Tn FLXopen: Markerless Transposons for Functional Fluorescent Fusion Proteins and Protein Interaction Prediction

Abstract

Fluorescence microscopy of cells expressing proteins translationally linked to a fluorophore can be a powerful tool to investigate protein localization dynamics in vivo. One major obstacle to reliably analyze biologically relevant localization is the construction of a fusion protein that is both fluorescent and functional. Here, we develop a strategy to construct fluorescent fusions at theoretically any location in the protein by using TnFLXopen random transposon mutagenesis to randomly insert a gene encoding a fluorescent protein. Moreover, insertions within a target gene are enriched by an inducible gene-trap strategy and selection by fluorescence activated cell sorting. Using this approach, we isolate a variety of fluorescent fusions to FtsZ that exhibit ring-like localization and a fusion to the flagellar stator protein that both is functional for supporting motility and localizes as fluorescent puncta. Finally, we further modify TnFLXopen to insert the coding sequence for the C-terminal half of mVenus for use in bimolecular fluorescence complementation (BiFC) and the in vivo detection of protein-protein interaction candidates. As proof-of-concept, the DivIVA polar scaffolding protein was fused to the N terminus of mVenus, the C terminus of mVenus was delivered by transposition, and a combination of fluorescence activated cell sorter (FACS) sorting and whole-genome sequencing identified the known self-interaction of DivIVA as well as other possible candidate interactors. We suggest that the FACS selection is a viable alternative to antibiotic selection in transposon mutagenesis that can generate new fluorescent tools for in vivo protein characterization. IMPORTANCE Transposon mutagenesis is a powerful tool for random mutagenesis, as insertion of a transposon and accompanying antibiotic resistance cassette often disrupt gene function. Here, we present a series of transposons with fluorescent protein genes which, when integrated in frame, may be selected with a fluorescence activated cell sorter (FACS). An open reading frame runs continuously through the transposon such that fluorescent protein fusions may be inserted theoretically anywhere in the primary sequence and potentially preserve function of the target protein. Finally, the transposons were further modified to randomly insert a partial fluorescent protein compatible with bimolecular fluorescence complementation (BiFC) to identify protein interaction candidates.

Keywords: Bacillus subtilis; BiFC; DivIVA; FACS; FtsZ; MotA; internal fluorescent protein fusion; protein-protein interactions; transposon mutagenesis.

FIG 1

TnFLXopen architecture. (A) The transposons of the TnFLXopen series share an architecture. A reporter construct, either mNeongreen or the coding sequence for the C-terminal 85 amino acids of mVenus (colored green), was inserted between two inverted terminal repeat (ITR) sequences for Himar transposase recognition (colored red). In each case, the reporter gene lacked a stop codon that would arrest translation within the transposon. (B to D) Left, a cartoon of the theoretical sequence in which the transposon was inserted boxed in orange. Bent arrow indicates a promoter, and stop sign indicates a stop codon. “N” indicates theoretically any nucleotide. Right, a cartoon of the theoretical sequence after the transposon had been inserted in frame. The sequence of the ITR is boxed in red, the sequence of the reporter is boxed in green, and bases added to preserve the open reading frame are boxed in blue. The TA sequence targeted for integration is bolded. Letters above the sequence line indicate the translated amino acid where X is theoretically any residue. (B) The TnFLXopen1 transposons were designed to maintain a continuous reading frame when inserted at an AT base doublet that is present at positions 2 and 3 of a codon. (C) The TnFLXopen2 transposons were designed to maintain a continuous reading frame when inserted at an AT base doublet that is present at positions 3 and 1, respectively, of two consecutive codons. (D) The TnFLXopen3 transposons were designed to maintain a continuous reading frame when inserted at an AT base doublet that is present at positions 1 and 2 of a codon. To avoid premature translation termination, the 5′ ITR was modified at three codon positions with a variety of substitutions. Multiple combinations of ITR substitutions were tested for their ability to permit transposition (Fig. S1). Ultimately, none of the TnFLXopen3 series resulted in functional transposition and the series was dropped from further use. (E) FACS sorting results for control (left) and TnFLXopen1mNeongreen mutagenized (right) populations. On the x axis is the side scatter width value (SSC-W), and on the y axis are the arbitrary fluorescence units. Each panel consists of 30,000 individual events. Red line indicates threshold gate value, and cells emitting fluorescence above this value were selected by FACS.

