Capacity of a Microbial Synbiotic To Rescue the In Vitro Metabolic Activity of the Gut Microbiome following Perturbation with Alcohol or Antibiotics.

The human gut microbiome contributes crucial bioactive metabolites that support human health and is sensitive to perturbations from the ingestion of alcohol and antibiotics. We interrogated the response and recovery of human gut microbes after acute alcohol or broad-spectrum antibiotic administration in a gut model simulating the luminal and mucosal colonic environment with an inoculated human microbiome. Both alcohol and antibiotic treatments reduced the production of major short-chain fatty acids (SCFAs) (acetate, propionate, and butyrate), which are established modulators of human health. Treatment with a microbial synbiotic restored and enhanced gut function. Butyrate and acetate production increased by up to 29.7% and 18.6%, respectively, relative to untreated, dysbiotic samples. In parallel, treatment led to increases in the relative abundances of beneficial commensal organisms not found in the synbiotic (e.g., Faecalibacterium prausnitzii and the urolithin-producing organism Gordonibacter pamelaeae) as well as species present in the synbiotic (e.g., Bifidobacterium infantis), suggesting synergistic interactions between supplemented and native microorganisms. These results lead us to conclude that functional shifts in the microbiome, evaluated by both metabolite production and specific taxonomic compositional changes, are an appropriate metric to assess microbiome "recovery" following a dysbiosis-inducing disruption. Overall, these findings support the execution of randomized clinical studies to determine whether a microbial synbiotic can help restore microbiome function after a disruption. IMPORTANCE The human gut microbiome is sensitive to disruptions by common stressors such as alcohol consumption and antibiotic treatment. In this study, we used an in vitro system modeling the gut microbiome to investigate whether treatment with a microbial synbiotic can help restore microbiome function after stress. We find that a complex gut community treated with alcohol or antibiotics showed reduced levels of production of short-chain fatty acids, which are critical beneficial molecules produced by a healthy gut microbiota. Treatment of stressed communities with a microbial synbiotic resulted in the recovery of SCFA production as well as an increase in the abundance of beneficial commensal organisms. Our results suggest that treatment with a microbial synbiotic has the potential to restore healthy gut microbiome function after stress and merits further investigation in clinical studies.

Leaf-FISH: Microscale Imaging of Bacterial Taxa on Phyllosphere.

Molecular methods for microbial community characterization have uncovered environmental and plant-associated factors shaping phyllosphere communities. Variables undetectable using bulk methods can play an important role in shaping plant-microbe interactions. Microscale analysis of bacterial dynamics in the phyllosphere requires imaging techniques specially adapted to the high autoflouresence and 3-D structure of the leaf surface. We present an easily-transferable method (Leaf-FISH) to generate high-resolution tridimensional images of leaf surfaces that allows simultaneous visualization of multiple bacterial taxa in a structurally informed context, using taxon-specific fluorescently labeled oligonucleotide probes. Using a combination of leaf pretreatments coupled with spectral imaging confocal microscopy, we demonstrate the successful imaging bacterial taxa at the genus level on cuticular and subcuticular leaf areas. Our results confirm that different bacterial species, including closely related isolates, colonize distinct microhabitats in the leaf. We demonstrate that highly related Methylobacterium species have distinct colonization patterns that could not be predicted by shared physiological traits, such as carbon source requirements or phytohormone production. High-resolution characterization of microbial colonization patterns is critical for an accurate understanding of microbe-microbe and microbe-plant interactions, and for the development of foliar bacteria as plant-protective agents.

The effects of variable sample biomass on comparative metagenomics.

