Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

Abstract

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3′,3′-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

Distinct chromophore–protein environments enable asymmetric activation of a bacteriophytochrome-activated diguanylate cyclase

“…This implies that dimers featuring one Pfr-like state and one Pfr state, but also combinations of one Pr protomer with a Pfr protomer, could provide the structural asymmetry of the phytochrome-sensory module that is sufficient for activation of the diguanylate cyclase output in solution (9). Considering the relevance of asymmetric intermediates as part of the GGDEF mechanism for formation of the symmetric final product cyclic dimeric-GMP (44), the observed asymmetry in all PadC members characterized so far (8) might play an important role for the efficient stimulation of the corresponding diguanylate cyclase activity. Although the formation of the bacterial second messenger cyclic dimeric-GMP, which is involved in the regulation of lifestyle decisions (44), might benefit from such an asymmetric molecular mechanism, other bacteriophytochrome-linked effector domains might not require this type of structural asymmetry.…”

Promoting bidirectional extracellular electron transfer of Shewanella oneidensis MR‐1 for hexavalent chromium reduction via elevating intracellular cAMP level

“…Similarly, cyclic diadenosine monophosphate (c‐di‐AMP) was also identified to regulate the biofilm formation in diverse bacteria (Peng, Zhang, Bai, Zhou, & Wu, 2016). Recently, GacA was found to specifically regulate the intracellular cyclic guanosine monophosphate–adenosine monophosphate (cyclic GMP‐AMP) level and was essential for Fe(III) particle‐associated growth for Geobacter sulfurreducensis (Hallberg et al, 2019). These results imply that increasing the intracellular levels of other second messengers such as c‐di‐GMP, c‐di‐AMP, and cyclic AMP‐GMP, should be the efficient approaches for constructing engineered Shewanella strains with the elevated bidirectional EET abilities.…”

Second messengers and divergent HD-GYP enzymes regulate 3’,3’-cGAMP signaling

“…GACs are structurally related to the GGDEF family of diguanylate cyclases (DGCs), which are classically associated with cyclic di-GMP signaling, but instead have 'Hypr' GGDEF domains harboring specific variations in the active site that favor cGAMP production (9). We recently demonstrated that the Hypr GGDEF GSU1658 (GsGacA) from Geobacter sulfurreducens regulates cGAMP but not cyclic di-GMP levels in vivo, resulting in a specific phenotype distinct from the biofilm-associated phenotype for the DGC EsnD (7).…”

“…While our focus to date has been on understanding the basis for cGAMP production by GACs with Hypr GGDEF domains (7,9), the regulation of GAC activity is relatively unexplored ( Fig. 1).…”

“…GacB could be subject to product autoinhibition by cGAMP, or it could be cross-regulated by cyclic di-GMP or cyclic di-AMP. Recent studies of GsGacA yielded phenotypic and transcriptomic evidence consistent with crossinhibition by cyclic di-GMP, but did not show a direct mechanism (7).…”