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Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis.

Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis.

The genera Catabacter (family ‘Catabacteraceae’) and Christensenella (family Christensenellaceae ) are close relatives within the phylum Firmicutes . Members of these genera are strictly anaerobic, non-spore-forming and short straight rods with diverse phenotypes. Phylogenetic analysis of 16S rRNA genes suggest that Catabacter splits Christensenella into a polyphyletic clade. In an effort to ensure that family/genus names represent monophyletic clades, we performed a whole-genome based analysis of the genomes available for the cultured representatives of these genera: four species of Christensenella and two strains of Catabacter hongkongensis . A concatenated alignment of 135 shared protein sequences of single-copy core genes present in the included strains indicates that C. hongkongensis is indeed nested within the Christensenella clade. Based on their evolutionary relationship, we propose the transfer of Catabacter hongkongensis to the genus Christensenella as Christensenella hongkongensis comb. nov.

The genera Catabacter (family ‘Catabacteraceae’) and Christensenella (family Christensenellaceae ) are close relatives within the phylum Firmicutes . Members of these genera are strictly anaerobic, non-spore-forming and short straight rods with diverse phenotypes. Phylogenetic analysis of 16S rRNA genes suggest that Catabacter splits Christensenella into a polyphyletic clade. In an effort to ensure that family/genus names represent monophyletic clades, we performed a whole-genome based analysis of the genomes available for the cultured representatives of these genera: four species of Christensenella and two strains of Catabacter hongkongensis . A concatenated alignment of 135 shared protein sequences of single-copy core genes present in the included strains indicates that C. hongkongensis is indeed nested within the Christensenella clade. Based on their evolutionary relationship, we propose the transfer of Catabacter hongkongensis to the genus Christensenella as Christensenella hongkongensis comb. nov.

Host-adapted microbiota are generally thought to have evolved from free-living ancestors. This process is in principle reversible, but examples are few. The genus Psychrobacter (family Moraxellaceae, phylum Gamma-Proteobacteria) includes species inhabiting diverse and mostly polar environments, such as sea ice and marine animals. To probe Psychrobacter’s evolutionary history, we analyzed 85 Psychrobacter strains by comparative genomics and phenotyping under 24 different growth conditions. Genome-based phylogeny shows Psychrobacter are derived from Moraxella, which are warm-adapted pathobionts. Psychrobacter strains form two ecotypes based on growth temperature: flexible (FE, growth at 4 - 37°C), and restricted (RE, 4 - 25°C). FE strains, which can be either phylogenetically basal or derived, have smaller genomes and higher transposon copy numbers. RE strains have larger genomes, and show genomic adaptations towards a psychrophilic lifestyle and are phylogenetically derived only. We then assessed Psychrobacter abundance in 86 mostly wild polar bear stools and tested persistence of select strains in germfree mice. Psychrobacter (both FE and RE) was enriched in stool of polar bears feeding on mammals, but only FE strains persisted in germfree mice. Together these results indicate growth at 37°C is ancestral in Psychrobacter, lost in many derived species, and likely necessary to colonize the mammalian gut.

The genera Catabacter (family Catabacteraceae) and Christensenella (family Christensenellaceae) are close relatives within the phylum Firmicutes. Members of these genera are strictly anaerobic, non-spore forming, short straight rods with diverse phenotypes. Phylogenetic analysis of 16S rRNA genes suggest that Catabacter splits Christensenella into a polyphyletic clade. In an effort to ensure that family/genus names represent monophyletic clades, we performed a whole-genome based analysis of the genomes available for the cultured representatives of these genera: four species of Christensenella and two strains of Catabacter hongkongensis. A concatenated alignment of 135 shared protein sequences indicates that C. hongkongensis is indeed nested within the Christensenella clade. Based on their evolutionary relationship, we propose the transfer of Catabacter hongkongensis to the new genus as Christensenella hongkongensis comb.nov.