Longitudinal studies that integrate samples with variable biomass are essential to understand microbial community dynamics across space or time. Shotgun metagenomics is widely used to investigate these communities at the functional level, but little is known about the effects of combining low and high biomass samples on downstream analysis. We investigated the interacting effects of DNA input and library amplification by polymerase chain reaction on comparative metagenomic analysis using dilutions of a single complex template from an Arabidopsis thaliana-associated microbial community. We modified the Illumina Nextera kit to generate high-quality large-insert (680 bp) paired-end libraries using a range of 50 pg to 50 ng of input DNA. Using assembly-based metagenomic analysis, we demonstrate that DNA input level has a significant impact on community structure due to overrepresentation of low-GC genomic regions following library amplification. In our system, these differences were largely superseded by variations between biological replicates, but our results advocate verifying the influence of library amplification on a case-by-case basis. Overall, this study provides recommendations for quality filtering and de-replication prior to analysis, as well as a practical framework to address the issue of low biomass or biomass heterogeneity in longitudinal metagenomic surveys.

Functional and phylogenetic assembly of microbial communities in the human microbiome.

Microbial communities associated with the human body, that is, the human microbiome, are complex ecologies critical for normal development and health. The taxonomic and phylogenetic composition of these communities tends to significantly differ among individuals, precluding the definition of a simple, shared set of 'core' microbes. Here, we review recent evidence and ecological theory supporting the assembly of host-associated microbial communities in terms of functional traits rather than specific organisms. That is, distinct microbial species may be responsible for specific host-associated functions and phenotypes in distinct hosts. We discuss how ecological processes (selective and stochastic forces) governing the assembly of metazoan communities can be adapted to describe microbial ecologies in host-associated environments, resulting in both niche-specific and 'core' metabolic and other pathways maintained throughout the human microbiome. The extent to which phylogeny and functional traits are linked in host-associated microbes, as opposed to unlinked by mechanisms, such as lateral transfer, remains to be determined. However, the definition of these functional assembly rules within microbial communities using controlled model systems and integrative 'omics' represents a fruitful opportunity for molecular systems ecology.

Ecological succession and stochastic variation in the assembly of Arabidopsis thaliana phyllosphere communities.

UNLABELLED: Bacteria living on the aerial parts of plants (the phyllosphere) are globally abundant and ecologically significant communities and can have significant effects on their plant hosts. Despite their importance, little is known about the ecological processes that drive phyllosphere dynamics. Here, we describe the development of phyllosphere bacterial communities over time on the model plant Arabidopsis thaliana in a controlled greenhouse environment. We used a large number of replicate plants to identify repeatable dynamics in phyllosphere community assembly and reconstructed assembly history by measuring the composition of the airborne community immigrating to plant leaves. We used more than 260,000 sequences from the v5v6 hypervariable region of the 16S rRNA gene to characterize bacterial community structure on 32 plant and 21 air samples over 73 days. We observed strong, reproducible successional dynamics: phyllosphere communities initially mirrored airborne communities and subsequently converged to a distinct community composition. While the presence or absence of particular taxa in the phyllosphere was conserved across replicates, suggesting strong selection for community composition, the relative abundance of these taxa was highly variable and related to the spatial association of individual plants. Our results suggest that stochastic events in early colonization, coupled with dispersal limitation, generated alternate trajectories of bacterial community assembly within the context of deterministic selection for community membership. IMPORTANCE: Commensal bacteria associated with plants help protect their hosts against infection and promote growth. Bacteria associated with plant leaves (the "phyllosphere") are highly abundant and diverse communities, but we have very limited information about their ecology. Here, we describe the formation of phyllosphere communities on the plant model organism Arabidopsis thaliana. We grew a large number of plants in a greenhouse and measured bacterial diversity in the phyllosphere throughout the Arabidopsis life cycle. We also measured the diversity of airborne microbes landing on leaves. Our findings show that plants develop distinctive phyllosphere bacterial communities drawn from low-abundance air populations, suggesting the plant environment is favorable for particular organisms and not others. However, we also found that the relative abundances of bacteria in the phyllosphere are determined primarily by the physical proximity of individual plants. This suggests that a mixture of selective and random forces shapes phyllosphere communities.

A semi-quantitative, synteny-based method to improve functional predictions for hypothetical and poorly annotated bacterial and archaeal genes.

