Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function

Abstract

Soil bacteria adapt to diverse and rapidly changing environmental conditions by sensing and responding to environmental cues using a variety of sensory systems. Two-component systems are a widespread type of signal transduction system present in all three domains of life and typically are comprised of a sensor kinase and a response regulator. Many two-component systems function by regulating gene expression in response to environmental stimuli. The bacterial chemotaxis system is a modified two-component system with additional protein components and a response that, rather than regulating gene expression, involves behavioral adaptation and results in net movement toward or away from a chemical stimulus. Soil bacteria generally have 20 to 40 or more chemoreceptors encoded in their genomes. To simplify the identification of chemoeffectors (ligands) sensed by bacterial chemoreceptors, we constructed hybrid sensor proteins by fusing the sensor domains of Pseudomonas putida chemoreceptors to the signaling domains of the Escherichia coli NarX/NarQ nitrate sensors. Responses to potential attractants were monitored by β-galactosidase assays using an E. coli reporter strain in which the nitrate-responsive narG promoter was fused to lacZ Hybrid receptors constructed from PcaY, McfR, and NahY, which are chemoreceptors for aromatic acids, tricarboxylic acid cycle intermediates, and naphthalene, respectively, were sensitive and specific for detecting known attractants, and the β-galactosidase activities measured in E. coli correlated well with results of chemotaxis assays in the native P. putida strain. In addition, a screen of the hybrid receptors successfully identified new ligands for chemoreceptor proteins and resulted in the identification of six receptors that detect propionate.IMPORTANCE Relatively few of the thousands of chemoreceptors encoded in bacterial genomes have been functionally characterized. More importantly, although methyl-accepting chemotaxis proteins, the major type of chemoreceptors present in bacteria, are easily identified bioinformatically, it is not currently possible to predict what chemicals will bind to a particular chemoreceptor. Chemotaxis is known to play roles in biodegradation as well as in host-pathogen and host-symbiont interactions, but many studies are currently limited by the inability to identify relevant chemoreceptor ligands. The use of hybrid receptors and this simple E. coli reporter system allowed rapid and sensitive screening for potential chemoeffectors. The fusion site chosen for this study resulted in a high percentage of functional hybrids, indicating that it could be used to broadly test chemoreceptor responses from phylogenetically diverse samples. Considering the wide range of chemical attractants detected by soil bacteria, hybrid receptors may also be useful as sensitive biosensors.

Keywords: Pseudomonas putida; biosensor; methyl-accepting chemotaxis protein; receptor; two-component system.

FIG 1

Results of ITC experiments to measure binding of ligands to the PcaY LBD. Binding of the aromatic compounds 4-hydroxybenzoate (4-HBA), benzoate, 4-nitrobenzoate (4-NBA), and vanillate and the hydroaromatic compounds quinate and shikimate, which are sensed by P. putida F1 via PcaY, was tested.

FIG 2

Design of PcaY-Nar hybrids. (A) Amino acid (aa) sequence alignments of the HAMP domains in Tar (from Salmonella enterica); Tsr, NarX, and NarQ (from E. coli); PcaY (from P. putida F1); and the constructed PcaY-NarX/Q hybrids. The HAMP domain consists of two amphipathic helices (AS1 and AS2) joined by an unstructured connecting sequence (3, 70). Sequences from NarX, NarQ, and PcaY are highlighted in blue, yellow, and green, respectively. (B) Schematic diagram displaying the elements of NarX, NarQ, and PcaY in the hybrid constructs. Segments from NarX, NarQ, and PcaY are shown in yellow, blue, and green, respectively. The red star denotes the approximate location of the L222Q amino acid substitution in PcaYHAMP-NarQ^L222Q.

FIG 3

Φ(narG-lacZ) expression in E. coli VJS5054 carrying the four PcaY-Nar hybrid constructs. Responses to 0.5 mM and 1 mM 4-HBA were tested. A schematic model of the four hybrids is shown, with elements from NarX, NarQ, and PcaY denoted in yellow, blue, and green, respectively. β-Galactosidase activity is reported in Miller units, and results are the averages from at least three independent experiments, with error bars representing standard deviations.

