scholarly journals Construction of an Integration-Proficient Vector Based on the Site-Specific Recombination Mechanism of Enterococcal Temperate Phage φFC1

2002 ◽  
Vol 184 (7) ◽  
pp. 1859-1864 ◽  
Author(s):  
Hee-Youn Yang ◽  
Young-Woo Kim ◽  
Hyo-Ihl Chang
Keyword(s):  
Pcr Amplification ◽  
Temperate Phage ◽  
Vector System ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Integration Vector ◽  
Site Specific Integration ◽  
A Site

ABSTRACT The genome of temperate phage φFC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination. In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage φFC1 was constructed. A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1. E. faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E. faecalis KBL 707 chromosome. This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage φFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 × 103 transformants/μg of DNA. In addition, a vector (pATTB1) containing the 290-bp attB region was constructed. pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage φFC1 can be used for genetic engineering in E. faecalis and in other hosts.

scholarly journals Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

2009 ◽  
Vol 191 (6) ◽  
pp. 1933-1940 ◽  
Author(s):  
André Larouche ◽  
Paul H. Roy
Keyword(s):  
Antibiotic Resistance Genes ◽  
Cysteine Residue ◽  
Wild Type ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Environmental Bacterium ◽  
Target Sites ◽  
A Site

ABSTRACT Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the β-4 strand is less important for the excision activity.

scholarly journals TPW22, a Lactococcal Temperate Phage with a Site-Specific Integrase Closely Related to Streptococcus thermophilus Phage Integrases

1999 ◽  
Vol 181 (22) ◽  
pp. 7034-7042 ◽  
Author(s):  
Anne Petersen ◽  
Jytte Josephsen ◽  
Mads G. Johnsen
Keyword(s):  
Attachment Site ◽  
Streptococcus Thermophilus ◽  
Temperate Phage ◽  
Sequence Information ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Integration ◽  
Phage Integrases ◽  
A Site ◽  
Encoding Genes

ABSTRACT The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp.cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5′-TAAGGCGACGGTCG-3′, of phage TPW22 was present on a 7.5-kbEcoRI fragment, a 3.4-kbEcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containingattP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages φLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the φLC3 lactococcal integrase with known Streptococcus thermophilus integrases.

scholarly journals Characterization of the Class 3 Integron and the Site-Specific Recombination System It Determines

2002 ◽  
Vol 184 (11) ◽  
pp. 3017-3026 ◽  
Author(s):  
Christina M. Collis ◽  
Mi-Jurng Kim ◽  
Sally R. Partridge ◽  
H. W. Stokes ◽  
Ruth M. Hall
Keyword(s):  
Terminal Inverted Repeat ◽  
Class 1 Integron ◽  
Recombination Mechanism ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Class 1 Integrons ◽  
Recombination System ◽  
Class 1 ◽  
A Site ◽  
Site Specific Recombination System

ABSTRACT Integrons capture gene cassettes by using a site-specific recombination mechanism. As only one class of integron and integron-determined site-specific recombination system has been studied in detail, the properties of a second class, the only known class 3 integron, were examined. The configuration of the three potentially definitive features of integrons, the intI3 gene, the adjacent attI3 recombination site, and the Pc promoter that directs transcription of the cassettes, was similar to that found in the corresponding region (5′ conserved segment) of class 1 integrons. The integron features are flanked by a copy of the terminal inverted repeat, IRi, from class 1 integrons on one side and a resolvase-encoding tniR gene on the other, suggesting that they are part of a transposable element related to Tn402 but with the integron module in the opposite orientation. The IntI3 integrase was active and able to recognize and recombine both known types of IntI-specific recombination sites, the attI3 site in the integron, and different cassette-associated 59-be (59-base element) sites. Both integration of circularized cassettes into the attI3 site and excision of integrated cassettes were also catalyzed by IntI3. The attI3 site was localized to a short region adjacent to the intI3 gene. Recombination between a 59-be and secondary sites was also catalyzed by IntI3, but at frequencies significantly lower than observed with IntI1, the class 1 integron integrase.

scholarly journals Characterization of the Integrase Gene and Attachment Site for the Myxococcus xanthus Bacteriophage Mx9

2003 ◽  
Vol 185 (21) ◽  
pp. 6325-6330 ◽  
Author(s):  
Bryan Julien
Keyword(s):  
Myxococcus Xanthus ◽  
Attachment Site ◽  
Temperate Phage ◽  
Bacterial Chromosome ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Single Protein

ABSTRACT Bacteriophage Mx9 is a temperate phage that infects Myxococcus xanthus. It lysogenizes the bacteria by integrating into the bacterial chromosome by site-specific recombination at one of two sites, attB1 or attB2. Integration at attB1 results in deletion of DNA between the two attB sites. The attB2 site lies within the 5′ region of the M. xanthus tRNAGly gene. Mx9 integration requires a single protein, Int. Analysis of integration revealed that the phage attachment site (attP) is contained in the int gene and that upon integration, the 3′ end of the int gene is altered. Plasmids containing fusions of the pilA or mgl promoter to lacZ integrated at either Mx9 attB site have higher levels of transcription than the same fusions integrated at the Mx8 attB site.

scholarly journals Amplification of the Tetracycline Resistance Determinant of pAMα1 in Enterococcus faecalis Requires a Site-Specific Recombination Event Involving Relaxase

2002 ◽  
Vol 184 (18) ◽  
pp. 5187-5193 ◽  
Author(s):  
M. Victoria Francia ◽  
Don B. Clewell
Keyword(s):  
Enterococcus Faecalis ◽  
Recombination Event ◽  
Tandem Repeats ◽  
Tetracycline Resistance ◽  
Multicopy Plasmid ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Rate Limiting Step ◽  
A Site ◽  
Rate Limiting

ABSTRACT The small multicopy plasmid pAMα1 (9.75 kb) encoding tetracycline resistance in Enterococcus faecalis is known to generate tandem repeats of a 4.1-kb segment carrying tet(L) when cells are grown extensively in the presence of tetracycline. Here we show that the initial (rate-limiting) step involves a site-specific recombination event involving plasmid-encoded relaxase activity acting at two recombination sequences (RS1 and RS2) that flank the tet determinant. We also present the complete nucleotide sequence of pAMα1.

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

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Innanurdiani Koko ◽  
Adelene Ai-Lian Song ◽  
Mas Jaffri Masarudin ◽  
Raha Abdul Rahim
Keyword(s):  
Signal Peptide ◽  
Heterologous Protein ◽  
Expression System ◽  
Surface Display ◽  
Host Genome ◽  
Integration System ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Integrative Vectors

Abstract 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.

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