During microbial evolution, genome rearrangement increases with increasing sequence divergence. If the relationship between synteny and sequence divergence can be modeled, gene clusters in genomes of distantly related organisms exhibiting anomalous synteny can be identified and used to infer functional conservation. We applied the phylogenetic pairwise comparison method to establish and model a strong correlation between synteny and sequence divergence in all 634 available Archaeal and Bacterial genomes from the NCBI database and four newly assembled genomes of uncultivated Archaea from an acid mine drainage (AMD) community. In parallel, we established and modeled the trend between synteny and functional relatedness in the 118 genomes available in the STRING database. By combining these models, we developed a gene functional annotation method that weights evolutionary distance to estimate the probability of functional associations of syntenous proteins between genome pairs. The method was applied to the hypothetical proteins and poorly annotated genes in newly assembled acid mine drainage Archaeal genomes to add or improve gene annotations. This is the first method to assign possible functions to poorly annotated genes through quantification of the probability of gene functional relationships based on synteny at a significant evolutionary distance, and has the potential for broad application.

Extended local similarity analysis (eLSA) of microbial community and other time series data with replicates.

BACKGROUND: The increasing availability of time series microbial community data from metagenomics and other molecular biological studies has enabled the analysis of large-scale microbial co-occurrence and association networks. Among the many analytical techniques available, the Local Similarity Analysis (LSA) method is unique in that it captures local and potentially time-delayed co-occurrence and association patterns in time series data that cannot otherwise be identified by ordinary correlation analysis. However LSA, as originally developed, does not consider time series data with replicates, which hinders the full exploitation of available information. With replicates, it is possible to understand the variability of local similarity (LS) score and to obtain its confidence interval. RESULTS: We extended our LSA technique to time series data with replicates and termed it extended LSA, or eLSA. Simulations showed the capability of eLSA to capture subinterval and time-delayed associations. We implemented the eLSA technique into an easy-to-use analytic software package. The software pipeline integrates data normalization, statistical correlation calculation, statistical significance evaluation, and association network construction steps. We applied the eLSA technique to microbial community and gene expression datasets, where unique time-dependent associations were identified. CONCLUSIONS: The extended LSA analysis technique was demonstrated to reveal statistically significant local and potentially time-delayed association patterns in replicated time series data beyond that of ordinary correlation analysis. These statistically significant associations can provide insights to the real dynamics of biological systems. The newly designed eLSA software efficiently streamlines the analysis and is freely available from the eLSA homepage, which can be accessed at http://meta.usc.edu/softs/lsa.

Community-wide analysis of microbial genome sequence signatures.

BACKGROUND: Analyses of DNA sequences from cultivated microorganisms have revealed genome-wide, taxa-specific nucleotide compositional characteristics, referred to as genome signatures. These signatures have far-reaching implications for understanding genome evolution and potential application in classification of metagenomic sequence fragments. However, little is known regarding the distribution of genome signatures in natural microbial communities or the extent to which environmental factors shape them. RESULTS: We analyzed metagenomic sequence data from two acidophilic biofilm communities, including composite genomes reconstructed for nine archaea, three bacteria, and numerous associated viruses, as well as thousands of unassigned fragments from strain variants and low-abundance organisms. Genome signatures, in the form of tetranucleotide frequencies analyzed by emergent self-organizing maps, segregated sequences from all known populations sharing < 50 to 60% average amino acid identity and revealed previously unknown genomic clusters corresponding to low-abundance organisms and a putative plasmid. Signatures were pervasive genome-wide. Clusters were resolved because intra-genome differences resulting from translational selection or protein adaptation to the intracellular (pH approximately 5) versus extracellular (pH approximately 1) environment were small relative to inter-genome differences. We found that these genome signatures stem from multiple influences but are primarily manifested through codon composition, which we propose is the result of genome-specific mutational biases. CONCLUSIONS: An important conclusion is that shared environmental pressures and interactions among coevolving organisms do not obscure genome signatures in acid mine drainage communities. Thus, genome signatures can be used to assign sequence fragments to populations, an essential prerequisite if metagenomics is to provide ecological and biochemical insights into the functioning of microbial communities.