Gut microbes are linked to host metabolism, but specific mechanisms remain to be uncovered. Ceramides, a type of sphingolipid (SL), have been implicated in the development of a range of metabolic disorders from insulin resistance (IR) to hepatic steatosis. SLs are obtained from the diet and generated by de novo synthesis in mammalian tissues. Another potential, but unexplored, source of mammalian SLs is production by Bacteroidetes, the dominant phylum of the gut microbiome. Genomes of Bacteroides spp. and their relatives encode serine palmitoyltransfease (SPT), allowing them to produce SLs. Here, we explore the contribution of SL-production by gut Bacteroides to host SL homeostasis. In human cell culture, bacterial SLs are processed by host SL-metabolic pathways. In mouse models, Bacteroides-derived lipids transfer to host epithelial tissue and the hepatic portal vein. Administration of B. thetaiotaomicron to mice, but not an SPT-deficient strain, reduces de novo SL production and increases liver ceramides. These results indicate that gut-derived bacterial SLs affect host lipid metabolism., Ceramides are a type of sphingolipid (SL) that have been shown to play a role in several metabolic disorders. Here, the authors investigate the effect of SL-production by gut Bacteroides on host SL homeostasis and show that microbiome-derived SLs enter host circulation and alter ceramide production.

Studies in mice using germfree animals as controls for microbial colonization have shown that the gut microbiome mediates diet-induced obesity. Such studies use diets rich in saturated fat, however, Western diets in the United States America are enriched in soybean oil, composed of unsaturated fatty acids, either linoleic or oleic acid. Here, we addressed whether the microbiome is a variable in fat metabolism in mice on a soybean oil diet. We used conventionally-raised, low-germ, and germfree mice fed for 10 weeks diets either high or low in high-linoleic-acid soybean oil as the sole source of fat. Conventional and germfree mice gained relative fat weight and all mice consumed more calories on the high fat vs. low fat soybean oil diet. Plasma fatty acid levels were generally dependent on diet, with microbial colonization status affecting iso-C18:0, C20:3n-6, C14:0, and C15:0 levels. Colonization status, but not diet, impacted levels of liver sphingolipids including ceramides, sphingomyelins, and sphinganine. Our results confirm that absorbed fatty acids are mainly a reflection of the diet and that microbial colonization influences liver sphingolipid pools regardless of diet.

ObjectiveInteractions between the gut microbiome and immunoglobulin (Ig) A in infancy are important for future health. IgM and IgG are also present, however, their interactions with the microbiome in the developing infant are less understood.DesignWe employed stool samples sampled 15 times in infancy from 32 healthy subjects at 4 locations in 3 countries (from the TEDDY study). We characterized patterns of microbiome development in relation to levels of IgA, IgG and IgM. For 8 infants from a single location, we FACS-sorted microbial cells from stool by Ig status. We used 16S rRNA gene profiling on full and sorted microbiomes to assess patterns of antibody coating in relation to age and other factors.ResultsAll antibodies decreased in concentration with age, but were augmented by breastmilk feeding regardless of infant age. Levels of IgA correlated with the relative abundances of OTUs belonging to the Bifidobacteria and Enterobacteriaceae, which dominated the early microbiome, and IgG levels correlated with Haemophilus. The diversity of Ig-coated microbiota was influenced by breastfeeding and age, but birth mode. IgA and IgM coated the same microbiota, while IgG targeted a different subset. Blautia generally evaded antibody coating, while members of the Bifidobacteria and Enterobacteriaceae were high in IgA/M.ConclusionIgA/M have similar dynamics with respect to microbiome development with age, and their interactions with the microbiome are influenced by breastfeeding status. IgG generally does not coat the commensal microbiota.SummaryWhat is already known on this subject?Secretory IgA coats ~50% of microbiota in the gutIgM and IgG are less prevalent and coat a lower fraction in the adult, dynamics in the infant gut are not well characterized.Breastmilk is a source of IgA to the infant gut and decreases with time.IgA coating of microbial cells in infant gut microbiome decreases over time.What are the new findings?Breastfeeding augments the IgA coating of the microbiome at all ages.IgA and IgM coat many of the same cells, whereas few are coated by IgG alone.Bifidobacteria, Enterobacteriaceae, Ruminococcus gnavus are enriched in IgA/M-coated cell fraction, Blautia is enriched in uncoated fraction.IgG levels correlated with Haemophilus.How might it impact on clinical practice in the foreseeable future?Ig-coated fraction of the gut microbiome could serve as a useful tool for tracking development of the infant gut microbiome, and/or identifying aberrations to immune sensing of the microbiome.

