Browse
Submit Data
Databases
API
Help

Dataset Information

42 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors.

ABSTRACT: Despite extensive similarities between the genomes of the Streptomyces temperate phages phiC31 and phiBT1, the attP-int loci are poorly conserved. Here we demonstrate that phiBT1 integrates into a different attachment site than phiC31. phiBT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein. Integration vectors based on phiBT1 integrase were shown to have a broad host range and are fully compatible with those based on the phiC31 attP-int locus.

SUBMITTER: Gregory MA

PROVIDER: S-EPMC180994 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors.

Gregory Matthew A MA Till Rob R Smith Margaret C M MC

Journal of bacteriology 20030901 17

Despite extensive similarities between the genomes of the Streptomyces temperate phages phiC31 and phiBT1, the attP-int loci are poorly conserved. Here we demonstrate that phiBT1 integrates into a different attachment site than phiC31. phiBT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein. Integration vectors based on phiBT1 integrase were shown to have a broad host range and are fully compatible with those based on the phiC31 attP-int locus. ...[more]

PMID: 12923110

Similar Datasets

Construction, Characterization, and Use of Two Listeria monocytogenes Site-Specific Phage Integration Vectors

Project description: Not available

| S-EPMC142897 | biostudies-literature

Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors.

Project description:Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L. monocytogenes, forms stable, single-copy integrants at a frequency of approximately 10(-4) per donor cell, and can be used in the L. monocytogenes 1/2 and 4b serogroups. Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed. pPL1 was used to introduce the hly and actA genes at comK-attBB' in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on the L. monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3' end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNA(Arg) gene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.

| S-EPMC135211 | biostudies-literature

Control of directionality in Streptomyces phage ?BT1 integrase-mediated site-specific recombination.

Project description:Streptomyces phage ?BT1 integrates its genome into the attB site of the host chromosome with the attP site to generate attL and attR. The ?BT1 integrase belongs to the large serine recombinase subfamily which directly binds to target sites to initiate double strand breakage and exchange. A recombination directionality factor (RDF) is commonly required for switching integration to excision. Here we report the characterization of the RDF protein for ?BT1 recombination. The RDF, is a phage-encoded gp3 gene product (28 KDa), which allows efficient active excision between attL and attR, and inhibits integration between attB and attP; Gp3 can also catalyze topological relaxation with the integrase of supercoiled plasmids containing a single excision site. Further study showed that Gp3 could form a dimer and interact with the integrase whether it bound to the substrate or not. The synapse formation of attL or attR alone with integrase and Gp3 showed that synapsis did not discriminate between the two sites, indicating that complementarity of central dinucleotides is the sole determinant of outcome in correct excision synapses. Furthermore, both in vitro and in vivo evidence support that the RDFs of ?BT1 and ?C31 were fully exchangeable, despite the low amino acid sequence identity of the two integrases.

| S-EPMC3836970 | biostudies-literature

Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis.

Project description:BACKGROUND:Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system. RESULTS:The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA. CONCLUSION:The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

| S-EPMC6882331 | biostudies-literature

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase.

Project description:We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

| S-EPMC87055 | biostudies-literature

Highly efficient in vitro site-specific recombination system based on streptomyces phage phiBT1 integrase.

Project description:The Streptomyces phage phiBT1 encodes a site-specific integrase of the large serine recombinase subfamily. In this report, the enzymatic activity of the phiBT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra- and intermolecular recombinations proceed with rapid kinetics. The recombination is highly specific, and no reactions are observed between pairs of sites including attB and attL, attB and attR, attP and attL, or attP and attR or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the phiBT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro.

| S-EPMC2565996 | biostudies-literature

Generation of induced pluripotent stem cells using site-specific integration with phage integrase.

Project description:To date, a large number of reports have described reprogramming many somatic cell types into induced pluripotent stem (iPS) cells, using different numbers of transcription factors and devising alternate methods of introducing the transcription factor genes or proteins into the somatic cells. Here, we describe a method using bacteriophage ?C31 integrase to reprogram mouse embryonic fibroblasts and human amniotic fluid cells into iPS cells. These iPS cells showed morphology, surface antigens, gene expression, and epigenetic states similar to ES cells and formed teratomas with three germ layers in nonobese diabetic/severely compromised immunodeficient mice. Importantly, these iPS cells have only a single integration site in each cell line. The locations of integration favor the intergenic regions, and their distances from the adjacent genes extended from several hundred to >1 million bp. The effect of the insertion on the expression of these genes can be studied by RT-PCR. No insertion into microRNA gene loci was detected. Hence, it is possible to select cells in which adjacent gene functions are not affected, or the inserts can be removed if necessary. We conclude that phage integrase-mediated site-specific recombination can produce iPS cells that have undisturbed endogenous gene function and could be safe for future human therapeutic application.

| S-EPMC2984165 | biostudies-literature

Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration.

Project description:The temperate lactococcal phage TP901-1, induced by UV light from Lactococcus lactis subsp. cremoris 901-1, was characterized. The restriction map was found to be circular, and the packaging of TP901-1 DNA was concluded to occur by a headful mechanism. The pac region was localized on the 38.4-kb phage genome. TP901-1 belongs to the class of P335 phages (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989). Evidence is presented that the phages TP936-1 (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989) and C3-T1 (A. W. Jarvis, V. R. Parker, and M. B. Bianchin, Can. J. Microbiol. 38:398-404, 1992) are very closely related to or are identical to TP901-1. The lytically propagated TP901-1 phages were able to lysogenize both indicator strains Lactococcus cremoris 3107 and Wg2. Lysogenization resulted in site-specific integration of the phage genome into the bacterial chromosome. Only one chromosomal attB site was found in 20 independent lysogens. The attP region of TP901-1 and the attL and attR regions were cloned and sequenced. The results showed a core region of only 5 bp, in which the recombination occurs, followed after a 1-bp mismatch by a 7-bp identical region, TCAAT(T/C)AAGGTAA. This result was further verified by sequencing of the attB region obtained by PCR. An integration vector was constructed with the 6.5-kb EcoRI fragment from TP901-1 containing attP. This vector also functions in the plasmid-free strains, MG1363 and LM0230 with only one specific attB site, strongly indicating a more general use of the TP901-1-based integration vector in lactococci.

| S-EPMC205158 | biostudies-other

Site-specific recombination of temperate Myxococcus xanthus phage Mx8: genetic elements required for integration.

Project description:Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination. The Mx8 int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3' ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M. xanthus genome. Although Int-mediated site-specific recombination occurs between attP and either attB1 (within trnD1) or attB2 (within trnD2), the attP x attB1 reaction is highly favored and often is accompanied by a deletion between attB1 and attB2. The int gene is the only Mx8 gene required in trans for attP x attB recombination. The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate attP x attB recombination efficiently. The attP core lies within the int coding sequence, and the product of integration is a prophage in which the 3' end of int is replaced by host sequences. The prophage intX gene is predicted to encode an integrase with a different C terminus.

| S-EPMC93896 | biostudies-literature

Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces.

Project description:Several strategies have been used to clone large DNA fragments directly from bacterial genome. Most of these approaches are based on different site-specific recombination systems consisting of a specialized recombinase and its target sites. In this study, a novel strategy based on phage ϕBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters. This method has been proved successful for the cloning of actinorhodin gene cluster from Streptomyces coelicolor M145, napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 at a frequency higher than 80%. Furthermore, the system could be used to increase the titer of antibiotics as we demonstrated with actinorhodin and daptomycin, and it will be broadly applicable in many Streptomyces.

| S-EPMC4349145 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data