A Minimal Threshold of c-di-GMP Is Essential for Fruiting Body Formation and Sporulation in Myxococcus xanthus

Abstract

Generally, the second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-di-GMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be—at least partially—functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus.

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

“…Radiolabeled c-di-GMP was produced as described (64). Briefly, 32 P-labeled c-di-GMP was synthesized using 10 μM purified C. crescentus His 6 -DgcA and 1 mM GTP/[α-32 P]GTP (0.1 μCi/μL) overnight at 30°C in a buffer containing 50 mM Tris·HCl, pH 8.0, 300 mM NaCl, and 10 mM MgCl 2 .…”

Highly Signal-Responsive Gene Regulatory Network Governing Myxococcus Development

“…Starvation triggers the Nla24, Mrp, and EBP cascade modules, but the molecular signals are largely unknown. For the recently discovered Nla24 module, cyclic diguanylate (c-di-GMP) acts as a second messenger of the unknown starvation signal, and genes for exopolysaccharide (EPS) production are induced, which is necessary for fruiting body formation [9] (Figure 2). Starvation also causes another guanine nucleotide second messenger to be made in cells – RelA activity causes guanosine penta- and tetraphosphates [(p)ppGpp)] to accumulate when ribosomes stall due to amino acid limitation [10, 11] (Figure 2).…”

“…This module was discovered recently [9], so much remains to be learned about its role in the GRN. So far, starvation has been shown to induce transcription of dmxB , which encodes a diguanylate cyclase that boosts the cellular level of c-di-GMP early in development.…”

“…So far, starvation has been shown to induce transcription of dmxB , which encodes a diguanylate cyclase that boosts the cellular level of c-di-GMP early in development. These events appear to be under control of the Dif chemosensory system [9], which responds not only to starvation, but to type IV pili of neighboring cells and lipids (perhaps released from lysing cells) [39]. c-di-GMP binds to Nla24 [9], an EBP encoded at a locus for EPS biosynthesis [40] but whose cognate protein kinase is unknown.…”

“…These events appear to be under control of the Dif chemosensory system [9], which responds not only to starvation, but to type IV pili of neighboring cells and lipids (perhaps released from lysing cells) [39]. c-di-GMP binds to Nla24 [9], an EBP encoded at a locus for EPS biosynthesis [40] but whose cognate protein kinase is unknown. Assuming that Nla24 is phosphorylated, it appears that a c-di-GMP·Nla24~P complex activates transcription of certain eps genes during development, increasing EPS production to enhance aggregation and sporulation [9] (Figure 3).…”