Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain.
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.
Affiliation: Bacterial Vaccines and Immune Sera, Department of Veterinary Medicine, Paul Ehrlich Institute, Langen, 63225, Germany.Show MeSH
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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.
Affiliation: Bacterial Vaccines and Immune Sera, Department of Veterinary Medicine, Paul Ehrlich Institute, Langen, 63225, Germany.