A Two-Component System That Modulates Cyclic di-GMP Metabolism Promotes Legionella pneumophila Differentiation and Viability in Low-Nutrient Conditions

During its life cycle, the environmental pathogen alternates between a replicative and transmissive cell type when cultured in broth, macrophages, or amoebae. Within a protozoan host, further differentiates into the hardy cell type known as the mature infectious form (MIF). The second messenger cyclic di-GMP coordinates lifestyle changes in many bacterial species, but its role in the life cycle is less understood. Using an broth culture model that approximates the intracellular transition from the replicative to the transmissive form, here we investigate the contribution to differentiation of a two-component system (TCS) that regulates cyclic di-GMP metabolism. The TCS is encoded by and is cotranscribed with , which encodes a protein upregulated in MIF cells. The promoter for this operon is RpoS dependent and induced in nutrient-limiting conditions that do not support replication, as demonstrated using a reporter and quantitative PCR (qPCR). The response regulator of the TCS (Lpg0277) is a bifunctional enzyme that both synthesizes and degrades cyclic di-GMP. Using a panel of site-directed point mutants, we show that cyclic di-GMP synthesis mediated by a conserved GGDEF domain promotes growth arrest of replicative , accumulation of pigment and poly-3-hydroxybutyrate storage granules, and viability in nutrient-limiting conditions. Genetic epistasis tests predict that the MIF protein Lpg0279 acts as a negative regulator of the TCS. Thus, is equipped with a regulatory network in which cyclic di-GMP stimulates the switch from a replicative to a resilient state equipped to survive in low-nutrient environments. Although an intracellular pathogen, has developed mechanisms to ensure long-term survival in low-nutrient aqueous conditions. Eradication of from contaminated water supplies has proven challenging, as outbreaks have been traced to previously remediated systems. Understanding the genetic determinants that support persistence in low-nutrient environments can inform design and assessment of remediation strategies. Here we characterize a genetic locus that encodes a two-component signaling system ( ) and a putative regulator protein ( ) that modulates the production of the messenger molecule cyclic di-GMP. We show that this locus promotes both cell differentiation and survival in nutrient-limiting conditions, thus advancing the understanding of the mechanisms that contribute to environmental resilience.