Functional and metagenomic level diversities of human gut symbiont-derived glycolipids.

Bioactive metabolites produced by symbiotic microbiota causally impact host health and disease, nonetheless, incomplete functional annotation of genes as well as complexities and dynamic nature of microbiota make understanding species-level contribution in production and actions difficult. Alpha-galactosylceramides produced by Bacteroides fragilis (BfaGC) are one of the first modulators of colonic immune development, but biosynthetic pathways and the significance of the single species in the symbiont community still remained elusive. To address these questions at the microbiota level, we have investigated the lipidomic profiles of prominent gut symbionts and the metagenome-level landscape of responsible gene signatures in the human gut. We first elucidated the chemical diversity of sphingolipid biosynthesis pathways of major bacterial species. In addition to commonly shared ceramide backbone synthases showing two distinct intermediates, alpha-galactosyltransferase (agcT), the necessary and sufficient component for BfaGC production and host colonic type I natural killer T (NKT) cell regulation by B. fragilis, was characterized by forward-genetics based targeted metabolomic screenings. Phylogenetic analysis of agcT in human gut symbionts revealed that only a few ceramide producers have agcT and hence can produce aGCs, on the other hand, structurally conserved homologues of agcT are widely distributed among species lacking ceramides. Among them, alpha-glucosyl-diacylglycerol(aGlcDAG)-producing glycosyltransferases with conserved GT4-GT1 domains are one of the most prominent homologs in gut microbiota, represented by Enterococcus bgsB . Of interest, aGlcDAGs produced by bgsB can antagonize BfaGC-mediated activation of NKT cells, showing the opposite, lipid structure-specific actions to regulate host immune responses. Further metagenomic analysis of multiple human cohorts uncovered that the agcT gene signature is almost exclusively contributed by B. fragilis , regardless of age, geographical and health status, where the bgsB signature is contributed by >100 species, of which abundance of individual microbes is highly variable. Our results collectively showcase the diversities of gut microbiota producing biologically relevant metabolites in multiple layers-biosynthetic pathways, host immunomodulatory functions and microbiome-level landscapes in the host.

A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae.

The bacterial second messenger bis-(3'-5')-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain-containing diguanylate cyclases and degraded by HD-GYP domain-containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain-containing PDE-As to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.

Sugar-mediated regulation of a c-di-GMP phosphodiesterase in Vibrio cholerae.

Biofilm formation protects bacteria from stresses including antibiotics and host immune responses. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae, but the underlying mechanisms remain unclear. Here, we show that EIIAGlc, a component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), regulates the intracellular concentration of the cyclic dinucleotide c-di-GMP, and thus biofilm formation. The availability of preferred sugars such as glucose affects EIIAGlc phosphorylation state, which in turn modulates the interaction of EIIAGlc with a c-di-GMP phosphodiesterase (hereafter referred to as PdeS). In a Drosophila model of V. cholerae infection, sugars in the host diet regulate gut colonization in a manner dependent on the PdeS-EIIAGlc interaction. Our results shed light into the mechanisms by which some nutrients regulate biofilm formation and host colonization.

An evolutionary optimization of a rhodopsin-based phototrophic metabolism in Escherichia coli.

BACKGROUND: The expression of the Gloeobacter rhodopsin (GR) in a chemotrophic Escherichia coli enables the light-driven phototrophic energy generation. Adaptive laboratory evolution has been used for acquiring desired phenotype of microbial cells and for the elucidation of basic mechanism of molecular evolution. To develop an optimized strain for the artificially acquired phototrophic metabolism, an ancestral E. coli expressing GR was adaptively evolved in a chemostat reactor with constant illumination and limited glucose conditions. This study was emphasized at an unexpected genomic mutation contributed to the improvement of microbial performance. RESULTS: During the chemostat culture, increase of cell size was observed, which were distinguished from that of the typical rod-shaped ancestral cells. A descendant ET5 strain was randomly isolated from the chemostat culture at 88-days. The phototrophic growth and the light-induced proton pumping of the ET5 strain were twofold and eightfold greater, respectively, than those of the ancestral E. coli strain. Single point mutation of C1082A at dgcQ gene (encoding diguanylate cyclase, also known as the yedQ gene) in the chromosome of ET5 strain was identified from whole genome sequencing analysis. An ancestral E. coli complemented with the same dgcQ mutation from the ET5 was repeated the subsequently enhancements of light-driven phototrophic growth and proton pumping. Intracellular c-di-GMP, the product of the diguanylate cyclase (dgcQ), of the descendant ET5 strain was suddenly increased while that of the ancestral strain was negligible. CONCLUSIONS: Newly acquired phototrophic metabolism of E. coli was further improved via adaptive laboratory evolution by the rise of a point mutation on a transmembrane cell signaling protein followed by increase of signal molecule that eventually led an increase proton pumping and phototrophic growth.