Heterologous Expression ofPseudomonas putidaMethyl-Accepting Chemotaxis Proteins YieldsEscherichia coliChemotactic to Aromatic Compounds

Abstract

25 26 27 28 Running title: Escherichia coli chemotactic to aromatic compounds 29 2 ABSTRACT (250 w.) 30 Escherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, 31 sugars and peptides. We envisioned modifying chemotaxis specificity of E. coli by expressing 32 heterologous chemoreceptors from Pseudomonas putida enabling attraction either to toluene 33 or benzoate. The mcpT gene encoding the type 40H methyl-accepting chemoreceptor for 34 toluene from Pseudomonas putida MT53 and the pcaY gene for the type 40H receptor for 35 benzoate and related molecules from P. putida F1 were expressed from the trg promoter on a 36 plasmid in motile wild-type E. coli MG1655. E. coli cells expressing McpT accumulated in 37 chemoattraction assays to sources with 60-200 µM toluene; less strongly than the response to 38 100 µM serine, but statistically significantly stronger than to sources without any added 39 attractant. An McpT-mCherry fusion protein was detectably expressed in E. coli and yielding 40 weak but distinguishable membrane and polar foci in 1% of cells. E. coli expressing PcaY 41 showed weak attraction to 0.1-1 mM benzoate but 50-70% of cells localized the PcaY-42 mCherry fusion to their membrane. We conclude that implementing heterologous receptors in 43 the E. coli chemotaxis network is possible and, upon improvement of the compatibility of the 44 type 40H chemoreceptors, may bear interest for biosensing . 45 46 IMPORTANCE (150 w.) 47 65Chemotaxis of Escherichia coli is strong and highly reproducible with known and 66 potent chemoattractants, such as serine or aspartate, and has been widely studied (4, 5). 67Unfortunately, E. coli does not naturally display chemotaxis towards molecules of potential 68 interest for environmental monitoring, such as aromatic or chlorinated solvents. Given its 69 relatively narrow native chemo-attractant range, it is interesting to investigate whether the E. 70 coli chemotaxis system can be complemented by heterologous chemoreceptors. One 71 important characteristic of methyl-accepting chemotaxis proteins (MCPs) and chemotaxis 72 effector proteins (e.g., CheY) is their structural conservation among bacteria (6-8). E. coli 73 possesses five chemotaxis receptors, but other environmental bacteria frequently encode 74 many more chemoreceptors albeit with often unknown effectors. For example, Pseudomonas 75 species can encode more than 20 MCPs in their genomes (9, 10). A few studies have 76 demonstrated successful expression of heterologous chemoreceptors in E. coli. For example, 77 several MCPs from Shewanella oneidensis could be expressed in E. coli, enabling energy 78 taxis with nitrate (11). Also, Aer-2, a soluble receptor from Pseudomonas aeruginosa 79 involved in aerotaxis, and PctApp, a putative MCP for amino acids from Pseudomonas putida 80 were shown to partially trigger chemotaxis response when expressed in E. coli (12, 13).81 However, no MCPs involved in sensing of environmental pollutants have to date been 82 functionally expressed in E. coli. 83 ...

Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

“…After drying during 5 min, 150 μl of washed exponentially-growing E. coli cell suspension in motility buffer were pipetted at the edge between the large coverslip and the glass slide. Cell accumulation was measured after 30 min by light microscopy close to the agarose plug source as outlined elsewhere (66).…”