Shotgun metagenomics and marker gene amplicon sequencing can be used to directly measure or predict the functional repertoire of the microbiota en masse, but current methods do not readily estimate the functional capability of individual microorganisms. Here we present BugBase, an algorithm that predicts organism-level coverage of functional pathways as well as biologically interpretable phenotypes such as oxygen tolerance, Gram staining and pathogenic potential, within complex microbiomes using either whole-genome shotgun or marker gene sequencing data. We find BugBase's organism-level pathway coverage predictions to be statistically higher powered than current 'bag-of-genes' approaches for discerning functional changes in both host-associated and environmental microbiomes.

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743 -7481. To investigate whether this stimulation was due to a soluble c1 subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein By dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that By subunits, purified from bovine retinal transducin (By,), markedly stimulated the hydrolysis of phosphatidylinositol4,Sbisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by by, was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin a subunit and was additive to activation of phospholipase C by guanosine 5'-[3-0-thio]triphosphate. byt also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to By,, but was also observed with highly purified subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in granulocytes. Only one of these forms was sensitive to stimulation by by,, demonstrating that stimulation of phospholipase C by by subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein By subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.The hydrolysis by phospholipase C of phosphatidylinositol 4,5-bisphosphate (PtdInsPJ to inositol 1,4,5-trisphosphate (InsP,) and diacylglycerol is a key mechanism by which many extracellular signalling molecules (hormones, growth factors and neurotransmitters) regulate the functions of their target cells [l, 21. There is ample evidence to suggest that many of the receptors interacting with these mediators stimulate phospholipase C via a guanine-nucleotide-binding protein (G protein) [3-51. In certain cell types, e.g.

Fecal microbiota transplantation (FMT) is now widely used to treat recurrent Clostridium difficile infection, but has been less studied as a means to restore microbiome diversity and composition following antibiotic or chemotherapy treatments. The purpose of our study was to assess the efficacy of FMT to reverse antibiotic- and chemotherapy-induced gut dysbiosis in a mouse model. C57BL/6J mice were treated with ampicillin for 1 week and/or received a single intraperitoneal injection of 5-Fluorouracil. Fresh stool was collected and analyzed using shotgun metagenomics and the Illumina sequencing platform. Ampicillin caused a significant and immediate decrease in bacterial species richness and diversity that persisted for one week. In mice that received FMT, disruption of the intestinal microbiota was reversed immediately. Antibiotic and chemotherapy administration caused significant alteration in species distribution, including a decrease in the relative proportions of Clostridium scindens and Faecalibacterium prausnitzii, and an increase in known pathogenic species. In mice receiving FMT, we observed a significant increase in species known to exhibit anti-inflammatory properties. Moreover, chemotherapy led to a critical decrease in key ‘health-promoting’ species and to an altered functional profile, especially when chemotherapy was administered in tandem with antibiotics, and that FMT can ameliorate these effects.

Bovine peripheral neutrophils contain high levels of a 40-kDa pertussis toxin substrate, which was found highly enriched in a light membrane fraction upon subcellular fractionation of neutrophil homogenates. The 40-kDa pertussis toxin substrate, referred to as a,, was purified to near homogeneity from t h s fraction by sequential ion-exchange, gel-filtration and hydrophobic chromatography. Purified a, was shown to interact with p,, subunits, undergo ADP-ribosylation by pertussis toxin, and bind guanine nucleotides with high affinity. The mobility of purified a, on SDS/polyacrylamide gels was intermediate between those of the a subunits of Gi and Go, purified from bovine brain, and slightly lower than the mobility of the a subunit of transducin (G,). Several polyclonal antisera against the a subunits of bovine GI and G, did not react with a, on immunoblots. CW 6, a polyclonal antiserum reactive against the bovine ai, reacted only minimally with a,.These results suggest that the major pertussis toxin substrate of bovine neutrophils, designated G,, is structurally different from previously identified pertussis toxin substrates and may represent a novel guanine-nucleotide-binding protein.