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1. Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function

   https://doi.org/10.1128/AEM.01626-19  

   Luu, Rita A.; Schomer, Rebecca A.; Brunton, Ceanne N.; Truong, Richard; Ta, Albert P.; Tan, Watumesa A.; Parales, Juanito V.; Wang, Yu-Jing; Huo, Yu-Wen; Liu, Shuang-Jiang; et al (November 2019, Applied and Environmental Microbiology)

   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. 

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2. Azospirillum brasilense AerC and Tlp4b Cytoplasmic Chemoreceptors Are Promiscuous and Interact with the Two Membrane-Bound Chemotaxis Signaling Clusters Mediating Chemotaxis Responses

   https://doi.org/10.1128/jb.00484-22  

   Ganusova, Elena E.; Rost, Madison; Aksenova, Anastasia; Abdulhussein, Mustafa; Holden, Alisha; Alexandre, Gladys (June 2023, Journal of Bacteriology)

   Maupin-Furlow, Julie A. (Ed.)

   ABSTRACT Chemotaxis in Bacteria and Archaea depends on the presence of hexagonal polar arrays composed of membrane-bound chemoreceptors that interact with rings of baseplate signaling proteins. In the alphaproteobacterium Azospirillum brasilense , chemotaxis is controlled by two chemotaxis signaling systems (Che1 and Che4) that mix at the baseplates of two spatially distinct membrane-bound chemoreceptor arrays. The subcellular localization and organization of transmembrane chemoreceptors in chemotaxis signaling clusters have been well characterized but those of soluble chemoreceptors remain relatively underexplored. By combining mutagenesis, microscopy, and biochemical assays, we show that the cytoplasmic chemoreceptors AerC and Tlp4b function in chemotaxis and localize to and interact with membrane-bound chemoreceptors and chemotaxis signaling proteins from both polar arrays, indicating that soluble chemoreceptors are promiscuous. The interactions of AerC and Tlp4b with polar chemotaxis signaling clusters are not equivalent and suggest distinct functions. Tlp4b, but not AerC, modulates the abundance of chemoreceptors within the signaling clusters through an unknown mechanism. The AerC chemoreceptor, but not Tlp4b, is able to traffic in and out of chemotaxis signaling clusters depending on its level of expression. We also identify a role of the chemoreceptor composition of chemotaxis signaling clusters in regulating their polar subcellular organization. The organization of chemotaxis signaling proteins as large membrane-bound arrays underlies chemotaxis sensitivity. Our findings suggest that the composition of chemoreceptors may fine-tune chemotaxis signaling not only through their chemosensory specificity but also through their role in the organization of polar chemotaxis signaling clusters. IMPORTANCE Cytoplasmic chemoreceptors represent about 14% of all chemoreceptors encoded in bacterial and archaeal genomes, but little is known about how they interact with and function in large polar assemblies of membrane-bound chemotaxis signaling clusters. Here, we show that two soluble chemoreceptors with a role in chemotaxis are promiscuous and interact with two distinct membrane-bound chemotaxis signaling clusters that control all chemotaxis responses in Azospirillum brasilense . We also found that any change in the chemoreceptor composition of chemotaxis signaling clusters alters their polar organization, suggesting a dynamic interplay between the sensory specificity of chemotaxis signaling clusters and their polar membrane organization. 

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3. McpT, a Broad-Range Carboxylate Chemoreceptor in Sinorhizobium meliloti

   https://doi.org/10.1128/JB.00216-21  

   Baaziz, Hiba; Compton, K. Karl; Hildreth, Sherry B.; Helm, Richard F.; Scharf, Birgit E. (August 2021, Journal of Bacteriology)

   Becker, Anke (Ed.)

