Abstract
Bacterial chemotaxis enables cells to move in gradients of environmental signals that are sensed by chemoreceptors. A major limitation in the field consists currently in the lacking information on the signal(s) or chemoeffectors recognized by the majority of chemoreceptors, which in turn hampers understanding of how the environment has shaped chemotactic capabilities. A number of different approaches for signal identification have been reported that are often labour‐intensive. Here, we developed a straightforward NADH‐coupled CheA ATPase assay to systematically screen potential chemoeffectors. Obtained results are complemented with differential scanning fluorimetry and isothermal titration calorimetry analyses, as well as structural protein modelling to study the chemoreceptors of Pseudomonas parafulva PSR09‐11288. Using this integrated approach, we identified L‐malate, citrate, L‐glutamine, and L‐asparagine as chemoattractants and revealed that chemoreceptor B2J77_03605 specifically recognizes L‐malate and citrate, whereas chemoreceptor B2J77_13170 binds L‐glutamine and L‐asparagine. The universality of this approach is shown by the fact that both chemoreceptors belong to different families and possess sensor domains that belong to the HBM and dCache families, respectively. This pipeline is a scalable workflow for chemoeffector identification, permitting systematic exploration of bacterial chemotactic capabilities.