Use of the Integration Elements Encoded by the Temperate Lactococcal Bacteriophage TP901-1 To Obtain Chromosomal Single-Copy Transcriptional Fusions in Lactococcus lactis

1999 ◽  
Vol 65 (2) ◽  
pp. 752-758 ◽  
Author(s):  
Lone Brøndsted ◽  
Karin Hammer
Keyword(s):  
Lactococcus Lactis ◽  
High Frequency ◽  
Attachment Site ◽  
Single Copy ◽  
Deletion Analysis ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Integration ◽  
Transcriptional Fusions ◽  
Reading Frames

ABSTRACT Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of Lactococcus lactis subsp.cremoris (B. Christiansen, L. Brøndsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164–5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when theorf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of anattP-carrying plasmid into the attB site on theL. lactis subsp. cremoris chromosome. Furthermore, the first detailed deletion analysis of anattP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of theattP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of L. lactis subsp. cremoriswhen the orf1 gene is donated in trans. The functional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages. Based on the deletion analysis, several repeats located within the attP region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (attP and orf1) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions inL. lactis was constructed. Two promoter-reporter integration vectors containing the reporter gene gusA orlacLM, encoding β-glucuronidase or β-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of L. lactis subsp.cremoris MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site attB used by bacteriophage TP901-1.

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

2002 ◽  
Vol 184 (15) ◽  
pp. 4177-4186 ◽  
Author(s):  
Peter Lauer ◽  
Man Yin Nora Chow ◽  
Martin J. Loessner ◽  
Daniel A. Portnoy ◽  
Richard Calendar
Keyword(s):  
Listeria Monocytogenes ◽  
Lethal Dose ◽  
Attachment Site ◽  
Donor Cell ◽  
Single Copy ◽  
Cell Extract ◽  
Site Specific ◽  
Specific Phage ◽  
Serotype 1 ◽  
Serotype 4B

ABSTRACT 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 ∼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 tRNAArg 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.

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.

Archaeal integrases and mechanisms of gene capture

2004 ◽  
Vol 32 (2) ◽  
pp. 222-226 ◽  
Author(s):  
Q. She ◽  
B. Shen ◽  
L. Chen
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Genome Evolution ◽  
Trna Genes ◽  
Gene Fragment ◽  
Integrase Gene ◽  
Site Specific ◽  
Conjugative Plasmids ◽  
Site Specific Integration ◽  
Target Sites

Archaeal integrases facilitate the formation of two distinctive types of integrated element within archaeal chromosomes: the SSV type and pNOB8 type. The former carries a smaller N-terminal and a larger C-terminal integrase gene fragment, and the latter an intact integrase gene. All integrated elements overlap tRNA genes that were target sites for integration. It has been demonstrated that SSV (Sulfolobus spindle virus) viruses, carrying an SSV-type integrase gene, and conjugative plasmids, carrying a pNOB8-type integrase, are integrative elements. Two mechanisms have been proposed for stably maintaining an integrated element within archaeal chromosomes. There is also evidence for changes having occurred in the captured integrated elements present in archaeal genomes. Thus we infer that site-specific integration constitutes an important mechanism for horizontal gene transfer and genome evolution.

scholarly journals Repetitive, Marker-Free, Site-Specific Integration as a Novel Tool for Multiple Chromosomal Integration of DNA

2013 ◽  
Vol 79 (12) ◽  
pp. 3563-3569 ◽  
Author(s):  
Kia Vest Petersen ◽  
Jan Martinussen ◽  
Peter Ruhdal Jensen ◽  
Christian Solem
Keyword(s):  
Low Frequency ◽  
Attachment Site ◽  
Plasmid Vector ◽  
Bacterial Attachment ◽  
Xylose Utilization ◽  
Site Specific ◽  
Content Type ◽  
Site Specific Integration ◽  
Attachment Sites ◽  
Marker Free

