Metabolic Value Chemoattractants Are Preferentially Recognized at Broad Ligand Range Chemoreceptor of Pseudomonas putida KT2440

Abstract

Bacteria have evolved a wide range of chemoreceptors with different ligand specificities. Typically, chemoreceptors bind ligands with elevated specificity and ligands serve as growth substrates. However, there is a chemoreceptor family that has a broad ligand specificity including many compounds that are not of metabolic value. To advance the understanding of this family, we have used the PcaY_PP (PP2643) chemoreceptor of Pseudomonas putida KT2440 as a model. Using Isothermal Titration Calorimetry we showed here that the recombinant ligand binding domain (LBD) of PcaY_PP recognizes 17 different C6-ring containing carboxylic acids with KD values between 3.7 and 138 μM and chemoeffector affinity correlated with the magnitude of the chemotactic response. Mutation of the pcaY_PP gene abolished chemotaxis to these compounds; phenotype that was restored following gene complementation. Growth experiments using PcaY_PP ligands as sole C-sources revealed functional relationships between their metabolic potential and affinity for the chemoreceptor. Thus, only 7 PcaY_PP ligands supported growth and their KD values correlated with the length of the bacterial lag phase. Furthermore, PcaY_PP ligands that did not support growth had significantly higher KD values than those that did. The receptor has thus binds preferentially compounds that serve as C-sources and amongst them those that rapidly promote growth. Tightest binding compounds were quinate, shikimate, 3-dehydroshikimate and protocatechuate, which are at the interception of the biosynthetic shikimate and catabolic quinate pathways. Analytical ultracentrifugation studies showed that ligand free PcaY_PP-LBD is present in a monomer-dimer equilibrium (KD = 57.5 μM). Ligand binding caused a complete shift to the dimeric state, which appears to be a general feature of four-helix bundle LBDs. This study indicates that the metabolic potential of compounds is an important parameter in the molecular recognition by broad ligand range chemoreceptors.

The ligand‐binding domain of a chemoreceptor from Comamonas testosteroni has a previously unknown homotrimeric structure

“…The individual LBD of Tar exists in a dynamic equilibrium between monomers and dimers and ligand binding induces dimerization since the ligand interacts with both monomers to stabilize the dimer (Milligan and Koshland, ; Yeh et al , ; Biemann and Koshland, ; Yeh et al , ). Similar to Tar, some other helical LBDs also alter the oligomeric state upon ligand binding, such as 4HB domains of PcaY_PP (Fernandez et al , ) and CtpH (Rico‐Jimenez et al , ), HBM domains of McpS (Lacal et al , ) and McpQ (Martin‐Mora et al , ), and PilJ domain of McpN (Martin‐Mora et al , ). On the contrary, the oligomerization of some dCACHE and sCACHE LBDs is not altered by ligand binding and their ligand binding sites are not located at the dimeric interface (Rico‐Jimenez et al , ; Martin‐Mora et al , ; Gavira et al , ).…”

The activity of the C4-dicarboxylic acid chemoreceptor of Pseudomonas aeruginosa is controlled by chemoattractants and antagonists

“…These experiments have resulted in two different scenarios. On one hand, individual 4HB or HBM domains are primarily monomeric in its ligand-free state and ligand binding induces dimerization 25 , 32 , 42 , 43 . In contrast, dCACHE domains were found to be monomeric in the absence and presence of ligands 15 .…”

“…Several previous studies have shown that the affinity of ligands for the LBD correlate with the magnitude of the chemosensory output 16 , 43 . However, this correlation was not observed for the PA2652 ligands (Figs 3 and 5 ).…”

“…Based on the hypothesis that ligand induced chemoreceptor dimerization is a prerequisite for signalling 41 , the effect of ligands on the oligomeric state of different LBD types has been investigated in the past 15 , 19 , 25 , 28 , 42 , 43 . The individual 4HB domains of receptors Tar, CtpH and PcaY_PP 19 , 42 , 43 and HBM LBDs (receptors McpS and McpQ) 25 , 28 were found to be largely monomeric in their ligand free state whereas in all cases the binding of the ligand induced complete LBD dimerization. This is due to the fact that ligands bind at the dimer interface and that amino acids from both monomers of the dimer establish contacts with the bound ligand 26 , 80 .…”

Sinorhizobium meliloti Chemoreceptor McpV Senses Short-Chain Carboxylates via Direct Binding

“…Similarly, when the PR of a Pseudomonas aeruginosa chemoreceptor was fused to the signaling domain of the Tar chemoreceptor to create a chimera in E. coli, correlations were found between ligand affinity and signal output or attractant utilization using a fluorescence resonance energy transfer (FRET) assay (41). However, this pattern does not hold for all systems, such as the Pseudomonas putida chemoreceptors for TCA intermediates (McpS) and cyclic carboxylates (PcaY_PP) that do not show a distinct difference in ligand-binding affinity for differently utilized attractants (42,43). In conclusion, small Combining DSF with Biolog PM plates is an effective and facile method for screening the ligand profile of a chemoreceptor (30,33).…”