Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

Abstract

words) 26Chemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein 27 interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the 28 development of rapid biosensor assays, but there is no consensus as to what the best readout of such 29 assays would have to be. Here we explore two potential fluorescent readouts of chemotactically active 30 Escherichia coli cells. In the first, we probed interactions between the chemotaxis signaling proteins 31 CheY and CheZ by fusing them individually with non-fluorescent parts of a 'split'-Green Fluorescent 32 Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced 33 distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the 34 chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were 35 attracted to serine sources, demonstrating measurable functional interactions between CheY~P and 36CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to 37 attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic 38 behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, 39 we expressed the pH-sensitive fluorophore pHluorin in the cyto-and periplasm of chemotactically 40 active E. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells 41 accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing 42 periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to 43 be further optimized in order to deliver practical biosensor assays. 44 45 IMPORTANCE (129 words) 46Bacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its 47 chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is 48 ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus 49 important to investigate different alternative ways to interrogate chemotactic response of cells. Here 50 we gauge the possibilities to measure dynamic response in the Escherichia coli chemotaxis pathway 51 resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. 52We further test whether pH differences between cyto-and periplasm as a result of chemotactic activity 53 3 can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches 54 conceptually function, but will need further improvement in terms of detection and assay types to be 55 practical for biosensing. 57 58Chemotaxis is the behaviour of cells to bias the direction of their motility in reaction to perceived 60 chemical gradients (1, 2). Chemotaxis by bacteria is rapid (ms-to s-scale) and does not require de 61 novo gene induction, since it is based on dynamic protein modifications and protein-p...