A novel locally c-di-GMP-controlled exopolysaccharide synthase required for N4 phage infection of E. coli

Abstract

A major target of c-di-GMP signaling is the production of biofilm-associated extracellular polymeric substances (EPS), which in Escherichia coli K-12 include amyloid curli fibres, phosphoethanolamine-modified (pEtN-)cellulose and poly-N-acetyl-glucosamine (PGA). However, the characterized c-di-GMP-binding effector systems are largely outnumbered by the 12 diguanylate cyclases (DGCs) and 13 phosphodiesterases (PDEs), which synthetize and degrade c-di-GMP, respectively. E. coli possesses a single protein with a potentially c-di-GMP-binding MshEN domain, NfrB, which - together with the outer membrane protein NfrA - is known to serve as a receptor system for phage N4. Here, we show that NfrB not only binds c-di-GMP with high affinity, but as a novel c-di-GMP-controlled glycosyltransferase synthesizes a secreted EPS, which can impede motility and is required as an initial receptor for phage N4 infection. In addition, a systematic screening of the 12 DGCs of E. coli K-12 revealed that specifically DgcJ is required for the infection with phage N4 and interacts directly with NfrB. This is in line with local signaling models, where specific DGCs and/or PDEs form protein complexes with particular c-di-GMP effector/target systems. Our findings thus provide further evidence that intracellular signaling pathways, which all use the same diffusible second messenger, can act in parallel in a highly specific manner. ImportanceKey findings in model organisms led to the concept of local signaling, challenging the dogma of a gradually increasing global intracellular c-di-GMP concentration driving the motile-sessile transition in bacteria. In our current model, bacteria dynamically combine global as well as local signaling modes, in which specific DGCs and/or PDEs team up with effector/target systems in multiprotein complexes. Our present study highlights a novel example of how specificity in c-di-GMP signaling can be achieved by showing NfrB as a novel c-di-GMP binding effector in E. coli, which is controlled in a local manner specifically by DgcJ. We further show that NfrB (which was initially found as a part of a receptor system for phage N4) is involved in the production of a novel exopolysaccharide. Finally, our data shine new light on host interaction of phage N4, which uses this exopolysaccharide as an initial receptor for adsorption.