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. IMPORTANCEMost 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.

Second messengers and divergent HD‐GYP phosphodiesterases regulate 3′,3′‐cGAMP signaling

“…We had expected the opposite trend upon addition of PdeH, which is a well‐studied cyclic di‐GMP‐specific PDE from E. coli previously called YhjH (Reinders et al , ; Sarenko et al , ). While PdeH did decrease the population of cyclic di‐GMP‐bright cells (Fig.…”

Cyclic di-GMP: second messenger extraordinaire

“…Despite of a total of 13 PDEs being encoded in the genome of this organism, only PdeH is able to reduce c-di-GMP levels and license motility in growing E. coli cells 35,36 (see below). The observations that most PDEs are readily expressed and that a large fraction of these enzymes can be genetically activated to substitute for PdeH in motility control implied that most of these enzymes simply lack the appropriate stimuli under laboratory conditions 37 . DGCs and PDEs also engage in downstream signaling through direct interactions with their target molecules thereby providing a platform for "spatially localized" control of cellular processes 24,38,39 .…”

High-throughput mapping of the phage resistance landscape inE. coli

“…dgcJ, one of the top scorers in both RB-TnSeq and CRISPRi LOF screens encodes diguanylate cyclase-J, which is involved in the biosynthesis of cyclic-di-GMP, whereas the seven c-di-GMP-specific phosphodiesterases (PDEs) that are involved in degradation of c-di-GMP showed the highest fitness scores in the Dub-seq screen. Though the signaling network of cyclic-di-GMP is complex, deletion of diguanylate cyclases (DGCs) or overexpression of PDEs is known to reduce c-di-GMP levels, inhibit curli and biofilm formation, while increasing cellular motility [144][145][146]166]. The high fitness scores for dgcJ in RB-TnSeq and CRISPRi screens is intriguing considering it is one of the 12 DGCs encoded on E. coli K-12 genome [167,168], and none of the other DGCs show phenotypes in our screens.…”