James Borneman

Microbial phyllosphere populations are more complex than previously realized

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97–100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.

Bacterial functional redundancy along a soil reclamation gradient

ABSTRACT A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates. Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest. Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU). BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site. The number and diversity of bacterial groups responding to four carbon substrates (l-serine, l-threonine, sodium citrate, and α-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate. These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils. They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning.

Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes

ABSTRACT One of the first steps in characterizing an ecosystem is to describe the organisms inhabiting it. For microbial studies, experimental limitations have hindered the ability to depict diverse communities. Here we describe oligonucleotide fingerprinting of rRNA genes (OFRG), a method that permits identification of arrayed rRNA genes (rDNA) through a series of hybridization experiments using small DNA probes. To demonstrate this strategy, we examined the bacteria inhabiting two different soils. Analysis of 1,536 rDNA clones revealed 766 clusters grouped into five major taxa: Bacillus, Actinobacteria, Proteobacteria, and two undefined assemblages. Soil-specific taxa were identified and then independently confirmed through cluster-specific PCR of the original soil DNA. Near-species-level resolution was obtained by this analysis as clones with average sequence identities of 97% were grouped in the same cluster. A comparison of these OFRG results with the results obtained in a denaturing gradient gel electrophoresis analysis of the same two soils demonstrated the significance of this methodological advance. OFRG provides a cost-effective means to extensively analyze microbial communities and should have applications in medicine, biotechnology, and ecosystem studies.

Abundant and Diverse Fungal Microbiota in the Murine Intestine

ABSTRACT Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.

Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism

Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn’s disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2−/− genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.

Integrative analysis of the microbiome and metabolome of the human intestinal mucosal surface reveals exquisite inter-relationships

BackgroundConsistent compositional shifts in the gut microbiota are observed in IBD and other chronic intestinal disorders and may contribute to pathogenesis. The identities of microbial biomolecular mechanisms and metabolic products responsible for disease phenotypes remain to be determined, as do the means by which such microbial functions may be therapeutically modified.ResultsThe composition of the microbiota and metabolites in gut microbiome samples in 47 subjects were determined. Samples were obtained by endoscopic mucosal lavage from the cecum and sigmoid colon regions, and each sample was sequenced using the 16S rRNA gene V4 region (Illumina-HiSeq 2000 platform) and assessed by UPLC mass spectroscopy. Spearman correlations were used to identify widespread, statistically significant microbial-metabolite relationships. Metagenomes for identified microbial OTUs were imputed using PICRUSt, and KEGG metabolic pathway modules for imputed genes were assigned using HUMAnN. The resulting metabolic pathway abundances were mostly concordant with metabolite data. Analysis of the metabolome-driven distribution of OTU phylogeny and function revealed clusters of clades that were both metabolically and metagenomically similar.ConclusionsThe results suggest that microbes are syntropic with mucosal metabolome composition and therefore may be the source of and/or dependent upon gut epithelial metabolites. The consistent relationship between inferred metagenomic function and assayed metabolites suggests that metagenomic composition is predictive to a reasonable degree of microbial community metabolite pools. The finding that certain metabolites strongly correlate with microbial community structure raises the possibility of targeting metabolites for monitoring and/or therapeutically manipulating microbial community function in IBD and other chronic diseases.

Functional diversity in resource use by fungi

Fungi influence nutrient cycling in terrestrial ecosystems, as they are major regulators of decomposition and soil respiration. However, little is known about the substrate preferences of individual fungal species outside of laboratory culture studies. If active fungi differ in their substrate preferences in situ, then changes in fungal diversity due to global change may dramatically influence nutrient cycling in ecosystems. To test the responses of individual fungal taxa to specific substrates, we used a nucleotide-analogue procedure in the boreal forest of Alaska (USA). Specifically, we added four organic N compounds commonly found in plant litter (arginine, glutamate, lignocellulose, and tannin-protein) to litterbags filled with decomposed leaf litter (black spruce and aspen) and assessed the responses of active fungal species using qPCR (quantitative polymerase chain reaction), oligonucleotide fingerprinting of rRNA genes, and sequencing. We also compared the sequences from our experiment with a concurrent warming experiment to see if active fungi that targeted more recalcitrant compounds would respond more positively to soil warming. We found that individual fungal taxa responded differently to substrate additions and that active fungal communities were different across litter types (spruce vs. aspen). Active fungi that targeted lignocellulose also responded positively to experimental warming. Additionally, resource-use patterns in different fungal taxa were genetically correlated, suggesting that it may be possible to predict the ecological function of active fungal communities based on genetic information. Together, these results imply that fungi are functionally diverse and that reductions in fungal diversity may have consequences for ecosystem functioning.

