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

Abstract

Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors. Mini-Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn7, present only once in most Gram-negative bacteria. However, integration of multiple mini-Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. 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. Integration of multiple copies of a mini-Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini-Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ-mini-Tn7, subsequent chromosomal insertion of a-attTn7 and a second round of transposition with lux-mini-Tn7. Mini-Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of Salmonella and possibly other bacteria.

Fig 1

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.

Fig 2

Consistency of simultaneous transposition of lux-mini-Tn7 into three loci of the S. Typhimurium chromosome.A. A SL7207 derivative with the natural attTn7 site (black square) and two additional a-attTn7 sites (black and white circles) was generated by two rounds of λ-Red-mediated recombination linked with chromosomal integration of the a-attTn7 sequence each time.B. Strain SL7207Δara::a-attTn7Δasd::a-attTn7 harbouring three sites for Tn7 transposition was used for transposition with lux-mini-Tn7 (white inversed triangle). lux-mini-Tn7 integration into the three sites was confirmed by colony PCR for 10 bioluminescent clones selected on LB plates containing streptomycin and kanamycin. The strain harbouring three empty Tn7 transposition sites was used as control template. The band size for lux-mini-Tn7 integration into attTn7 is 332 bp and for integrations into a-attTn7 are ara 378 bp and asd 629 bp, band positions are indicated by black arrow heads.

Fig 3

Modulation of GFP synthesis by chromosomal integration of either one, two or three copies of gfp-mini-Tn7 and consecutive chromosomal integration of two different mini-Tn7 encoding lacZ and lux into S. Typhimurium strain SL7207.A. Strain SL7207 with the natural attTn7 site (black square) or derivatives with one or two additional a-attTn7 sites integrated into ara and asd loci (black and white circles) were used for transposition with gfp-mini-Tn7 harbouring the L-arabinose inducible gfp expression cassette (grey inversed triangle). gfp-mini-Tn7 integration into each site was verified by colony PCR and expected bands were obtained at 332 bp for integration into attTn7, 378 bp for integration into a-attTn7 at the ara locus and 629 bp for integration into a-attTn7 at the asd locus. Bands are indicated by black arrow heads.B. Strains SL7207 and derivatives with one, two or three chromosomal copies of the gfp cassette were grown in the absence or presence of L-arabinose prior to flow cytometric analysis. Depicted mean fluorescence intensities (MFI) represent three experimental repetitions and error bars indicate standard deviation.C. Strain SL7207 was used for transposition with lacZ-mini-Tn7 or lux-mini-Tn7 (black and white inversed triangles). Recipient strains carry either lacZ or lux within attTn7 (black square) and are designated attTn7::lacZ and attTn7::lux respectively. Subsequently, the rha operon of strain attTn7::lacZ was replaced by a-attTn7 (white square) giving rise to strain attTn7::lacZΔrha::a-attTn7. This strain was employed for another transposition round with lux-mini-Tn7 yielding strain attTn7::lacZΔrha::lux. Colony PCR verified transposon integrations into the chromosome of each strain. Band sizes for integration into attTn7 are 332 bp and into a-attTn7 at the rha locus 1523 bp.D. lacZ expression of bacteria was observed on X-Gal plates by blue dye formation and lux expression by detection of bioluminescence from the same plate.