11During its life cycle, the environmental pathogen Legionella pneumophila alternates 12 between a replicative and a transmissive cell type when cultured in broth, macrophages, or 13 amoebae. Within a protozoan host, L. pneumophila further differentiates into the hardy cell type 14 known as the Mature Infectious Form (MIF). The second messenger cyclic-di-GMP coordinates 15 lifestyle changes in many bacterial species, but its role in the L. pneumophila life cycle is less 16 understood. Using an in vitro broth culture model that approximates the intracellular transition 17 from the replicative to transmissive form, here we investigate the contribution to L. 18 pneumophila differentiation of a two-component system (TCS) that regulates cyclic-di-GMP 19 metabolism. The TCS is encoded by lpg0278-lpg0277 and is co-transcribed with lpg0279, which 20 encodes a protein upregulated in MIF cells. Using a gfp-reporter, we demonstrate that the 21 promoter for this operon is RpoS-dependent and induced in nutrient-limiting conditions that do 22Although an intracellular pathogen, L. pneumophila has developed mechanisms to ensure 33 long-term survival in low-nutrient aqueous conditions. Eradication of L. pneumophila from 34 contaminated water supplies has proven challenging, as outbreaks have been traced to previously 35 remediated systems. Understanding the genetic determinants that support L. 36 pneumophila persistence in low-nutrient environments can inform design of remediation 37 methods. Here we characterize a genetic locus that encodes a two-component signaling system 38 (lpg0278-lpg0277) and a putative regulator protein (lpg0279) that modulates production of the 39 messenger molecule cyclic-di-GMP. We show that this locus promotes both L. pneumophila cell 40 differentiation and survival in nutrient-limiting conditions, thus advancing our understanding of 41 the mechanisms that contribute to L. pneumophila environmental resilience. 42 43 infectious Mature Infectious Form (MIF), a cell-type believed to be prevalent in the environment 55 (5, 6). 56To alternate between replication within phagocytes and persistence within nutrient-poor 57 aquatic environments, L. pneumophila relies on multiple regulatory mechanisms that coordinate 58 rapid adaption to changing conditions (3). For example, replication in broth requires amino acids 59 as the primary carbon source (7, 8), and a reduction in amino acid availability activates 60 regulatory pathways that trigger conversion from the exponential (E) phase to the post-61 exponential (PE) transmissive phase (9, 10). The L. pneumophila life cycle is governed by a 62 sophisticated regulatory network that includes the stringent response enzymes RelA and SpoT, 63 multiple alternative sigma factors including RpoS and FliA, two-component regulatory systems, 64 small regulatory RNAs, and CsrA post-transcriptional repressors (3, 11, 12). Driving the E to PE 65 differentiation is a stringent response pathway coordinated by the alarmone guanosine penta-and 66 tetraphosphate (abbreviated here as pp...