The dynamic genetic repertoire of microbial communities.

Community genomic data have revealed multiple levels of variation between and within microbial consortia. This variation includes large-scale differences in gene content between ecosystems as well as within-population sequence heterogeneity. In the present review, we focus specifically on how fine-scale variation within microbial and viral populations is apparent from community genomic data. A major unresolved question is how much of the observed variation is due to neutral vs. adaptive processes. Limited experimental data hint that some of this fine-scale variation may be in part functionally relevant, whereas sequence-based and modeling analyses suggest that much of it may be neutral. While methods for interpreting population genomic data are still in their infancy, we discuss current interpretations of existing datasets in the light of evolutionary processes and models. Finally, we highlight the importance of virus-host dynamics in generating and shaping within-population diversity.

Population genomic analysis of strain variation in Leptospirillum group II bacteria involved in acid mine drainage formation.

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth approximately 20x). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types ( approximately 94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination.

Population dynamics of marine magnetotactic bacteria in a meromictic salt pond described with qPCR.

Magnetotactic bacteria (MTB) contain membrane-bound magnetic iron minerals and are globally abundant in the suboxic/anoxic portions of chemically stratified marine and freshwater environments. However, their population dynamics and potential quantitative contribution to the biogeochemical cycles that they influence (iron, sulfur, carbon) have not been previously considered. Here we report the first quantitative description of the distribution of individual species of magnetite- and greigite-producing MTB in a natural system. We developed a quantitative polymerase chain reaction assay targeting 16s rRNA genes to enumerate four major groups of marine MTB, and applied the assay to samples collected with respect to geochemical parameters during summer 2003 in seasonally stratified Salt Pond, MA. Using catalysed reporter deposition-fluorescent in situ hybridization, we also show that a large greigite-producing bacterium is distantly related to Thiomicrospira pelophila in the Gammaproteobacteria. Ribosomal RNA copy numbers obtained with quantitative polymerase chain reaction indicate that MTB comprise up to 10% of total Bacteria and that each organism has a characteristic distributional profile with respect to the chemocline.

Unexpected diversity in populations of the many-celled magnetotactic prokaryote.

The many-celled magnetotactic prokaryote (MMP) is an uncultivated, highly motile aggregate of 10-30 cells containing numerous chains of greigite (Fe(3)S(4)) magnetosomes. It is unique to marine environments and is abundant in slightly sulfidic sediments of the Little Sippewissett salt marsh (Falmouth, MA). We sequenced 16s rDNA genes from a natural population of MMP and found five lineages separated by at least 5% sequence divergence. Fluorescent in situ hybridization probes for three of these lineages showed significant variation in their relative abundances across a seasonal cycle in marsh productivity. The MMP should therefore be considered a separate genus in the delta-proteobacteria rather than a single species as previously thought. All cells in each aggregate express identical SSU rRNAs, suggesting that the aggregates are composed of a single MMP phylotype. This observation supports a model of the MMP as comprised of clonal cells which reproduce by binary fission of the aggregate.

South-seeking magnetotactic bacteria in the Northern Hemisphere.

Magnetotactic bacteria contain membrane-bound intracellular iron crystals (magnetosomes) and respond to magnetic fields. Polar magnetotactic bacteria in vertical chemical gradients are thought to respond to high oxygen levels by swimming downward into areas with low or no oxygen (toward geomagnetic north in the Northern Hemisphere and geomagnetic south in the Southern Hemisphere). We identified populations of polar magnetotactic bacteria in the Northern Hemisphere that respond to high oxygen levels by swimming toward geomagnetic south, the opposite of all previously reported magnetotactic behavior. The percentage of magnetotactic bacteria with south polarity in the environment is positively correlated with higher redox potential. The coexistence of magnetotactic bacteria with opposing polarities in the same redox environment conflicts with current models of the adaptive value of magnetotaxis.