Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient.

IMPORTANCE: The Gram-negative bacterium Bacteroides fragilis is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation.

Microbial-enrichment method enables high-throughput metagenomic characterization from host-rich samples.

Host-microbe interactions have been linked to health and disease states through the use of microbial taxonomic profiling, mostly via 16S ribosomal RNA gene sequencing. However, many mechanistic insights remain elusive, in part because studying the genomes of microbes associated with mammalian tissue is difficult due to the high ratio of host to microbial DNA in such samples. Here we describe a microbial-enrichment method (MEM), which we demonstrate on a wide range of sample types, including saliva, stool, intestinal scrapings, and intestinal mucosal biopsies. MEM enabled high-throughput characterization of microbial metagenomes from human intestinal biopsies by reducing host DNA more than 1,000-fold with minimal microbial community changes (roughly 90% of taxa had no significant differences between MEM-treated and untreated control groups). Shotgun sequencing of MEM-treated human intestinal biopsies enabled characterization of both high- and low-abundance microbial taxa, pathways and genes longitudinally along the gastrointestinal tract. We report the construction of metagenome-assembled genomes directly from human intestinal biopsies for bacteria and archaea at relative abundances as low as 1%. Analysis of metagenome-assembled genomes reveals distinct subpopulation structures between the small and large intestine for some taxa. MEM opens a path for the microbiome field to acquire deeper insights into host-microbe interactions by enabling in-depth characterization of host-tissue-associated microbial communities.

Dynamic genetic adaptation of Bacteroides thetaiotaomicron during murine gut colonization.

To understand how a bacterium ultimately succeeds or fails in adapting to a new host, it is essential to assess the temporal dynamics of its fitness over the course of colonization. Here, we introduce a human-derived commensal organism, Bacteroides thetaiotaomicron (Bt), into the guts of germ-free mice to determine whether and how the genetic requirements for colonization shift over time. Combining a high-throughput functional genetics assay and transcriptomics, we find that gene usage changes drastically during the first days of colonization, shifting from high expression of amino acid biosynthesis genes to broad upregulation of diverse polysaccharide utilization loci. Within the first week, metabolism becomes centered around utilization of a predominant dietary oligosaccharide, and these changes are largely sustained through 6 weeks of colonization. Spontaneous mutations in wild-type Bt also evolve around this locus. These findings highlight the importance of considering temporal colonization dynamics in developing more effective microbiome-based therapies.

Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient.

Bacteroides fragilis comprises 1-5% of the gut microbiota in healthy humans but can expand to >50% of the population in ulcerative colitis (UC) patients experiencing inflammation. The mechanisms underlying such microbial blooms are poorly understood, but the gut of UC patients has physicochemical features that differ from healthy patients and likely impact microbial physiology. For example, levels of the secondary bile acid deoxycholate (DC) are highly reduced in the ileoanal J-pouch of UC colectomy patients. We isolated a B. fragilis strain from a UC patient with pouch inflammation (i.e. pouchitis) and developed it as a genetic model system to identify genes and pathways that are regulated by DC and that impact B. fragilis fitness in DC and crude bile. Treatment of B. fragilis with a physiologically relevant concentration of DC reduced cell growth and remodeled transcription of one-quarter of the genome. DC strongly induced expression of chaperones and select transcriptional regulators and efflux systems and downregulated protein synthesis genes. Using a barcoded collection of ≈50,000 unique insertional mutants, we further defined B. fragilis genes that contribute to fitness in media containing DC or crude bile. Genes impacting cell envelope functions including cardiolipin synthesis, cell surface glycosylation, and systems implicated in sodium-dependent bioenergetics were major bile acid fitness factors. As expected, there was limited overlap between transcriptionally regulated genes and genes that impacted fitness in bile when disrupted. Our study provides a genome-scale view of a B. fragilis bile response and genetic determinants of its fitness in DC and crude bile.

Metabolic independence drives gut microbial colonization and resilience in health and disease.

BACKGROUND: Changes in microbial community composition as a function of human health and disease states have sparked remarkable interest in the human gut microbiome. However, establishing reproducible insights into the determinants of microbial succession in disease has been a formidable challenge. RESULTS: Here we use fecal microbiota transplantation (FMT) as an in natura experimental model to investigate the association between metabolic independence and resilience in stressed gut environments. Our genome-resolved metagenomics survey suggests that FMT serves as an environmental filter that favors populations with higher metabolic independence, the genomes of which encode complete metabolic modules to synthesize critical metabolites, including amino acids, nucleotides, and vitamins. Interestingly, we observe higher completion of the same biosynthetic pathways in microbes enriched in IBD patients. CONCLUSIONS: These observations suggest a general mechanism that underlies changes in diversity in perturbed gut environments and reveal taxon-independent markers of "dysbiosis" that may explain why widespread yet typically low-abundance members of healthy gut microbiomes can dominate under inflammatory conditions without any causal association with disease.

