Delivering "Chromatic Bacteria" Fluorescent Protein Tags to Proteobacteria Using Conjugation

Abstract

Recently, we published a large and versatile set of plasmids, the chromatic bacteria toolbox, to deliver eight different fluorescent protein genes and four combinations of antibiotic resistance genes to Gram-negative bacteria. Fluorescent tags are important tools for single-cell microbiology, synthetic community studies, biofilm, and host-microbe interaction studies. Using conjugation helper strain E. coli S17-1 as a donor, we show how plasmid conjugation can be used to deliver broad host range plasmids, Tn5 transposons delivery plasmids, and Tn7 transposon delivery plasmids into species belonging to the Proteobacteria. To that end, donor and recipient bacteria are grown under standard growth conditions before they are mixed and incubated under non-selective conditions. Then, transconjugants or exconjugant recipients are selected on selective media. Mutant colonies are screened using a combination of tools to ensure that the desired plasmids or transposons are present and that the colonies are not containing any surviving donors. Through conjugation, a wide range of Gram-negative bacteria can be modified without prior, often time-consuming, establishment of competent cell and electroporation procedures that need to be adjusted for every individual strain. The here presented protocol is not exclusive for the delivery of Chromatic bacteria plasmids and transposons, but can also be used to deliver other mobilizable plasmids to bacterial recipients.

Keywords: Tn5 transposon; Tn7 transposon; Conjugation; Fluorescent proteins; GFP; Mobilisable plasmids; Plasmid.

Figure 1.. Flowsheet of protocol

Figure 2.. Bacterial colonies expressing fluorescent proteins.

A and B. Bacteria were streaked as crude streaks on agar plates. Expressed fluorescent proteins were visualized on a blue light gel reader. Green, yellow, orange, red and far-red fluorescent colonies were easily detectable on gel readers, blue and cyan fluorescent colonies were difficult to distinguish from non-fluorescent wild type colonies. C. Expressed fluorescent proteins were visualized using a UV light tray with attached gel documentation system. Fluorescent protein expressing colonies appear brighter than the wild type.

Figure 3.. Overlay of fluorescent and brightfield microscope channels.

A. Example of unsuccessful conjugation that yielded colonies. The colonies are mixes of a Sphingomonas species and red fluorescent protein expressing E. coli S17-1. B. Clonal colony of P. eucalypti 299R::MRE-Tn7-145. Scale bars = 500 µm.

Figure 4.. Colony PCR of uidA for detection of Escherichia coli.

uidA is an E. coli and Shigella sp. specific gene, thereby amplification of this gene fragment indicates mixed colony or false positive colony after restreak and colony isolation. Primers used were FWD_uidA and REV_uidA (Table 3) and yield a 500 bp product. Lane M: Molecular marker (HyperLadder^TM, Bioline); Sample 1: P. eucalypti 299R::MRE-Tn5-145; Sample 2: E. coli (pMRE-Tn5-145); Sample 3: P. eucalypti 299R::MRE-Tn7-145; Sample 4: E. coli (pMRE-Tn7-145); C-: water control. In this example, samples #1 and #3 showed no detection of E. coli, while sample #2 and #4 showed presence of uidA.

Figure 5.. Colony PCR to confirm Tn5 insertions.

Multiplex PCR result using primers FWD_Tn5_gt, REV_Tn5_gt, FWD_Tn5/7_gt, and REV_Tn5/7_gt. M: Molecular marker (HyperLadder, Bioline); Sample 1: P. eucalypti 299R::MRE-Tn5-145; Sample 2: E. coli (pMRE-Tn5-145); Sample 3: pMRE-Tn5-145; C-: water control.

Figure 6.. Colony PCRs to confirm Tn7 insertions.

PCR result using primers FWD_Tn5/7_gt and REV_Tn5/7_gt for the amplification of Tn7-delivered insert (MRE-Tn7 insert), and PCR result using primers Tn7_gt1, Tn7_gt2, and Tn7_gt3 for amplification of a fragment of plasmid pMRE-Tn7 backbone (MRE-Tn7 backbone). M: Molecular marker. Sample 1: P. eucalypti 299R::MRE-Tn7-145; Lane 2: E. coli (pMRE-Tn7-145); Lane 3: pMRE-Tn7-145; C-: water control.