title: Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli.
abstract: Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell.
  
  
  OBJECTIVE
  Most bacteria possess multiple diguanylate cyclases and phosphodiesterases. Genetic studies have proposed that these enzymes show signaling specificity by contributing to distinct cellular processes without much cross talk. Thus, spatial separation of individual c-di-GMP signaling units was postulated. However, since most cyclases and phosphodiesterases harbor N-terminal signal input domains, it is equally possible that most of these enzymes lack their activating signals under laboratory conditions, thereby simulating signaling specificity on a genetic level. We demonstrate that a subset of E. coli phosphodiesterases can be activated genetically to affect the global c-di-GMP pool and thus influence different c-di-GMP-dependent processes. Although this does not exclude spatial confinement of individual phosphodiesterases, this study emphasizes the importance of environmental signals for activation of phosphodiesterases.
authors.affiliation: Focal Area of Infection Biology, Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Alberto
authors.initials: A
authors.lastname: Reinders
authors.affiliation: Focal Area of Structural Biology and Biophysics, Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Chee-Seng
authors.initials: CS
authors.lastname: Hee
authors.affiliation: Focal Area of Infection Biology, Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Shogo
authors.initials: S
authors.lastname: Ozaki
authors.affiliation: Research IT Department, Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Adam
authors.initials: A
authors.lastname: Mazur
authors.affiliation: Focal Area of Infection Biology, Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Alex
authors.initials: A
authors.lastname: Boehm
authors.affiliation: Focal Area of Structural Biology and Biophysics, Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Tilman
authors.initials: T
authors.lastname: Schirmer
authors.affiliation: Focal Area of Infection Biology, Biozentrum, University of Basel, Basel, Switzerland urs.jenal@unibas.ch.
authors.forename: Urs
authors.initials: U
authors.lastname: Jenal
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D009068:Movement; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D014741:Video Recording
chemical_list: D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP