Study of NIT domain‐containing chemoreceptors from two global phytopathogens and identification of NIT domains in eukaryotes

Abstract

Bacterial signal transduction systems are typically activated by the binding of signal molecules to receptor ligand binding domains (LBDs), such as the NIT LBD. We report here the identification of the NIT domain in more than 15,000 receptors that were present in 30 bacterial phyla, but also in 19 eukaryotic phyla, expanding its known phylogenetic distribution. The NIT domain formed part of seven receptor families that either control transcription, mediate chemotaxis or regulate second messenger levels. We have produced the NIT domains from chemoreceptors of the bacterial phytopathogens Pectobacterium atrosepticum (PacN) and Pseudomonas savastanoi (PscN) as individual purified proteins. High‐throughput ligand screening using compound libraries revealed a specificity for nitrate and nitrite binding. Isothermal titration calorimetry experiments showed that PacN‐LBD bound preferentially nitrate ( K D = 1.9 μM), whereas the affinity of PscN‐LBD for nitrite ( K D = 2.1 μM) was 22 times higher than that for nitrate. Analytical ultracentrifugation experiments indicated that PscN‐LBD is monomeric in the presence and absence of ligands. The R182A mutant of PscN did not bind nitrate or nitrite. This residue is not conserved in the NIT domain of the Pseudomonas aeruginosa chemoreceptor PA4520, which may be related to its failure to bind nitrate/nitrite. The magnitude of P. atrosepticum chemotaxis towards nitrate was significantly greater than that of nitrite and pacN deletion almost abolished responses to both compounds. This study highlights the important role of nitrate and nitrite as signal molecules in life and advances our knowledge on the NIT domain as universal nitrate/nitrite sensor module.

Differential CheR affinity for chemoreceptor C-terminal pentapeptides biases chemotactic responses

“…Typhimurium (14,32). SCRI1043 has a single chemosensory pathway (14), and the chemoreceptors analyzed thus far respond to amino acids (PacB, PacC), quaternary amines (PacA), and nitrate (PacN) (37)(38)(39). We show here that there are very significant differences in the affinity of the individual pentapeptides for CheR that are reflected in differences in the magnitude of the chemotactic responses.…”

Sensing the environment by bacterial plant pathogens: What do their numerous chemoreceptors recognize?

“…This lack of knowledge is partly due to the fact that the major model species in chemotaxis research have alternative lifestyles. Research has so far shown that phytopathogen chemoreceptors respond to a number of compounds of the general metabolism like amino acids (Cerna‐Vargas et al., 2019 ; Kumar Verma et al., 2018 ; McKellar et al., 2015 ; Tumewu et al., 2021 ; Velando et al., 2023 ), organic acids (Hida et al., 2015 , 2019 ), quaternary amines (Matilla, Velando, Tajuelo, et al., 2022 ) or nitrate (Gálvez‐Roldán et al., 2023 ; Monteagudo‐Cascales et al., 2023 ). Although these molecules are not plant‐specific compounds, they are present at high concentrations in plant exudates (Lohaus, 2022 ); Vives‐Peris et al., 2020 ) and are therefore major signals for host recognition.…”

“…Although this method identifies ligands that bind to LBDs, it fails to monitor chemoreceptor activation by non‐canonical mechanisms or stimulation by ligand‐loaded solute binding proteins. However, the combined use of in vivo and in vitro approaches is a powerful set‐up to identify the function of chemoreceptors in plant pathogens, as illustrated by a number of examples (Cerna‐Vargas et al., 2019 ; Gálvez‐Roldán et al., 2023 ; Monteagudo‐Cascales et al., 2023 ; Santamaría‐Hernando et al., 2022 ; Velando et al., 2023 ).…”

Journey to the center of the phage; revealing the ejectosome of Pectobacterium bacteriophage ΦM1

“…Phage ΦM1 encodes a DNA genome approximately 44 kb long, organized into 52 putative genes. The host bacterium for ΦM1, P. atrosepticum, is a globalGram-negative potato pathogen found in both temperate and tropical regions, and is known to colonize the intercellular spaces of plant cells and degrade the cell wall, causing blackleg disease and aerial stem rot (22,23). Recent studies have focused on identifying biocontrol agents against P. atrosepticum, including antimicrobial chemicals, zinc/silver nanoparticles, and antagonistic bacterial isolates (24)(25)(26)(27).…”