Levels of Bacteroidetes in the gut microbiome are positively associated with insulin resistance (IR) in humans. Considering that IR is promoted by elevations in hepatic sphingolipids (SL), particularly ceramides, and that Bacteroidetes are the only microbiome phylum possessing genes encoding serine palmitoyltransferase (SPT), which mediates SL synthesis, we investigated a potential link between bacterial SL production, host SL metabolism, and IR.In vitro, bacterial SLs entered colonocytes and were metabolized into complex SL, including ceramides. In mice, administration of WTBacteroides thetaiotaomicron, but not a SPT-deficient mutant, resulted in elevated levels of liver ceramides and reduced responsiveness to exogenously administered insulin. This work establishes bacterial SLs as a new class of microbiome-derived molecule capable of impacting host metabolism.One Sentence SummarySL production by gut Bacteroidetes regulates liver ceramide levels and insulin sensitivity.

Toll-like receptor (TLR) 5-deficient mice display aberrantly low levels of flagellin-specific antibodies (Flic-IgA) secreted into the gut, combined with excess bacterial flagellin in the gut, and together these attributes define microbiome dysbiosis (T5-dysbiosis). How TLR5 signaling deficiency results in T5-dysbiosis is unclear. Here, we address the role of B cells in T-dysbiosis. We observed that B cells do not express TLR5, and that B cell transplantation from TLR5−/− mouse donors into B-cell deficient mice resulted in a slight reduction in Flic-IgA levels compared to B-cells from WT donors. Bone marrow transplants from WT and TLR5−/− donors into recipients of both genotypes confirmed that TLR5 signaling by non-hematopoietic cells is required for T5-dysbiosis. We observed TLR5 deficiency was associated with an expanded population of IgA+ B cells. TLR5−/− mice tended to have higher richness for the IgA gene hypervariable region (CDR3 gene) variants. Transplantation of microbiomes from TLR5−/− and WT microbiomes donors into germfree mice resulted in a higher proportion of IgA-secreting B cells, and higher overall fecal IgA and anti-Flic IgA for TLR5−/− microbiome recipients. This observation indicated that the TLR5−/− mouse microbiome elicits an anti-flagellin antibody response that requires TLR5 signaling. Together these results indicate that TLR5 signaling on epithelial cells influences B cell populations and antibody repertoire.

Recent studies in human populations and mouse models reveal notable congruences in gut microbial taxa whose abundances are partly regulated by host genotype. Host genes associating with these taxa are related to diet sensing, metabolism, and immunity. These broad patterns are further validated in similar studies of nonmammalian microbiomes. The next generation of genome-wide association studies will expand the size of the data sets and refine the microbial phenotypes to fully capture these intriguing signatures of host-microbiome coevolution.

Host genetic variation influences microbiome composition. While studies have focused on associations between the gut microbiome and specific alleles, gene copy number (CN) also varies. We relate microbiome diversity to CN variation of the AMY1 locus, which encodes salivary amylase, facilitating starch digestion. After imputing AMY1-CN for ∼1,000 subjects, we identified taxa differentiating fecal microbiomes of high and low AMY1-CN hosts. In a month-long diet intervention study, we show that diet standardization drove gut microbiome convergence, and AMY1-CN correlated with oral and gut microbiome composition and function. The microbiomes of low-AMY1-CN subjects had enhanced capacity to break down complex carbohydrates. High-AMY1-CN subjects had higher levels of salivary Porphyromonas; their gut microbiota had increased abundance of resistant starch-degrading microbes, produced higher levels of short-chain fatty acids, and drove higher adiposity when transferred to germ-free mice. This study establishes AMY1-CN as a genetic factor associated with microbiome composition and function.