   ABSTRACT Chemoreceptors enable the legume symbiont Sinorhizobium meliloti to detect and respond to specific chemicals released from their host plant alfalfa, which allows the establishment of a nitrogen-fixing symbiosis. The periplasmic region (PR) of transmembrane chemoreceptors act as the sensory input module for chemotaxis systems via binding of specific ligands, either directly or indirectly. S. meliloti has six transmembrane and two cytosolic chemoreceptors. However, the function of only three of the transmembrane receptors have been characterized so far, with McpU, McpV, and McpX serving as general amino acid, short-chain carboxylate, and quaternary ammonium compound sensors, respectively. In the present study, we analyzed the S. meliloti chemoreceptor McpT. High-throughput differential scanning fluorimetry assays, using Biolog phenotype microarray plates, identified 15 potential ligands for McpT PR , with the majority classified as mono-, di-, and tricarboxylates. S. meliloti exhibited positive chemotaxis toward seven selected carboxylates, namely, α-ketobutyrate, citrate, glyoxylate, malate, malonate, oxalate, and succinate. These carboxylates were detected in seed exudates of the alfalfa host. Deletion of mcpT resulted in a significant decrease of chemotaxis to all carboxylates except for citrate. Isothermal titration calorimetry revealed that McpT PR bound preferentially to the monocarboxylate glyoxylate and with lower affinity to the dicarboxylates malate, malonate, and oxalate. However, no direct binding was detected for the remaining three carboxylates that elicited an McpT-dependent chemotaxis response. Taken together, these results demonstrate that McpT is a broad-range carboxylate chemoreceptor that mediates chemotactic response via direct ligand binding and an indirect mechanism that needs to be identified. IMPORTANCE Nitrate pollution is one of the most widespread and challenging environmental problems that is mainly caused by the agricultural overapplication of nitrogen fertilizers. Biological nitrogen fixation by the endosymbiont Sinorhizobium meliloti enhances the growth of its host Medicago sativa (alfalfa), which also efficiently supplies the soil with nitrogen. Establishment of the S. meliloti - alfalfa symbiosis relies on the early exchange and recognition of chemical signals. The present study contributes to the disclosure of this complex molecular dialogue by investigating the underlying mechanisms of carboxylate sensing in S. meliloti . Understanding individual steps that govern the S. meliloti -alfalfa molecular cross talk helps in the development of efficient, commercial bacterial inoculants that promote the growth of alfalfa, which is the most cultivated forage legume in the world, and improves soil fertility. 

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4. An Azospirillum brasilense chemoreceptor that mediates nitrate chemotaxis has conditional roles in the colonization of plant roots

   https://doi.org/10.1128/aem.00760-24  

   Ganusova, Elena E; Russell, Matthew H; Patel, Siddhi; Seats, Terry; Alexandre, Gladys (June 2024, Applied and Environmental Microbiology)

   Reguera, Gemma (Ed.)

   ABSTRACT Motile plant-associated bacteria use chemotaxis and dedicated chemoreceptors to navigate gradients in their surroundings and to colonize host plant surfaces. Here, we characterize a chemoreceptor that we named Tlp2 in the soil alphaproteobacteriumAzospirillum brasilense. We show that the Tlp2 ligand-binding domain is related to the 4-helix bundle family and is conserved in chemoreceptors found in the genomes of many soil- and sediment-dwelling alphaproteobacteria. The promoter oftlp2is regulated in an NtrC- and RpoN-dependent manner and is most upregulated under conditions of nitrogen fixation or in the presence of nitrate. Using fluorescently tagged Tlp2 (Tlp2-YFP), we show that this chemoreceptor is present in low abundance in chemotaxis-signaling clusters and is prone to degradation. We also obtained evidence that the presence of ammonium rapidly disrupts Tlp2-YFP localization. Behavioral experiments using a strain lacking Tlp2 and variants of Tlp2 lacking conserved arginine residues suggest that Tlp2 mediates chemotaxis in gradients of nitrate and nitrite, with the R159 residue being essential for Tlp2 function. We also provide evidence that Tlp2 is essential for root surface colonization of some plants (teff, red clover, and cowpea) but not others (wheat, sorghum, alfalfa, and pea). These results highlight the selective role of nitrate sensing and chemotaxis in plant root surface colonization and illustrate the relative contribution of chemoreceptors to chemotaxis and root surface colonization.IMPORTANCEBacterial chemotaxis mediates host-microbe associations, including the association of beneficial bacteria with the roots of host plants. Dedicated chemoreceptors specify sensory preferences during chemotaxis. Here, we show that a chemoreceptor mediating chemotaxis to nitrate is important in the beneficial soil bacterium colonization of some but not all plant hosts tested. Nitrate is the preferred nitrogen source for plant nutrition, and plants sense and tightly control nitrate transport, resulting in varying nitrate uptake rates depending on the plant and its physiological state. Nitrate is thus a limiting nutrient in the rhizosphere. Chemotaxis and dedicated chemoreceptors for nitrate likely provide motile bacteria with a competitive advantage to access this nutrient in the rhizosphere. 

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5. Arsenite Oxidase Also Functions as an Antimonite Oxidase

   https://doi.org/10.1128/AEM.02981-14  

   Wang, Qian; Warelow, Thomas P.; Kang, Yoon-Suk; Romano, Christine; Osborne, Thomas H.; Lehr, Corinne R.; Bothner, Brian; McDermott, Timothy R.; Santini, Joanne M.; Wang, Gejiao; et al (February 2015, Applied and Environmental Microbiology)

   ABSTRACT Arsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work with Agrobacterium tumefaciens strain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show with in vivo experiments that a mutation in aioA [encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further, in vitro studies with the purified As(III) oxidase from Rhizobium sp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA of A. tumefaciens ) provide direct evidence of Sb(III) oxidation but also show a significantly decreased V max compared to that of As(III) oxidation. The aioBA genes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereas arsR gene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium. 

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