Probing chemotaxis activity in Escherichia coli using fluorescent protein fusions

Abstract

Bacterial chemotaxis signaling may be interesting for the development of rapid biosensor assays, but is difficult to quantify. Here we explore two potential fluorescent readouts of chemotactically active Escherichia coli cells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of stable or unstable ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically active E. coli . Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise for biosensor assays based on bacterial chemotaxis activity.

The Divergent Key Residues of Two Agrobacterium fabrum (tumefaciens) CheY Paralogs Play a Key Role in Distinguishing Their Functions

“…If both CheY1 and CheY2 of A. fabrum were involved in the chemotaxis signaling transduction, the co-localization of CheA and CheY1 (or CheY2) at cell poles could be observed. To visualize the co-localization of CheA and CheY1 (or CheY2) in the A. fabrum cell, eGFP was split into two non-fluorescent parts (N-terminal part and C-terminal part) at the peptide bond between residues 158 and 159 [ 39 ]. The C-terminal part (C egfp ) of eGFP was fused to the C-terminus of CheA to form the CheA-C egfp fusion protein and the N-terminal part (N egfp ) of eGFP was fused to the C-terminus of CheY1 (or CheY2) to form the CheY1-N egfp (or CheY2-N egfp ) fusion protein.…”

“…To test the interaction between CheA and CheY in vivo, eGFP protein was split at the peptide bond between amino acid residues 158 and 159 [ 39 ]. The C-terminal part of the eGFP protein (residues 158–238) was fused to the C-terminus of CheA with a seven-residue linker (GTSGGSG) between two fused parts to generate CheA-linker-C egfp fusion protein.…”

Spatial structure, chemotaxis and quorum sensing shape bacterial biomass accumulation in complex porous media

“…Microfluidic chip experiments were performed using E. coli strain MG1655 and its derivatives: the motile wild type (WT) E. coli MG1655 (CGSC#: 8237; Yale E. coli genetic stock center) tagged with GFP (pME6012-P tac -gfp) 75 ; the non-flagellated mutant E. coli MG1655 ( ΔfliM ) tagged with mCherry (pME6012-P tac -mCherry) 76 ; the mutant lacking the AI-2 synthase gene luxS , E. coli MG1655 ( ΔluxS ) tagged with GFP (pME6012-P tac -gfp, this work); the mutant lacking the gene cheA , E. coli MG1655 ( ΔcheA ) 77 ; the mutant lacking the gene tsr , E. coli MG1655 ( Δtsr ) 77 and AI-2 reporter E. coli MG1655 (pMSs201-P lsrR -gfp) 53 . The inoculum for the microfluidic chip experiments was grown overnight in 4 mL of lysogeny broth Miller (LB) with 25 µg/mL of kanamycin at 37 °C and 180 rpm shaking, followed by 1:100 dilution in fresh LB and incubation until exponential growth phase ( ~ 3 hours).…”

Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis

“…Eventually, further improvement was obtained by Blakeley et al [ 16 ] who devised a “super positive” GFP (spGFP) featuring a much higher net charge (+34) than that of sg100 (–8), based on the observation that increasing protein charge is associated with increased solubility. In addition to sg100, frGFP and spGFP, split enhanced GFP (eGFP [ 17 ]) and split eYFP [ 18 ] have also been used. In the case of sg100, fr and spGFP, the bipartite reporter was generated by cutting the loop linking β-strands 7 and 8 between residues 157 and 158, which yields two moieties, respectively made of the first seven (NGFP) and the last four (CGFP) β-strands.…”