A Modular, Tn7-Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

Abstract

Our goal was to develop a robust tagging method that can be used to track bacterial strains in vivo To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonella and Escherichia coli and Tn7 transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ(70)-dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7 system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10(-8) to 10(-10) in Salmonella enterica serovar Typhimurium and E. coli APEC O1, whereas the existing Tn7 system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivo animal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics.

Importance: This article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system in Salmonella and E. coli, and we predict that it will be widely applicable to other bacterial strains within the Enterobacteriaceae This technology will allow for improved in vivo analysis of bacterial pathogens.

FIG 1

Use of modular pCS26 plasmids in a modified Tn7 transposition system. (A) The antibiotic resistance marker (Kn^r or Cm^r), sig70 promoter (or new promoter cloned with XhoI/BamHI), and luxCDABE, mCherry, or GFP (or new reporter cloned with NotI/NotI) in the modular pCS26 plasmid are chosen. The synthetic, σ⁷⁰-dependent promoters (shown in magenta) allow for constitutive expression at differing strengths; the nucleotide differences are outlined, and −35 and −10 regions are recognized by σ⁷⁰. (B) pCS26 constructs are digested with PacI and ligated into the pUC18R6K-mini-Tn7T-PacI delivery plasmid. pHSG415-tnsABCD is a new helper plasmid that is replication proficient and can be conditionally maintained in most Enterobacteriaceae strains. The tnsABCD operon is expressed under the control of the preexisting cat promoter, leading to elevated expression; two black arrows represent truncated aph (Kn^r) and cat (Cm^r) genes. (C) One hundred nanograms of mini-Tn7T-pCS26 delivery plasmid is transformed into cells containing pHSG415-tnsABCD, resulting in orientation-specific integration of Kn^r- or Cm^r-promoter-reporter constructs into the attTn7 site (shown as a black box) present in the chromosome of Salmonella or E. coli, downstream of the glmS gene. The details of Tn7 transposition were adapted from reference . Chromosomal insertion can be verified by PCR, followed by growth at 37°C to cure the pHSG415 helper plasmid. All plasmid maps were generated using Geneious v. 9.0.4 (Biomatters Inc., Newark, NJ).

FIG 2

Chromosome-based expression of luciferase in S. Typhimurium and E. coli. Synthetic, σ⁷⁰-dependent promoter-luxCDABE operon fusions were integrated into the chromosome of S. Typhimurium 14028 ΔcsgD or E. coli APEC O1 using the modified Tn7 transposition system. (A and D) Luciferase expression from each strain was measured during growth at 37°C for 48 h; each line represents the expression curve from one individual reporter strain. (B and E) Box plots show the mean and range of maximum expression values for the different chromosomal reporter strains. Numerical values above the boxes represent the stepwise increase in mean expression levels between promoters. Statistical significance was calculated using unpaired t tests with Welch's correction (***, P < 0.001). (C and F) Maximum expression values for Cm^r and Kn^r strains within each sig70 promoter group are shown, with histogram bars representing the mean and error bars representing the standard deviation. Statistical significance (*) was determined using multiple t tests, corrected for multiple comparisons using the Holm-Sidak method (alpha = 5%).

FIG 3

Fluorescent reporter strains of Salmonella serovar Typhimurium 14028. (A) S. Typhimurium reporter strains with a sig70_16, sig70c10, or sig70c35 promoter controlling luxCDABE, mCherry, or GFP expression were grown on LB agar at 37°C. Expression from the different strains was visualized using an IVIS Lumina II whole-animal imaging system (Perkin-Elmer). (B) Peak luciferase (cps) or fluorescence (radiant efficiency) was measured using the region-of-interest (ROI) module in Living Image software ver. 4.2 (PerkinElmer). (C) Cells of the Kn^r sig70_16 GFP reporter strain were visualized on a Leica TCS SP5 confocal microscope, using the 63× oil immersion objective with the 180 laser operating at 488 nm. The scale bar is displayed in the bottom right corner.

FIG 4

Bacterial counts and competitive indices from C57BL/6 mice after oral infection with S. Typhimurium sig70_16 lux operon fusion strains. (A to D) The spleen (A), liver (B), cecum (C), and MLN (D) were collected from euthanized mice, homogenized, and plated on LB agar supplemented with 50 μg ml⁻¹ Kn or 10 μg ml⁻¹ Cm to determine the levels of each strain present (measured as CFU per whole organ). (E) Competitive index (CI) values were calculated for each mouse in each organ: CI = (CFU Kn^r OUT/CFU Cm^r OUT)/(CFU Kn^r IN/CFU Cm^r IN). A CI value of 1.0, which indicates equal virulence, is represented by a horizontal dotted line. Statistical significance from a value of 1 was determined using a Wilcoxon signed rank test (ns, not significant [P > 0.05]).

FIG 5

S. Typhimurium reporter strains have various levels of luciferase expression in vivo. C57BL/6 mice were pretreated with streptomycin and then challenged with S. Typhimurium luxCDABE reporter strains containing promoter sig70_16 (A and D), sig70c10 (B and E), or sig70c35 (C and F). Mice were visualized on a whole-animal imager at 1 day postinfection (A, B, and C). At 4 to 5 days postinfection, mice were euthanized and their organs (spleen, liver, gastrointestinal [GI] tract [stomach, intestine, cecum, and colon], and MLN) were recovered and imaged (D, E, and F). Peak luciferase values (measured in cps) are indicated.

FIG 6

Chromosome-based complementation of an S. Typhimurium ΔcsgD mutant strain. (A and B) S. Typhimurium ATCC 14028 (WT), ΔcsgD, csgD⁺, and pACYC-csgD strains were grown on 1% tryptone agar at 28°C or 37°C (A) or in 1% tryptone broth at 28°C (B). (C) csgDEFG, csgBAC, and adrA expression was measured during growth using promoter-luciferase (luxCDABE) fusion plasmids, designed to measure gene expression by light production (cps). RpoS activity was measured using the sig38H4::lux reporter plasmid, which contains a synthetic, RpoS-dependent promoter (26). Each line represents the expression curve from one biological replicate culture performed in triplicate, where cps was measured at half-hour intervals during growth. Cells were grown in 1% tryptone broth at 28°C with agitation; expression was measured in a Victor X³ multilabel plate reader (PerkinElmer).