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Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain.

Roos K, Werner E, Loessner H - Microb Biotechnol (2014)

Bottom Line: Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors.Here we demonstrate that integration of a 9.6 kb mini-Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ-Red recombinase to contain one or two additional artificial attTn7 sites (a-attTn7).Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity.

View Article: PubMed Central - PubMed

Affiliation: Bacterial Vaccines and Immune Sera, Department of Veterinary Medicine, Paul Ehrlich Institute, Langen, 63225, Germany.

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Integration of two lux-mini-Tn7 into attTn7 and in addition one a-attTn7 site inserted into selected chromosomal loci of S. Typhimurium.A. Strain SL7207 was modified in two steps for each of the indicated genes. λ-Red-mediated deletion and concurrent integration of a-attTn7 (black circle) is depicted for recF. Strains harbouring the natural attTn7 site (black square) and in addition a-attTn7 were used for transposition with lux-mini-Tn7 9.6 kb in size (white inversed triangle).B. Approximate distances between attTn7 and the newly introduced a-attTn7 sites in respective strains.C. Integration of lux-mini-Tn7 into attTn7 or a-attTn7 (indicated by combined symbols) was confirmed by the bioluminescent phenotype (data not shown) and by colony PCR with primers homologous to sequences of the right end of lux-mini-Tn7 (Tn7R) and the neighbouring genomic location (Supporting Information Table S1). The expected band sizes for lux-mini-Tn7 integration into attTn7 is 332 bp, for integrations into a-attTn7 sites in mutant strains are recF 586 bp, rha 1523 bp, asd 629 bp, endA 526 bp, ara 378 bp and sifA 543 bp respectively. Δ indicates a gene deletion and double colon indicates a chromosomal insertion. PCR data for representative clones are shown.
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fig01: Integration of two lux-mini-Tn7 into attTn7 and in addition one a-attTn7 site inserted into selected chromosomal loci of S. Typhimurium.A. Strain SL7207 was modified in two steps for each of the indicated genes. λ-Red-mediated deletion and concurrent integration of a-attTn7 (black circle) is depicted for recF. Strains harbouring the natural attTn7 site (black square) and in addition a-attTn7 were used for transposition with lux-mini-Tn7 9.6 kb in size (white inversed triangle).B. Approximate distances between attTn7 and the newly introduced a-attTn7 sites in respective strains.C. Integration of lux-mini-Tn7 into attTn7 or a-attTn7 (indicated by combined symbols) was confirmed by the bioluminescent phenotype (data not shown) and by colony PCR with primers homologous to sequences of the right end of lux-mini-Tn7 (Tn7R) and the neighbouring genomic location (Supporting Information Table S1). The expected band sizes for lux-mini-Tn7 integration into attTn7 is 332 bp, for integrations into a-attTn7 sites in mutant strains are recF 586 bp, rha 1523 bp, asd 629 bp, endA 526 bp, ara 378 bp and sifA 543 bp respectively. Δ indicates a gene deletion and double colon indicates a chromosomal insertion. PCR data for representative clones are shown.

Mentions: Mini-Tn7 transposons are frequently used for single-copy integration of DNA fragments into chromosomes of Gram-negative bacteria, with few exceptions (Choi et al., 2005; Crepin et al., 2012). However, consecutive integration of multiple mini-Tn7 copies into additional attTn7 sites present in the E. coli chromosome was until now thought to be suppressed in a distance-dependent manner by Tn7 target immunity (Arciszewska et al., 1989; DeBoy and Craig, 1996; Stellwagen and Craig, 1997; Choi et al., 2014). Here we investigated the possibility of simultaneous integration of two mini-Tn7 copies into attTn7 as well as an additional a-attTn7 site inserted at genomic positions at varying distances apart from attTn7 in the S. Typhimurium vaccine strain SL7207. The a-attTn7 was derived from the S. Typhimurium strain SL1344 genomic sequence and contains 105 bp of the 3′ end of glmS, 140 bp intergenic region and 45 bp of the 3′ end of gene SL1344_3827. The glmS sequence harbours the entire TnsD binding site and the intergenic region attTn7 downstream of glmS (Mitra et al., 2010). We successively replaced chromosomal genes ara, asd, endA, recF, rha and sifA of strain SL7207 with a-attTn7 one at a time by λ-Red-mediated homologous recombination (Fig. 1A). The targeted loci were located at varying distances below or above 190 kb apart from attTn7 (Fig. 1B). We then subjected the six different mutant strains carrying attTn7 as well as one additional a-attTn7 site to transposition with lux-mini-Tn7, which contains a constitutive lux-cassette and a kanamycin marker. Bioluminescent Salmonella colonies with chromosomal insertions of lux-mini-Tn7 were selected on LB plates containing streptomycin and kanamycin. Colony PCR of all mutant strains revealed that integration of lux-mini-Tn7 occurred into both integration sites (Fig. 1C). This was even the case for strains carrying a-attTn7 in the recF and the rha loci, which are less than 190 kb away from attTn7. Therefore, we did not observe immunity-related inhibition of lux-mini-Tn7 integration into either one of the two transposon integration sites present in the genome of all tested mutant strains.


Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain.

