Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense

Abstract

Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state.IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and modulates their contribution to the overall chemotaxis response. Here, we used an optogenetic system to perturb intracellular concentrations of the bacterial second messenger c-di-GMP to show that in some chemotaxis receptors, c-di-GMP functions in a similar feedback loop to connect the metabolic status of the cells to the sensory activity of chemotaxis receptors.

Optogenetic Module for Dichromatic Control of c-di-GMP Signaling

“…However, previous studies revealed that the half-lives of truncated BLUF proteins are often longer than those of the full-length proteins (31). Interestingly, this seemingly long half-life observed in vitro proved inconsequential in in vivo studies (49).…”

“…It can also be used for studying how transient changes in c-di-GMP levels affect motility, the cell cycle, predation, and numerous other phenotypes regulated by this ubiquitous second messenger (19,20). While the experimental setups of this study employed static conditions and "bulk" phenotypes, we anticipate that some of the most exciting applications of the engineered module will involve the fast modulation of c-di-GMP levels in cell populations, as demonstrated in the accompanying paper (49) and in single cells.…”

The MapZ-Mediated Methylation of Chemoreceptors Contributes to Pathogenicity of Pseudomonas aeruginosa

“…The finding that a functional defect in MapZ affects the methylation of the aerotaxis chemoreceptors Aer indicates that MapZ-mediated mechanism is likely to govern the taxis toward O 2 -rich sources. In a recent study, it was found that the aerotaxis of the alpha-proteobacterium Azospirillum brasilense is regulated by intracellular c-di-GMP concentrations (O'Neal et al, 2017). However, unlike P. aeruginosa that relies on the discrete single PilZ domain protein MapZ, c-di-GMP mediates the changes in the aerotaxis response of A. brasilense by binding to the C-terminal PilZ domain of the chemotaxis receptors Tlp1.…”

A PilZ-Containing Chemotaxis Receptor Mediates Oxygen and Wheat Root Sensing in Azospirillum brasilense

“…Only two of the chemotaxis systems, Che1 and Che4, are directly involved in chemotaxis, and the other two systems have functions outside of chemotaxis, including a likely role in flagellum biosynthesis [Che2 (unpublished data)] and cyst development [Che3 (Bible et al, 2015)]. To date, the function of only two of the chemotaxis receptors have been characterized in this bacterial species: Tlp1 and AerC (Greer-Phillips et al, 2004; Xie et al, 2010; Russell et al, 2013; O’Neal et al, 2017). Both are important in aerotaxis (taxis in an oxygen gradient), but only Tlp1 is necessary for plant root colonization (Greer-Phillips et al, 2004; Xie et al, 2010).…”