Tlr1612 is the major repressor of cell aggregation in the light-color-dependent c-di-GMP signaling network of Thermosynechococcus vulcanus

Gen Enomoto,Yukiko Okuda,Masahiko Ikeuchi

doi:10.1038/s41598-018-23628-4

Gen Enomoto, Yukiko Okuda + Show 1 more

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https://doi.org/10.1038/s41598-018-23628-4

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Abstract

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger involved in sessile/motile lifestyle transitions. We previously reported that c-di-GMP is a crucial inducer of cell aggregation of the cyanobacterium Thermosynechococcus vulcanus. The three cooperating cyanobacteriochrome photoreceptors (SesA/B/C) regulate cell aggregation in a light color–dependent manner by synthesizing/degrading c-di-GMP. Although a variety of c-di-GMP signaling proteins are encoded in cyanobacterial genomes, how c-di-GMP signaling networks are organized remains elusive. Here we experimentally demonstrate that the cellulose synthase Tll0007, which is essential for cell aggregation, binds c-di-GMP although the affinity is low (Kd = 63.9 ± 5.1 µM). We also show that SesA—the main trigger of cell aggregation—is subject to strict product feedback inhibition (IC50 = 1.07 ± 0.13 µM). These results suggest that SesA-produced c-di-GMP may not directly bind to Tll0007. We therefore systematically analyzed all 10 of the genes encoding proteins containing a c-di-GMP synthesis/degradation domain. We identified Tlr1612, harboring both domains, as the major repressor of cell aggregation under the repressing teal-green light irradiation. tlr1612 acts downstream of sesA and is not regulated transcriptionally by light color, suggesting that Tlr1612 may be involved in c-di-GMP amplification in the signaling cascade. Post-transcriptional control is likely crucial for the light-regulated c-di-GMP signaling.

Highlights

Cyclic diguanylate, or bis-(3′-5′)-cyclic diguanylic acid (c-di-GMP) is a second messenger that is most commonly found in bacteria
We previously reported that both c-di-GMP and the PilZ domain of Tll0007 are essential for inducing cell aggregation of T. vulcanus at 31 °C21,22
To assess c-di-GMP binding to Tll0007, in the present study we designed c-di-GMP biosensor proteins using a fluorescence resonance energy transfer (FRET)-based system by concatenating two fluorescent proteins with the PilZ domain of Tll0007 in between (Fig. 1A), according to[28,29]

Summary

Introduction

Bis-(3′-5′)-cyclic diguanylic acid (c-di-GMP) is a second messenger that is most commonly found in bacteria. Bacterial genomes often contain multiple genes encoding DGCs and c-di-GMP PDEs that encode GGDEF/EAL/HD-GYP domain proteins, which contrasts with the number of genes that govern other second messengers such as cAMP14,15. This suggests that a complex c-di-GMP–based signaling network operates in diverse bacterial cells. The Thermosynechococcus genome contains a total of 10 genes encoding DGCs and c-di-GMP PDEs (Fig. S1) This complexity is much less than that of other cyanobacteria (e.g., 28 genes in Synechocystis), the relationship and interdependence of the c-di-GMP DGC and PDE proteins remain to be explored in T. vulcanus

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