FIG 2

Some TnFLXopen insertions of mNeongreen within FtsZ produce subcellular fluorescence patterns consistent with FtsZ localization. (A) A flow diagram of the selection strategy used to enrich TnFLXopenmNeongreen insertions within ftsZ. Cells in a population are indicated by a red outline. After transposition, cells grown in the presence of IPTG were selected for fluorescence by FACS (dotted box), and the population exhibited a diverse variety of fluorescence localization patterns (green). The enriched population was regrown in the absence of IPTG, FACS sorted for nonfluorescent cells (dotted box), and plated. The resulting colonies were regrown in the presence of IPTG, colonies that produced fluorescent cells were retained, and the transposon insertion site within ftsZ was determined. (B) Fluorescence microscopy images of the indicated TnFLXopenmNeongreen insertions labeled with the corresponding insertion site. Membranes were stained with FM4-64 (false colored magenta) and FtsZ-mNeongreen (false colored green). The following strains were used to generate this panel: FtsZ^Garner (DK5094), FtsZ⁻⁴ (DK8445), FtsZ⁵⁷ (DK8441), FtsZ¹¹⁴ (DK8440), FtsZ¹⁹⁹ (DK8444), FtsZ²⁸⁰ (DK8443), FtsZ³⁷² (DK8439), and FtsZ⁹⁹⁰ (DK8442). (C) Top, location of transposon insertion sites within the ftsZ gene. Numbers indicate the distance (in bp) from the transposon insertion relative to the translation start site. Red carets indicate insertions predicted to position the mNeongreen fusion on the surface of the protein, blue caret indicates an insertion predicted to position mNeongreen in the FtsZ core region, and black carets indicate insertions that fall within unstructured domains not present in the FtsZ 3-dimensional structure. Bottom, ribbon diagram of the FtsZ protein (PDB 2VAM [60]) from B. subtilis with four of the mNeongreen insertion sites that led to distinct localization patterns space filled in red. Only diffuse localization was observed when insertion of the transposon led to fusion of mNeongreen to the internal core of the FtsZ protein (space filled in blue). AlphaFold2 was used to model each of the resulting fusion proteins (Fig. S2). Each fusion that gave rise to FtsZ-like localization patterns positioned mNeongreen such that it did not interfere with protofilament oligomerization, whereas the one fusion that abolished localization positioned mNeongreen in a steric clash with the adjacent monomer. (D) Localization analysis of cells expressing the various FtsZ-mNeongreen fusion proteins. Green bars represent percentage of events in which the FtsZ ring was observed at the predivisional midcell, orange bars indicate the percentage of events in which the FtsZ ring was observed adjacent to a nascent pole, and cyan bars indicate the percentage of events in which FtsZ adopted a spiral-like intermediate.

FIG 3

An mNeongreen-MotA fusion complements the lack of the wild-type motA gene. (A) A flow diagram of the selection strategy used to enrich TnFLXopenmNeongreen insertions within motAB. Cells in a population are indicated by a red outline. After transposition, cells grown in the presence of IPTG were selected for fluorescence by FACS (dotted box) and the population exhibited a diverse variety of fluorescence localization patterns (green). The enriched population was regrown in the absence of IPTG and FACS sorted for nonfluorescent cells (dotted box) and plated. The resulting colonies were regrown in the presence of IPTG, spotted on IPTG-containing swarm agar plates, and cells from motile flares were retained. (B) Quantitative swarm expansion assays. Top, the following strains were measured for motility on swarm agar plates in the presence of IPTG: motAB (open circles, DK2530), motAB P_IPTG-motAB (gray circles, DK801), motAB P_IPTG-mNGmotAB (closed circles, DK6666). Bottom, the following strains were measured for motility on swarm agar plates: motAB (open circles, DK2530), motAB P_motA-motAB (gray circles, DK8678), motAB P_motA-mNGmotAB (closed circles, DK7154). mNG, mNeongreen. Each data point is the average of three replicates. (C) Fluorescence microscopy images of the mNeongreen-MotA (mNG-MotA) fusion expressed from either the IPTG-inducible P_IPTG promoter (top, DK6832) or the native P_motA promoter (bottom, DK7104). Cell membrane was fluorescently stained with FM4-64 (false colored magenta) and mNG-MotA (false colored green). (D) Top, schematic representation of the TnFLXopenmNeongreen transposon inserted 15 bp upstream of the MotA translation start site. Bent arrow represents the IPTG-inducible promoter, red boxes represent inverted terminal repeat regions and the green box represents the mNeonGreen gene. Brown boxes represents the motA and motB genes, respectively, and the black caret indicates the location of the in-frame fusion event generated by transposon insertion. Bottom, model of the mNeongreen-MotA/MotB motor complex in the bacterial membrane. mNeongreen-MotA is represented in light brown with the fused mNeongreen highlighted as a green circle. MotB is colored in dark brown. Lipid bilayer is represented in dark gray, the cell wall in light gray. N and C indicate respective protein termini.

FIG 4

Transposon-delivered BiFC identified candidate DivIVA interactors. (A) Cartoon of bimolecular fluorescence complementation (BiFC) strategy. One half of a fluorophore mVenusN154 is fused to one protein of interest (protein X), and the other half of mVenusC155 is fused to another protein with which protein X interacts (protein Y). The interaction of proteins X and Y bring both mVenus halves into close spatial proximity, thereby restoring fluorophore structure and fluorescence emission. (B) Fluorescence micrographs of TnFLXopenmVenusC155 fusion candidates cloned under the control of an IPTG-inducible promoter and inserted at an ectopic locus of a strain expressing DivIVA-mVenusN154 at the native locus. Membrane stained with FM4-64 (false colored in magenta) and mVenus (false colored in green). The following strains were used to generate each panel: ahpC (DK8247), divIVA (DK8062), ezrA (DK8478), glnA (DK8179), msmX (DK8249), rocD (DK8479), and ybbC (DK8248). Bar represents 5 μm. (C) Cartoon of the TnFLXopenmVenusC155 fusion in the divIVA-mVenusN154 reporter gene. The transposon inserted the C-terminal half of mVenus into the reporter upstream of the N-terminal half of mVenus. Blue box indicates divIVA sequence, yellow boxes indicate the mVenus sequence, and red boxes indicate the TnFLXopen Himar recognition ITR sequences. Numbers above the diagram indicate base pair locations. Bent arrow indicates the divIVA promoter.