ABSTRACTWe present a tool for repetitive, marker-free, site-specific integration inLactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attBmin), two mutatedloxPsequences (lox66andlox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection ofloxPrecombination on the pKV6 vector. When transformed intoL. lactisexpressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked byattLandattRhybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate inL. lactis,loxPrecombinants can be selected for by using 5-fluoroorotic acid. The introducedattBminsite can subsequently be used for a second round of integration. To examine ifattPrecombination was specific to theattBsite, integration was performed in strains containing theattB,attL, andattRsites or theattLandattRsites only. OnlyattP-attBrecombination was observed when all three sites were present. In the absence of theattBsite, a low frequency ofattP-attLrecombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABRandxylT) fromL. lactisstrain KF147 were integrated into the chromosome ofL. lactisstrain MG1363 in two steps.

Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2443-2453 ◽  
Author(s):  
Dylan C. Alexander ◽  
David J. Devlin ◽  
Duane D. Hewitt ◽  
Ann C. Horan ◽  
Thomas J. Hosted
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Integration Site ◽  
Attachment Site ◽  
Bacterial Attachment ◽  
Temperate Bacteriophage ◽  
Site Specific ◽  
Sequence Identity ◽  
Site Specific Integration ◽  
Chromosome Sequence

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4·4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3′ end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.

scholarly journals Site-Specific Recombination of TemperateMyxococcus xanthus Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification

1999 ◽  
Vol 181 (13) ◽  
pp. 4062-4070 ◽  
Author(s):  
Vincent Magrini ◽  
Michael L. Storms ◽  
Philip Youderian
Keyword(s):  
Specific Activity ◽  
Active Form ◽  
Attachment Site ◽  
Covalent Modification ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Site Specific Integration ◽  
C Terminus ◽  
Primary Amino ◽  
Reversible Covalent

ABSTRACT Temperate Myxococcus xanthus phage Mx8 integrates into the attB locus of the M. xanthus genome. The phage attachment site, attP, is required in cisfor integration and lies within the int (integrase) coding sequence. Site-specific integration of Mx8 alters the 3′ end ofint to generate the modified intX gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes attP × attB recombination more efficiently than attR × attB, attL × attB, or attB × attB recombination. The attP and attBsites share a common core. Sequences flanking both sides of theattP core within the int gene are necessary forattP function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the attP core. Expression of theuoi gene immediately upstream of int inhibits integrative (attP × attB) recombination, supporting the idea that uoi encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.

scholarly journals Conjugative Transfer of the Integrative Conjugative Elements ICESt1 and ICESt3 from Streptococcus thermophilus

2009 ◽  
Vol 191 (8) ◽  
pp. 2764-2775 ◽  
Author(s):  
Xavier Bellanger ◽  
Adam P. Roberts ◽  
Catherine Morel ◽  
Frédéric Choulet ◽  
Guillaume Pavlovic ◽  
...  
Keyword(s):  
Streptococcus Thermophilus ◽  
Genomic Islands ◽  
Conjugative Transfer ◽  
Integrase Gene ◽  
Site Specific ◽  
Integrative And Conjugative Elements ◽  
Conjugative Transposons ◽  
Site Specific Integration ◽  
The One ◽  
Integrative Conjugative Elements

ABSTRACT Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in Streptococcus thermophilus, ICESt1 and ICESt3. Mitomycin C, a DNA-damaging agent, derepresses ICESt3 conjugative transfer almost 25-fold. The ICESt3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICESt3 transconjugants of Streptococcus pyogenes and Enterococcus faecalis were recovered, only one transconjugant of Lactococcus lactis was obtained. The newly incoming ICEs, except the one from L. lactis, are site-specifically integrated into the 3′ end of the fda gene and are still able to excise in these transconjugants. Furthermore, ICESt3 was retransferred from E. faecalis to S. thermophilus. Recombinant plasmids carrying different parts of the ICESt1 recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.

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