Commensal Microbiota and CD8+ T Cells Shape the Formation of Invariant NKT Cells

Commensal bacteria play an important role in formation of the immune system, but the mechanisms involved are incompletely understood. In this study, we analyze CD1d-restricted invariant NKT (iNKT) cells in germfree mice and in two colonies of C57BL/6 mice termed conventional flora and restricted flora (RF), stably bearing commensal microbial communities of diverse but distinct composition. In germfree mice, iNKT cells were moderately reduced, suggesting that commensal microbiota were partially required for the antigenic drive in maintaining systemic iNKT cells. Surprisingly, even greater depletion of iNKT cell population occurred in RF mice. This was in part attributable to reduced RF levels of intestinal microbial taxa (Sphingomonas spp.) known to express antigenic glycosphingolipid products. However, memory and activated CD8+ T cells were also expanded in RF mice, prompting us to test whether CD8+ T cell activity might be further depleting iNKT cells. Indeed, iNKT cell numbers were restored in RF mice bearing the CD8α−/− genotype or in adult wild-type RF mice acutely depleted with anti-CD8 Ab. Moreover, iNKT cells were restored in RF mice bearing the Prf1−/− phenotype, a key component of cytolytic function. These findings indicate that commensal microbiota, through positive (antigenic drive) and negative (cytolytic depletion by CD8+ T cells) mechanisms, profoundly shape the iNKT cell compartment. Because individuals greatly vary in the composition of their microbial communities, enteric microbiota may play an important epigenetic role in the striking differences in iNKT cell abundance in humans and therefore in their potential contribution to host immune status.

HIV Infection is associated with compositional and functional shifts in the rectal mucosal microbiota.

BackgroundRegardless of infection route, the intestine is the primary site for HIV-1 infection establishment and results in significant mucosal CD4+ T lymphocyte depletion, induces an inflammatory state that propagates viral dissemination, facilitates microbial translocation, and fosters establishment of one of the largest HIV reservoirs. Here we test the prediction that HIV infection modifies the composition and function of the mucosal commensal microbiota.ResultsRectal mucosal microbiota were collected from human subjects using a sponge-based sampling methodology. Samples were collected from 20 HIV-positive men not receiving combination anti-retroviral therapy (cART), 20 HIV-positive men on cART and 20 healthy, HIV-negative men. Microbial composition of samples was analyzed using barcoded 16S Illumina deep sequencing (85,900 reads per sample after processing). Microbial metagenomic information for the samples was imputed using the bioinformatic tools PICRUST and HUMAnN. Microbial composition and imputed function in HIV-positive individuals not receiving cART was significantly different from HIV-negative individuals. Genera including Roseburia, Coprococcus, Ruminococcus, Eubacterium, Alistipes and Lachnospira were depleted in HIV-infected subjects not receiving cART, while Fusobacteria, Anaerococcus, Peptostreptococcus and Porphyromonas were significantly enriched. HIV-positive subjects receiving cART exhibited similar depletion and enrichment for these genera, but were of intermediate magnitude and did not achieve statistical significance. Imputed metagenomic functions, including amino acid metabolism, vitamin biosynthesis, and siderophore biosynthesis differed significantly between healthy controls and HIV-infected subjects not receiving cART.ConclusionsHIV infection was associated with rectal mucosal changes in microbiota composition and imputed function that cART failed to completely reverse. HIV infection was associated with depletion of some commensal species and enrichment of a few opportunistic pathogens. Many imputed metagenomic functions differed between samples from HIV-negative and HIV-positive subjects not receiving cART, possibly reflecting mucosal metabolic changes associated with HIV infection. Such functional pathways may represent novel interventional targets for HIV therapy if normalizing the microbial composition or functional activity of the microbiota proves therapeutically useful.

A Modular Organization of the Human Intestinal Mucosal Microbiota and Its Association with Inflammatory Bowel Disease

Abnormalities of the intestinal microbiota are implicated in the pathogenesis of Crohns disease (CD) and ulcerative colitis (UC), two spectra of inflammatory bowel disease (IBD). However, the high complexity and low inter-individual overlap of intestinal microbial composition are formidable barriers to identifying microbial taxa representing this dysbiosis. These difficulties might be overcome by an ecologic analytic strategy to identify modules of interacting bacteria (rather than individual bacteria) as quantitative reproducible features of microbial composition in normal and IBD mucosa. We sequenced 16S ribosomal RNA genes from 179 endoscopic lavage samples from different intestinal regions in 64 subjects (32 controls, 16 CD and 16 UC patients in clinical remission). CD and UC patients showed a reduction in phylogenetic diversity and shifts in microbial composition, comparable to previous studies using conventional mucosal biopsies. Analysis of weighted co-occurrence network revealed 5 microbial modules. These modules were unprecedented, as they were detectable in all individuals, and their composition and abundance was recapitulated in an independent, biopsy-based mucosal dataset 2 modules were associated with healthy, CD, or UC disease states. Imputed metagenome analysis indicated that these modules displayed distinct metabolic functionality, specifically the enrichment of oxidative response and glycan metabolism pathways relevant to host-pathogen interaction in the disease-associated modules. The highly preserved microbial modules accurately classified IBD status of individual patients during disease quiescence, suggesting that microbial dysbiosis in IBD may be an underlying disorder independent of disease activity. Microbial modules thus provide an integrative view of microbial ecology relevant to IBD.