The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure–function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF–RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand–receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor–synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.
Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia cenocepacia in a biofilm model, mutations were most frequently selected in the conserved gene rpfR. RpfR uniquely integrates two major signaling systems—quorum sensing and the motile–sessile switch mediated by cyclic-di-GMP—by two domains that sense, respond to, and control the synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The BDSF response in turn regulates the activity of diguanylate cyclase and phosphodiesterase domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these domains to produce unique life history strategies by regulating cyclic-di-GMP levels, global transcriptional responses, biofilm production, and polysaccharide composition. These phenotypes translated into distinct ecology and biofilm structures that enabled mutants to coexist and produce more biomass than expected from their constituents grown alone. This study shows that when bacterial populations are selected in environments challenging the limits of their plasticity, the evolved mutations not only alter genes at the nexus of signaling networks but also reveal the scope of their regulatory functions.
25Many bacteria cycle between sessile and motile forms in which they must sense and 26 respond to internal and external signals to coordinate appropriate physiology. Maintaining 27 fitness requires genetic networks that have been honed in variable environments to 28 integrate these signals. The identity of the major regulators and how their control 29 mechanisms evolved remain largely unknown in most organisms. During four different 30 evolution experiments with the opportunist betaproteobacterium Burkholderia 31 cenocepacia in a biofilm model, mutations were most frequently selected in the conserved 32 gene rpfR. RpfR uniquely integrates two major signaling systems --quorum sensing and 33 the motile-sessile switch mediated by cyclic-d-GMP --by two domains that sense, 34 respond to, and control synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The 35 BDSF response in turn regulates activity of diguanylate cyclase and phosphodiesterase 36 domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these 37 domains to produce unique life history strategies by regulating cyclic-di-GMP levels, 38 global transcriptional responses, biofilm production, and polysaccharide composition. 39These phenotypes translated into distinct ecology and biofilm structures that enabled 40 mutants to coexist and produce more biomass than expected from their constituents 41 grown alone. This study shows that when bacterial populations are selected in 42 environments challenging the limits of their plasticity, the evolved mutations not only alter 43 genes at the nexus of signaling networks but also reveal the scope of their regulatory 44 functions. 45 46 Significance statement 47 ecological strategies that can coexist and even increase net growth. This study 55demonstrates that a single gene may coordinate complex life histories in biofilm-dwelling 56 bacteria and that selection in defined environments can reshape niche breadth by single 57 mutations. 58 59 60 Bacteria have experienced strong selection over billions of generations to efficiently and 62 reversibly switch from free-swimming to surface-bound life. The record of this selection is 63 etched in the genomes of thousands of species, many of which have tens or even 64 hundreds of genes that govern this lifestyle switch (1). At the nexus of this switch in the 65 majority of bacteria is the second messenger molecule cyclic diguanylate monophosphate 66 (c-di-GMP). Many genes synthesize, degrade, or directly bind and respond to c-di-GMP 67 that in high concentrations promotes a sessile lifestyle and biofilm production and in low 68 concentrations promotes a solitary, motile life. Those genomes with the greatest apparent 69 redundancy in this signaling network demonstrate the highest plasticity along this motile-70 sessile axis (2). For instance, in Vibrio cholerae, there are 41 distinct diguanylate cyclases 71 (DGCs) that synthesize c-di-GMP and 31 different phosphodiesterases (PDEs) that 72 degrade this molecule (1). 73 74Recent theory and experiments...
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