SummaryHost genetic variation influences the composition of the human microbiome. While studies have focused on associations between the microbiome and single nucleotide polymorphisms in genes, their copy number (CN) can also vary. Here, in a study of human subjects including a 2-week standard diet, we relate oral and gut microbiome to CN at theAMY1locus, which encodes the gene for salivary amylase, active in starch degradation. We show that although diet standardization drove gut microbiome convergence,AMY1-CN influenced oral and gut microbiome composition and function. The gut microbiomes of low-AMY1-CN subjects had an enhanced capacity for breakdown of complex carbohydrates. Those of high-AMY1subjects were enriched in microbiota linked to resistant starch fermentation, had higher fecal SCFAs, and drove higher adiposity when transferred to germfree mice. Gut microbiota results were validated in a larger separate population. This study establishesAMY1-CN as a genetic factor patterning microbiome composition and function.

Over the past century, soybean oil (SBO) consumption in the United States increased dramatically. The main SBO fatty acid, linoleic acid (18:2), inhibits in vitro the growth of lactobacilli, beneficial members of the small intestinal microbiota. Human-associated lactobacilli have declined in prevalence in Western microbiomes, but how dietary changes may have impacted their ecology is unclear. Here, we compared the in vitro and in vivo effects of 18:2 on Lactobacillus reuteri and L. johnsonii. Directed evolution in vitro in both species led to strong 18:2 resistance with mutations in genes for lipid biosynthesis, acid stress, and the cell membrane or wall. Small-intestinal Lactobacillus populations in mice were unaffected by chronic and acute 18:2 exposure, yet harbored both 18:2- sensitive and resistant strains. This work shows that extant small intestinal lactobacilli are protected from toxic dietary components via the gut environment as well as their own capacity to evolve resistance., eLife digest Though “you are what you eat” may just be a figure of speech, it is clear that what we eat does affect our own cells and the microbes that live in our gut. During the 20th century, the American diet changed dramatically and now includes a lot more vegetable oil from soybeans, the main component of which – a fat called linoleic acid – is toxic to many microbes. Among the microbes inhibited by linoleic acid is a beneficial bacterium called Lactobacillus reuteri. This microbe has become less common in Western populations, and the timing of its decline approximately follows when the consumption of soybean oil began increasing. However, L. reuteri and other related microbes still exist in people who eat a Western diet. This suggests that these bacteria must be protected from linoleic acid in the gut, or that they can become resistant to this toxic molecule. Now, Di Rienzi et al. report that both protection in the gut and resistance could explain how L. reuteri can persist in the presence of linoleic acid. First, experiments in the laboratory showed that these microbes could indeed become resistant to linoleic acid, either by gaining mutations in genes involved in creating fats, by growing in an acid, and by forming a cell wall. Further experiments involving mice then showed that the gut protects also L. reuteri from this molecule: linoleic acid did not inhibit L. reuteri within the mouse, but those same L. reuteri were inhibited when grown outside of a mouse. Di Rienzi et al. went on to recover some resistant L. reuteri from mice, implying that there is a mix of resistant and non-resistant strains in the mouse gut. However, notably, the resistant bacteria recovered from the mice did not have mutations in the genes that had been identified from the earlier experiments. Together these findings show that gut bacteria have several means of surviving the high levels of potentially toxic fat molecules. Also, the specific finding that linoleic acid does not inhibit L. reuteri within the gut may help scientists to understand how a high fat diet affects microbes; for example, it is possible that the decrease in carbohydrates or protein that occurs in high fat diets may explain why such diets cause microbes to be lost. Lastly, and on a practical level, linoleic acid-resistant L. reuteri may in the future be used as a probiotic in foods rich in vegetable oil.

SummaryHost genetics and the gut microbiome can both influence metabolic phenotypes. However, whether host genetic variation shapes the gut microbiome and interacts with it to affect host phenotype is unclear. Here, we compared microbiotas across >1,000 fecal samples obtained from the TwinsUK population, including 416 twin pairs. We identified many microbial taxa whose abundances were influenced by host genetics. The most heritable taxon, the family Christensenellaceae, formed a co-occurrence network with other heritable Bacteria and with methanogenic Archaea. Furthermore, Christensenellaceae and its partners were enriched in individuals with low body mass index (BMI). An obese-associated microbiome was amended with Christensenella minuta, a cultured member of the Christensenellaceae, and transplanted to germ-free mice. C. minuta amendment reduced weight gain and altered the microbiome of recipient mice. Our findings indicate that host genetics influence the composition of the human gut microbiome and can do so in ways that impact host metabolism.

