RESUMEN
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 ß-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 ß-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.
Asunto(s)
Proteínas Bacterianas/genética , Quimiotaxis/genética , Escherichia coli/genética , Pseudomonas putida/genética , Transducción de Señal , Proteínas de Escherichia coli/genética , Expresión Génica , Genoma Bacteriano , Proteínas Quimiotácticas Aceptoras de Metilo/genéticaRESUMEN
The study of chemotaxis to xenobiotic chemicals in soil bacteria has revealed that the core mechanism for transduction of chemotactic signals is conserved. Responses to chemicals degraded by specialized catabolic pathways are often coordinately regulated with degradation genes, and in some cases auxiliary processes such as transport are integrated into the sensory process. In addition, degradation genes and associated chemotaxis genes carried on transmissible plasmids may facilitate the dissemination and evolution of catabolic and sensory systems. However, the strategies and receptors used by bacteria to sense chemicals are difficult to predict solely by bioinformatics, and much work is needed to uncover the range of chemicals detected and the specific functions of the numerous chemoreceptors present in catabolically versatile soil bacteria.
Asunto(s)
Quimiotaxis , Xenobióticos/metabolismo , Bacterias/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Células Quimiorreceptoras/metabolismo , Quimiotaxis/genética , Plásmidos , Xenobióticos/químicaRESUMEN
Aromatic and hydroaromatic compounds that are metabolized through the ß-ketoadipate catabolic pathway serve as chemoattractants for Pseudomonas putidaâ F1. A screen of P. putidaâ F1 mutants, each lacking one of the genes encoding the 18 putative methyl-accepting chemotaxis proteins (MCPs), revealed that pcaY encodes the MCP required for metabolism-independent chemotaxis to vanillate, vanillin, 4-hydroxybenzoate, benzoate, protocatechuate, quinate, shikimate, as well as 10 substituted benzoates that do not serve as growth substrates for P. putidaâ F1. Chemotaxis was induced during growth on aromatic compounds, and an analysis of a pcaY-lacZ fusion revealed that pcaY is expressed in the presence of ß-ketoadipate, a common intermediate in the pathway. pcaY expression also required the transcriptional activator PcaR, indicating that pcaY is a member of the pca regulon, which includes three unlinked gene clusters that encode five enzymes required for the conversion of 4-hydroxybenzoate to tricarboxylic acid cycle intermediates as well as the major facilitator superfamily transport protein PcaK. The 4-hydroxybenzoate permease PcaK was shown to modulate the chemotactic response by facilitating the uptake of 4-hydroxybenzoate, which leads to the accumulation of ß-ketoadipate, thereby increasing pcaY expression. The results show that chemotaxis, transport and metabolism of aromatic compounds are intimately linked in P. putida.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzoatos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Pseudomonas putida/fisiología , Adipatos/metabolismo , Secuencia de Aminoácidos , Antioxidantes/metabolismo , Benzaldehídos/metabolismo , Transporte Biológico , Quimiotaxis , Genes Bacterianos , Proteínas Quimiotácticas Aceptoras de Metilo , Familia de Multigenes , Mutación , Parabenos/metabolismo , Pseudomonas putida/genética , RegulónRESUMEN
Soil bacteria are generally capable of growth on a wide range of organic chemicals, and pseudomonads are particularly adept at utilizing aromatic compounds. Pseudomonads are motile bacteria that are capable of sensing a wide range of chemicals, using both energy taxis and chemotaxis. Whilst the identification of specific chemicals detected by the ≥26 chemoreceptors encoded in Pseudomonas genomes is ongoing, the functions of only a limited number of Pseudomonas chemoreceptors have been revealed to date. We report here that McpC, a methyl-accepting chemotaxis protein in Pseudomonas putida F1 that was previously shown to function as a receptor for cytosine, was also responsible for the chemotactic response to the carboxylated pyridine nicotinic acid.
Asunto(s)
Proteínas Bacterianas/metabolismo , Quimiotaxis , Citosina/metabolismo , Proteínas de la Membrana/metabolismo , Niacina/metabolismo , Pseudomonas putida/fisiología , Proteínas Quimiotácticas Aceptoras de MetiloRESUMEN
Previous studies have demonstrated that Pseudomonas putida strains are not only capable of growth on a wide range of organic substrates, but also chemotactic towards many of these compounds. However, in most cases the specific chemoreceptors that are involved have not been identified. The complete genome sequences of P. putida strains F1 and KT2440 revealed that each strain is predicted to encode 27 methyl-accepting chemotaxis proteins (MCPs) or MCP-like proteins, 25 of which are shared by both strains. It was expected that orthologous MCPs in closely related strains of the same species would be functionally equivalent. However, deletion of the gene encoding the P. putida F1 orthologue (locus tag Pput_4520, designated mcfS) of McpS, a known receptor for organic acids in P. putida KT2440, did not result in an obvious chemotaxis phenotype. Therefore, we constructed individual markerless MCP gene deletion mutants in P. putida F1 and screened for defective sensory responses to succinate, malate, fumarate and citrate. This screen resulted in the identification of a receptor, McfQ (locus tag Pput_4894), which responds to citrate and fumarate. An additional receptor, McfR (locus tag Pput_0339), which detects succinate, malate and fumarate, was found by individually expressing each of the 18 genes encoding canonical MCPs from strain F1 in a KT2440 mcpS-deletion mutant. Expression of mcfS in the same mcpS deletion mutant demonstrated that, like McfR, McfS responds to succinate, malate, citrate and fumarate. Therefore, at least three receptors, McfR, McfS, and McfQ, work in concert to detect organic acids in P. putida F1.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Carboxílicos/metabolismo , Quimiotaxis , Proteínas de la Membrana/metabolismo , Pseudomonas putida/fisiología , Proteínas Bacterianas/genética , Eliminación de Gen , Proteínas de la Membrana/genética , Proteínas Quimiotácticas Aceptoras de Metilo , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/genética , Especificidad por SustratoRESUMEN
The phenylacetic acid (PAA) degradation pathway is a widely distributed funneling pathway for the catabolism of aromatic compounds, including the environmental pollutants styrene and ethylbenzene. However, bacterial chemotaxis to PAA has not been studied. The chemotactic strain Pseudomonas putida F1 has the ability to utilize PAA as a sole carbon and energy source. We identified a putative PAA degradation gene cluster (paa) in P. putida F1 and demonstrated that PAA serves as a chemoattractant. The chemotactic response was induced during growth with PAA and was dependent on PAA metabolism. A functional cheA gene was required for the response, indicating that PAA is sensed through the conserved chemotaxis signal transduction system. A P. putida F1 mutant lacking the energy taxis receptor Aer2 was deficient in PAA taxis, indicating that Aer2 is responsible for mediating the response to PAA. The requirement for metabolism and the role of Aer2 in the response indicate that P. putida F1 uses energy taxis to detect PAA. We also revealed that PAA is an attractant for Escherichia coli; however, a mutant lacking a functional Aer energy receptor had a wild-type response to PAA in swim plate assays, suggesting that PAA is detected through a different mechanism in E. coli. The role of Aer2 as an energy taxis receptor provides the potential to sense a broad range of aromatic growth substrates as chemoattractants. Since chemotaxis has been shown to enhance the biodegradation of toxic pollutants, the ability to sense PAA gradients may have implications for the bioremediation of aromatic hydrocarbons that are degraded via the PAA pathway.