Roos K, Werner E, Loessner H - Microb Biotechnol (2014)

Integration of two lux-mini-Tn7 into attTn7 and in addition one a-attTn7 site inserted into selected chromosomal loci of S. Typhimurium.A. Strain SL7207 was modified in two steps for each of the indicated genes. λ-Red-mediated deletion and concurrent integration of a-attTn7 (black circle) is depicted for recF. Strains harbouring the natural attTn7 site (black square) and in addition a-attTn7 were used for transposition with lux-mini-Tn7 9.6 kb in size (white inversed triangle).B. Approximate distances between attTn7 and the newly introduced a-attTn7 sites in respective strains.C. Integration of lux-mini-Tn7 into attTn7 or a-attTn7 (indicated by combined symbols) was confirmed by the bioluminescent phenotype (data not shown) and by colony PCR with primers homologous to sequences of the right end of lux-mini-Tn7 (Tn7R) and the neighbouring genomic location (Supporting Information Table S1). The expected band sizes for lux-mini-Tn7 integration into attTn7 is 332 bp, for integrations into a-attTn7 sites in mutant strains are recF 586 bp, rha 1523 bp, asd 629 bp, endA 526 bp, ara 378 bp and sifA 543 bp respectively. Δ indicates a gene deletion and double colon indicates a chromosomal insertion. PCR data for representative clones are shown.
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Related In: Results  -  Collection

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fig01: Integration of two lux-mini-Tn7 into attTn7 and in addition one a-attTn7 site inserted into selected chromosomal loci of S. Typhimurium.A. Strain SL7207 was modified in two steps for each of the indicated genes. λ-Red-mediated deletion and concurrent integration of a-attTn7 (black circle) is depicted for recF. Strains harbouring the natural attTn7 site (black square) and in addition a-attTn7 were used for transposition with lux-mini-Tn7 9.6 kb in size (white inversed triangle).B. Approximate distances between attTn7 and the newly introduced a-attTn7 sites in respective strains.C. Integration of lux-mini-Tn7 into attTn7 or a-attTn7 (indicated by combined symbols) was confirmed by the bioluminescent phenotype (data not shown) and by colony PCR with primers homologous to sequences of the right end of lux-mini-Tn7 (Tn7R) and the neighbouring genomic location (Supporting Information Table S1). The expected band sizes for lux-mini-Tn7 integration into attTn7 is 332 bp, for integrations into a-attTn7 sites in mutant strains are recF 586 bp, rha 1523 bp, asd 629 bp, endA 526 bp, ara 378 bp and sifA 543 bp respectively. Δ indicates a gene deletion and double colon indicates a chromosomal insertion. PCR data for representative clones are shown.
Mentions: Mini-Tn7 transposons are frequently used for single-copy integration of DNA fragments into chromosomes of Gram-negative bacteria, with few exceptions (Choi et al., 2005; Crepin et al., 2012). However, consecutive integration of multiple mini-Tn7 copies into additional attTn7 sites present in the E. coli chromosome was until now thought to be suppressed in a distance-dependent manner by Tn7 target immunity (Arciszewska et al., 1989; DeBoy and Craig, 1996; Stellwagen and Craig, 1997; Choi et al., 2014). Here we investigated the possibility of simultaneous integration of two mini-Tn7 copies into attTn7 as well as an additional a-attTn7 site inserted at genomic positions at varying distances apart from attTn7 in the S. Typhimurium vaccine strain SL7207. The a-attTn7 was derived from the S. Typhimurium strain SL1344 genomic sequence and contains 105 bp of the 3′ end of glmS, 140 bp intergenic region and 45 bp of the 3′ end of gene SL1344_3827. The glmS sequence harbours the entire TnsD binding site and the intergenic region attTn7 downstream of glmS (Mitra et al., 2010). We successively replaced chromosomal genes ara, asd, endA, recF, rha and sifA of strain SL7207 with a-attTn7 one at a time by λ-Red-mediated homologous recombination (Fig. 1A). The targeted loci were located at varying distances below or above 190 kb apart from attTn7 (Fig. 1B). We then subjected the six different mutant strains carrying attTn7 as well as one additional a-attTn7 site to transposition with lux-mini-Tn7, which contains a constitutive lux-cassette and a kanamycin marker. Bioluminescent Salmonella colonies with chromosomal insertions of lux-mini-Tn7 were selected on LB plates containing streptomycin and kanamycin. Colony PCR of all mutant strains revealed that integration of lux-mini-Tn7 occurred into both integration sites (Fig. 1C). This was even the case for strains carrying a-attTn7 in the recF and the rha loci, which are less than 190 kb away from attTn7. Therefore, we did not observe immunity-related inhibition of lux-mini-Tn7 integration into either one of the two transposon integration sites present in the genome of all tested mutant strains.

Bottom Line: Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors.Here we demonstrate that integration of a 9.6 kb mini-Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ-Red recombinase to contain one or two additional artificial attTn7 sites (a-attTn7).Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity.

View Article: PubMed Central - PubMed

Affiliation: Bacterial Vaccines and Immune Sera, Department of Veterinary Medicine, Paul Ehrlich Institute, Langen, 63225, Germany.

Show MeSH
Related in: MedlinePlus