FIG 4

Φ(narG-lacZ) expression in E. coli VJS5054 carrying the native NarQ and PcaY-NarQ hybrid receptors. β-Galactosidase activity in response to the presence of 40 mM nitrate or 1 mM 4-HBA, quinate, 3-nitrobenzoate (3-NBA), 2-chlorobenzoate (2-CBA), or p-coumarate was monitored. β-Galactosidase activity is reported in Miller units, and the results are the averages from at least three independent experiments, with error bars representing standard deviations.

FIG 5

Comparison of the sensitivity of the hybrid PcaY-NarQ receptor in E. coli VJS5054 with that of the native chemoreceptor PcaY in P. putida F1. (A) Responses of the E. coli reporter strain carrying PcaY-NarQ to 4-HBA, benzoate, PCA, vanillate, vanillin, quinate, and shikimate measured using β-galactosidase assays. Colored triangles below the results indicate increasing concentrations of the ligand (0, 0.01, 0.1, 1.0, and 10 mM, respectively). Results are averages from at least three independent experiments, with error bars representing standard deviations. (B) Responses of wild-type P. putida F1 to 4-HBA, benzoate, PCA, vanillate, vanillin, quinate, and shikimate measured using quantitative capillary assays. The average number of cells in capillaries containing buffer only (170 ± 54 CFU/ml) was subtracted from each data set. Results shown are the averages from at least three independent experiments, and error bars represent the standard errors of the means. Results shown for quinate and 4-HBA were reported previously (21).

FIG 6

Φ(narG-lacZ) expression in E. coli VJS5054 carrying McpR-NarQ. β-Galactosidase assays were carried out on cultures grown with the indicated concentrations of succinate, fumarate, malate, and citrate. Results are averages from at least three independent experiments, with error bars representing standard deviations.

FIG 7

Φ(narG-lacZ) expression in E. coli VJS5054 carrying the NahY-NarQ hybrid reporter. β-Galactosidase assays were carried out on cultures grown with the indicated concentrations of naphthalene. Results are averages from at least three independent experiments, with error bars representing standard deviations.

FIG 8

Φ(narG-lacZ) expression in E. coli VJS5054 carrying six different MCP-NarQ hybrid receptors in response to propionate. β-Galactosidase assays were carried out on cultures grown with the indicated concentrations of propionate. Results are averages from at least three independent experiments, with error bars representing standard deviations.

FIG 9

Chemotactic responses of P. putida mutants to 1 mM propionate in quantitative swim plate assays. (A) Responses of the P. putida F1 Δaer-2 deletion mutant (black bar, control) were compared to those of double mutants lacking aer-2 and each of the putative propionate receptor genes identified in the MCP-NarQ hybrid reporter screen (gray bars, strains listed in Table 3). The Δaer-2 ΔmcfP mutant (light-gray bar on the far right) is the ortholog of the P. putida KT2440 propionate chemoreceptor. The Δaer-2 ΔPput_0342 mutant (light-gray bar) was not expected to respond to propionate based on the absence of a response of the E. coli reporter strain carrying the Pput_0342-NarQ hybrid to propionate. (B) Responses of the P. putida F1Δ11 deletion mutant RPF018 harboring either the empty vector pRK415 or pRK415Km (black bars) were compared to those of strains carrying the indicated wild-type MCP genes in the respective vectors (plasmid names listed in Table 4). The gray bars represent complementation of propionate chemotaxis with the receptors identified in the hybrid receptor propionate screen. The light-gray bars represent complementation with Pput_0342 (not expected to complement) and mcfP (expected to complement). Results are averages from at least three independent experiments, with error bars representing standard deviations. Means with the same letter are not significantly different (P < 0.05 by one-way analysis of variance [ANOVA] with a Tukey multiple-comparison test).