SUMMARY The gastrointestinal (GI) tract contains much of the body’s serotonin (5-hydroxytryptamine, 5-HT), but mechanisms controlling the metabolism of gut-derived 5-HT remain unclear. Here we demonstrate that the microbiota plays a critical role in regulating host 5-HT. Indigenous spore-forming bacteria (Sp) from the mouse and human microbiota promote 5-HT biosynthesis from colonic enterochromaffin cells (ECs), which supply 5-HT to the mucosa, lumen and circulating platelets. Importantly, microbiota-dependent effects on gut 5-HT significantly impact host physiology, modulating GI motility and platelet function. We identify select fecal metabolites that are increased by Sp and that elevate 5-HT in chromaffin cell cultures, suggesting direct metabolic signaling of gut microbes to ECs. Furthermore, elevating luminal concentrations of particular microbial metabolites increases colonic and blood 5-HT in germ-free mice. Altogether, these findings demonstrate that Sp are important modulators of host 5-HT, and further highlight a key role for host-microbiota interactions in regulating fundamental 5-HT-related biological processes.

PREFACE Animals assemble and maintain a diverse, yet host-specific gut microbial community. In addition to characteristic microbial compositions along the longitudinal axis of the intestines, discrete bacterial communities form in microhabitats, such as the gut lumen, colon mucus layers and colon crypts. In this Review, we examine how spatial distribution of symbiotic bacteria among physical niches in the gut impacts the development and maintenance of a resilient microbial ecosystem. We consider novel hypotheses for how nutrient selection, immune activation and other mechanisms control the biogeography of bacteria in the gut and discuss the relevance of this spatial heterogeneity to health and disease.

Cell 161, 264-276; April 9, 2015) In Figure S5D of this article, the representative flow cytometry plot of forward versus side scatter for unstimulated platelets was incorrectly duplicated during the final formatting of the paper for SPF+PCPA and GF conditions. The figure has been corrected online, and the originally published descriptions of the results in the text and figure legend are accurate.In Figure 3A, the ''GF+conv'' bar represents germ-free (GF) mice conventionalized with standard pathogen-free (SPF) microbiota on postnatal day 21 (P21). The published main text incorrectly referred to conventionalization on P42. Though we show in Figure 1B very similar levels of colonic serotonin after conventionalization on P21 versus P42, the ''GF+conv'' data in Figure 3A is specifically from GF mice conventionalized on P21. This error in the text has also been corrected online.Overall, these changes have no bearing on the experimental results or conclusions presented in the manuscript. We apologize for any inconvenience that these errors have caused.

Mammals harbor a complex gut microbiome, comprised of bacteria that confer immunologic, metabolic and neurologic benefits1. Despite advances in sequence-based microbial profiling and myriad studies defining microbiome composition during health and disease, little is known about the molecular processes employed by symbiotic bacteria to stably colonize the gastrointestinal (GI) tract. We sought to define how mammals assemble and maintain the Bacteroides, one of the most numerically prominent genera of the human microbiome. While the gut normally contains hundreds of bacterial species2,3, we surprisingly find that germ-free mice mono-associated with a single Bacteroides are resistant to colonization by the same, but not different, species. To identify bacterial mechanisms for species-specific saturable colonization, we devised an in vivo genetic screen and discovered a unique class of Polysaccharide Utilization Loci (PUL) that are conserved among intestinal Bacteroides. We named this genetic locus the commensal colonization factors (ccf). Deletion of the ccf genes in the model symbiont, Bacteroides fragilis, results in colonization defects in mice and reduced horizontal transmission. The ccf genes of B. fragilis are up-regulated during gut colonization, preferentially at the colonic surface. When we visualize microbial biogeography within the colon, B. fragilis penetrates the colonic mucus and resides deep within crypt channels, while ccf mutants are defective in crypt association. Remarkably, the CCF system is required for B. fragilis colonization following microbiome disruption with Citrobacter rodentium infection or antibiotic treatment, suggesting the niche within colonic crypts represents a reservoir for bacteria to maintain long-term colonization. These findings reveal that intestinal Bacteroides have evolved species-specific physical interactions with the host that mediate stable and resilient gut colonization, and the CCF system represents a novel molecular mechanism for symbiosis.