Recent scientific studies are providing increasing evidence for how microbes living in and on us are essential to our good health. However, many students still think of microbes only as germs that harm us. The classroom activities presented here are designed to shift student thinking on this topic. In these guided inquiry activities, students investigate human–microbe interactions as they work together to interpret and analyze authentic data from published articles and develop scientific models. Through the activities, students learn and apply ecological concepts as they come to see the human body as a fascinatingly complex ecosystem.

CTnDOT is a 65-kb conjugative transposon (CTn) in Bacteroides spp. that confers resistance to the antibiotics erythromycin and tetracycline (Tc). Conjugative transfer of CTnDOT is regulated upon exposure of cells to Tc. In the absence of Tc, no transfer is detectable; however, a cascade of regulatory events results in the conjugative transfer of CTnDOT upon Tc induction. Previous studies addressing regulation of CTnDOT conjugative transfer focused primarily on the 13-kb transfer ( tra ) operon, which encodes the proteins required for assembly of the mating apparatus. We report here that the mob operon that encodes the relaxase and coupling proteins required for mobilization of CTnDOT are regulated at the transcriptional level upon Tc induction. The Xis2d and Exc excision proteins are required for the upregulation of mob transcription upon Tc induction, and yet a deletion of xis2c has no effect. We also show preliminary evidence suggesting that the integrase, IntDOT, may play a regulatory role, as pLYL72 transfer is not detectable when intDOT is provided in trans .

CTnDOT is a 65-kb conjugative transposon that is found in Bacteroides spp., which are one of the more abundant members within the lower human gastrointestinal tract. CTnDOT encodes resistance to the antibiotics erythromycin and tetracycline (Tc). An interesting feature of CTnDOT is that exposure to low levels of Tc induces a cascade of events that ultimately results in CTnDOT conjugative transfer. However, Tc is apparently not a switch that activates transfer but rather a signal that appears to override a series of negative regulators that inhibit premature excision and transfer of CTnDOT. In this minireview, we summarize over 20 years of research that focused on elucidating the highly coordinated regulation of excision, mobilization, and transfer of CTnDOT., IMPORTANCE Bacteroides spp. are abundant commensals in the human colon, but they are also considered opportunistic pathogens, as they can cause life-threatening infections if they should escape the colon. Bacteroides spp. are the most common cause of anaerobic infections and are rather difficult to treat due to the prevalence of antibiotic resistance within this genus. Today over 80% of Bacteroides are resistant to tetracycline (Tc), and a study looking at both clinical and community isolates demonstrated that this resistance was specifically due to the conjugative transposon CTnDOT.

CTnDOT is a 65-kb conjugative transposon present in Bacteroides spp. that confers resistance to erythromycin [ erm (F)] and tetracycline [ tet (Q)]. An interesting feature of CTnDOT is that both excision from the chromosome and transfer of CTnDOT are stimulated by exposure to tetracycline. However, when no tetracycline is present, transfer of CTnDOT is not detectable. Previous studies suggested that a region containing a small RNA, RteR, appeared to mediate repression of CTnDOT transfer; however, virtually nothing was known about RteR. We have demonstrated that RteR is a 90-nucleotide transcript that is not further processed. RteR inhibits conjugative transfer of CTnDOT by targeting the transfer region, a 13-kb operon that encodes the tra genes required to assemble the mating apparatus. We report here that RteR interacts with the region downstream of traA . Levels of the downstream tra mRNA are dramatically reduced when RteR is present. Further, RteR does not appear to decrease the half-life of the tra mRNA transcript, suggesting that RteR does not bind to the transcript to initiate RNase-dependent decay, similar to other trans -acting small RNAs. We predict that RteR may act to enhance termination of the tra operon within traB , which could account for the decreased abundance of the tra transcript downstream of traA and explain why the tra mRNA has the same half-life whether or not RteR is present. RteR is the only small RNA that has been characterized so far within the Bacteroidetes phylum.