Genetic and physiological insights into the diazotrophic activity of a non-cyanobacterial marine diazotroph.

Nitrogen (N2 ) fixation, or diazotrophy, supports a large part of primary production in oceans. Culture-independent approaches highlighted the presence in abundance of marine non-cyanobacterial diazotrophs (NCD), but their ecophysiology remains elusive, mostly because of the low number of isolated NCD and because of the lack of available genetic tools for these isolates. Here, a dual genetic and functional approach allowed unveiling the ecophysiology of a marine NCD affiliated to the species Vibrio diazotrophicus. Physiological characterization of the first marine NCD mutant obtained so far was performed using a soft-gellan assay, demonstrating that a ΔnifH mutant is not able to grow in nitrogen-free media. Furthermore, we demonstrated that V. diazotrophicus produces a thick biofilm under diazotrophic conditions, suggesting biofilm production as an adaptive response of this NCD to cope with the inhibition of nitrogen fixation by molecular oxygen. Finally, the genomic signature of V. diazotrophicus is essentially absent from metagenomic data of Tara Ocean expeditions, despite having been isolated from various marine environments. We think that the genetically tractable V. diazotrophicus strain used in this study may serve as an ideal model to study the ecophysiology of these overlooked procaryotic group.

The mosquito microbiome includes habitat-specific but rare symbionts.

Microbial communities are known to influence mosquito lifestyles by modifying essential metabolic and behavioral processes that affect reproduction, development, immunity, digestion, egg survival, and the ability to transmit pathogens. Many studies have used 16S rRNA gene amplicons to characterize mosquito microbiota and investigate factors that influence host-microbiota dynamics. However, a relatively low taxonomic resolution due to clustering methods based on arbitrary threshold and the overall dominance of Wolbachia or Asaia symbionts obscured the investigation of rare members of mosquito microbiota in previous studies. Here, we used high resolution Shannon entropy-based oligotyping approach to analyze the microbiota of Culex pipiens, Culex quinquefasciatus and Aedes individuals from continental Southern France and overseas Guadeloupe as well as from laboratories with or without antibiotics treatment. Our experimental design that resulted in a series of mosquito samples with a gradient of Wolbachia density and relative abundance along with high-resolution analyses of amplicon sequences enabled the recovery of a robust signal from typically less accessible bacterial taxa. Our data confirm species-specific mosquito-bacteria associations with geography as a primary factor that influences bacterial community structure. But interestingly, they also reveal co-occurring symbiotic bacterial variants within single individuals for both Elizabethkingia and Erwinia genera, distinct and specific Asaia and Chryseobacterium in continental and overseas territories, and a putative rare Wolbachia variant. Overall, our study reveals the presence of previously overlooked microdiversity and multiple closely related symbiotic strains within mosquito individuals with a remarkable habitat-specificity.

High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes.

By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long-read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long-read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short-read sequencing. The challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long-read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol-chloroform (PC) extraction and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions, as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read-size distribution, assembly performance and the number of circularized elements found in sequencing results suggest that column-based kits with enzyme supplementation, rather than PC methods, may be more appropriate for long-read sequencing of metagenomes.

Electroactive Bacteria Associated With Stainless Steel Ennoblement in Seawater.

Microorganisms can increase the open-circuit potential of stainless steel immersed in seawater of several hundred millivolts in a phenomenon called ennoblement. It raises the chance of corrosion as the open-circuit potential may go over the pitting corrosion potential. Despite the large impact of the ennoblement, no unifying mechanisms have been described as responsible for the phenomenon. Here we show that the strict electrotroph bacterium "Candidatus Tenderia electrophaga" is detected as an ennoblement biomarker and is only present at temperatures at which we observe ennoblement. This bacterium was previously enriched in biocathode systems. Our results suggest that "Candidatus Tenderia electrophaga," and its previously described extracellular electron transfer metabolism coupled to oxygen reduction activity, could play a central role in modulating stainless steel open-circuit potential and consequently mediating ennoblement.