Production of 3′,3′-cGAMP by a Bdellovibrio bacteriovorus promiscuous GGDEF enzyme, Bd0367, regulates exit from prey by gliding motility

Bacterial second messengers play a crucial role in regulating a wide range of bacterial behaviors and lifestyles. One such messenger, cyclic di-GMP (c-di-GMP), is synthesized by diguanylate cyclase enzymes known as GGDEF proteins, which are broadly distributed across bacterial species. Recently, a subset of these enzymes—referred to as hybrid promiscuous (Hypr) GGDEF proteins—has been identified in certain bacteria. These enzymes not only produce c-di-GMP but also generate another second messenger, 3′,3′-cyclic-GMP-AMP (cGAMP), a more recently discovered signaling molecule.

One such Hypr GGDEF protein, Bd0367, is found in the predatory bacterium Bdellovibrio bacteriovorus. A strain lacking the bd0367 gene was observed to invade prey cells normally but failed to exit the prey remnants after replication, due to an inability to perform gliding motility on solid surfaces. However, the specific signaling mechanism controlling this exit behavior remained unclear.

In this study, we demonstrate that cGAMP signaling is functionally active in B. bacteriovorus and that Bd0367 serves as a major in vivo producer of cGAMP. Although Bd0367 also synthesizes c-di-GMP and small amounts of c-di-AMP, cGAMP production is a key functional output. Site-directed mutagenesis of Bd0367, substituting serine 214 with aspartate (S214D), shifted its activity toward primarily producing c-di-GMP. The resulting bd0367^S214D mutant strain exhibited the same phenotype as the deletion strain—loss of gliding motility and inability of progeny to exit prey cells—thereby confirming that this process is regulated specifically by cGAMP.

Interestingly, deletion of bd0367 also disrupted flagellar biogenesis. This was linked to a secondary, compensatory mutation in fliS, a gene encoding a flagellin chaperone homolog, suggesting that c-di-GMP plays a role in regulating swimming motility. Thus, Bd0367 represents the first known example of a single enzyme that synthesizes and regulates multiple bacterial second messengers—c-di-GMP and cGAMP—via the same GGDEF domain in vivo.

Unlike previously described roles for these signaling molecules in other bacterial species, in B. bacteriovorus, c-di-GMP and cGAMP govern two distinct motility systems: flagellar swimming and surface gliding, respectively. Both forms of motility are critical for completing the bacterium’s predatory lifecycle, including replication within prey and the subsequent exit from the prey cell.