High specificity in CheR methyltransferase function: CheR2 of Pseudomonas putida is essential for chemotaxis, whereas CheR1 is involved in biofilm formation

J Biol Chem. 2013 Jun 28;288(26):18987-99. doi: 10.1074/jbc.M113.472605. Epub 2013 May 15.

Abstract

Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (KD of 0.14-2.2 μM) than the substrate S-adenosylmethionine (KD of 22-43 μM), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.

Keywords: Calorimetry; CheR Methyltransferase; Chemoreceptor; Chemotaxis; Protein Methylation; Pseudomonas; Receptor Modification; Receptors; S-Adenosylmethionine (SAM); Signal Transduction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / metabolism*
  • Biofilms*
  • Calorimetry
  • Chemotaxis / physiology*
  • Methyltransferases / metabolism*
  • Molecular Sequence Data
  • Mutation
  • Oligonucleotides / genetics
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Pseudomonas putida / enzymology*
  • S-Adenosylmethionine / metabolism
  • Sequence Homology, Amino Acid
  • Signal Transduction
  • Substrate Specificity
  • Ultracentrifugation

Substances

  • Bacterial Proteins
  • Oligonucleotides
  • S-Adenosylmethionine
  • CheR protein, Pseudomonas putida
  • Methyltransferases