segment, whereas both attl and att2 sites of PAK remained unutilized. These results exemplify an extensive genomic variation of Pfl-related islands involving differential genetic organizations and differential att site utilizations.

• KSBB Journal
• /
• v.25 no.2
• /
• pp.167-172
• /
• 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
• /
• v.5 no.3
• /
• pp.132-137
• /
• 1995

The lambda integrase (lnt) is believed to bind to several arm and core sites of attP DNA in order to facilitate intasome formation. We have done systematic mutagenic analysis on all 5 arm sites and found that P1 is absolutely required for integration while P2 is not. We also found that all 3 P' arm sites(P'1, P'2, and P'3) are required for efficient integrative recombination. P'1, which is an important binding site for excision, also seems to be crucial for integration when preincubation of attP DNA with Int and IHF is performed before recombination. Preincubation assay revealed that preincubation with Int and IHF improved the efficiency of recombination of wild type attP DNA and demolished recombinations of P'1 mutant attP DNAs.

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

• BMB Reports
• /
• v.29 no.5
• /
• pp.448-454
• /
• 1996

Bacteriophage ${\phi}FC1$ is a temperate phage which was identified as a prophage in the Enterococcus faecalis KBL703 chromosome. Phage ${\phi}FC1$ integrates into the host chromosome by site-specific recombination. The phage attachment site P (attP) was localized within the 0.65-kb XhoI-HindIII fragment and the nucleotide sequence of the region was determined. An open reading frame (mj1) which adjoined the phage attachment site encoded a deduced protein related to the site-specific recombinase family. The organization of this region was comparable to other site-specific recombination systems. The molecular weight of the expressed MJ1 in E. coli was in good agreement with the predicted 53,537 Da of the mj1 gene product. Elucidation of the phage-specific integration process in this study would provide useful genetic tools such as a chromosomal integration system.

• Genomics & Informatics
• /
• v.12 no.3
• /
• pp.80-86
• /
• 2014

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as $180^{\circ}$ and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.