Modified Tn 7 transposon vectors for controlled chromosomal gene expression

Abstract

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.IMPORTANCEChromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology.

Keywords: Tn7; gene expression; transcriptional regulation; transposon.

FIG 1

Schematic diagram of the mini-Tn7 derivative vector pGP-Tn7-Cm and the modified vectors comprising the inducible cumate (Pcym) promoter and the constitutive PcL and PrpsM promoters. The promoters were cloned into the MCS in Tn7, thereby generating pGP-Tn7-Pcym, pGP-Tn7-PcL, and pGP-Tn7-PrpsM. The cat gene is flanked by FRTs, which allow excision of the selection marker by FLP recombinase (not shown). Selected commonly used single-cutter restriction sites are shown. Abbreviations: bla, beta-lactamase; cat; chloramphenicol acetyl transferase (resistance); CuO, cymR operator; cymR, repressor of p-cymene catabolism; mob RP4, conjugative transfer Mob RP4; ori R6K, origin of replication; Tn7L, Tn7 left end; Tn7R, Tn7 right end.

FIG 2

Schematic representation of the insertion of Tn7 harboring a promoter and gfp into MG1655 attTn7. The promoter represents Pcym (cymR repressor gene not shown), PcL or PrpsM. Tn7 is orientation-specific and inserts specifically into the chromosomal attTn7 site downstream of glmS. The temperature-sensitive plasmid pSTNSK encodes the TnsABCD transposase. Abbreviations: attTn7, attachment site; attTn7 half, 5 bp half of attTn7 resulting from duplication; bla, beta-lactamase; cat; chloramphenicol acetyl transferase (resistance); Cm30, chloramphenicol 30 µg/mL; gfp, green fluorescent protein; glmS, glutamine synthetase; mob RP4, conjugative transfer Mob RP4; ori R6K, origin of replication; pstS, phosphate ABC transporter; Tn7L, Tn7 left end; Tn7R, Tn7 right end. The original pGP-Tn7-Cm vector was designed by Crépin et al. (13).

FIG 3

Fluorescence of MG1655 strains harboring promoters and gfp at the chromosomal attTn7 site as shown by (A) kinetics and (B) area under the curve. All strains other than MG1655 have the prefix “MG1655attTn7::Tn7-” (not shown). MG1655attTn7::Tn7-Pcym-GFP was supplemented with cumate at time = 0 h. Biological replicates are shown as dots and fluorescence kinetics were performed using enhanced dynamic range. One-way ANOVA and Tukey’s multiple comparisons test were performed for area under the curve analysis.

FIG 4

Growth of MG1655 and MG1655attTn7::Tn7-Pcym-impCAB on LB agar with and without cumate supplementation. Overnight cultures were standardized to OD₆₀₀ 0.5, then serially diluted 10-fold, and spotted on LB agar or LB agar+cumate (75 µM). Photos are representative of three biological replicates.