The immune system responds vigorously to microbial infection, while permitting life-long colonization by the microbiome. Mechanisms that facilitate the establishment and stability of the gut microbiota remain poorly described. We discovered that a sensor/regulatory system in the prominent human commensal Bacteroides fragilis modulates its surface architecture to invite binding of immunoglobulin A (IgA). Specific immune recognition facilitated bacterial adherence to cultured intestinal epithelial cells and intimate association with the gut mucosal surface in vivo. The IgA response was required for B. fragilis, and other commensal species, to occupy a defined mucosal niche that mediated stable colonization of the gut through exclusion of exogenous competitors. Therefore, in addition to its role in pathogen clearance, we propose that IgA responses can be co-opted by the microbiome to engender robust host-microbial symbiosis.

The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A from Pseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two-to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and Δorn strains of P. aeruginosa PA14 for pGpG stability. The lysates from Δorn showed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the Δorn strain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP-regulated pel promoter. Additionally, the c-di-GMP-governed auto-aggregation and biofilm phenotypes were elevated in the Δorn strain in a pel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the Δorn strain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.cyclic di-GMP | oligoribonuclease | pGpG | PDE-B | nanoRNase

Germinal centers (GCs), structures normally associated with B cell immunoglobulin (Ig) hypermutation and development of high-affinity antibodies upon infection or immunization, are present in gut-associated lymphoid organs of humans and mice under steady state. Gut-associated (ga)GCs can support antibody responses to enteric infections and immunization 1 . However, whether B cell selection and antibody affinity maturation can take place in face of the chronic and diverse antigenic stimulation characteristic of steady-state gaGCs is less clear 2 – 8 . Combining multicolor “Brainbow” fate-mapping and single-cell Ig sequencing, we find that 5–10% of gaGCs from specific pathogen-free (SPF) mice contained highly-dominant “winner” clones at steady state, despite rapid turnover of GC B cells. Monoclonal antibodies (mAbs) derived from these clones showed increased binding to commensal bacteria compared to their unmutated ancestors, consistent with antigen-driven selection and affinity maturation. Frequency of highly-selected gaGCs was markedly higher in germ-free (GF) than in SPF mice, and winner B cells in GF gaGCs were enriched in public IgH clonotypes found across multiple individuals, indicating strong B cell receptor (BCR)-driven selection in the absence of microbiota. Vertical colonization of GF mice with a defined microbial consortium (Oligo-MM 12 ) did not eliminate GF-associated clonotypes, yet induced a concomitant commensal-specific, affinity-matured B cell response. Thus, positive selection can take place in steady-state gaGCs, at a rate that is tunable over a wide range by the presence and composition of the microbiota.

The cyclic dinucleotide c-di-GMP is a master regulator of bacterial virulence and biofilm formation. The activations of c-di-GMP metabolism proteins, diguanylate cyclases (DGCs) and phosophodiesterases (PDEs), usually lead to diametrically opposite phenotypes in bacteria. Analogues of c-di-GMP, which can selectively modulate the activities of c-di-GMP processing proteins, will be useful chemical tools for studying and altering bacterial behavior. Herein we report that a conservative modification of one of the phosphate groups in c-di-GMP with a bridging sulfur in the phosphodiester linkage affords an analogue called endo-S-c-di-GMP. Computational, NMR (including DOSY), and CD experiments all reveal that, unlike c-di-GMP, endo-S-c-di-GMP does not readily form higher aggregates. The lower propensity of endo-S-c-di-GMP to form aggregates (as compared to that of c-di-GMP) is probably due to a higher activation barrier to convert from the "open" conformer (where the two guanines are on opposite faces) to the "closed" conformer (where the two guanines are on the same face). Consequently, endo-S-c-di-GMP has selectivity for proteins that bind monomeric but not dimeric c-di-GMP, which form from the "closed" conformer. For example, endo-S-c-di-GMP can inhibit the hydrolysis of c-di-GMP by RocR (a PDE enzyme that binds monomeric c-di-GMP) but did not bind to Alg44 (a PilZ protein) or regulate WspR (a DGC enzyme that has been shown to bind to dimeric c-di-GMP). This work demonstrates that selective binding to different classes of c-di-GMP binding proteins could be achieved by altering analogue conformer populations (conformational steering). We provide important design principles for the preparation of selective PDE inhibitors and reveal the role played by the c-di-GMP backbone in c-di-GMP polymorphism and binding to processing proteins.