The small things can matter.

In the context of biology as a whole and of our own personal lives, seemingly small things can prove surprisingly influential. Here, I consider the powerful impact of small organisms-the inhabitants of the microbial world-and the small events that shaped my own development as a scientist. I reflect on the early days of the fields of molecular biology and microbial ecology and my own role in the origin story of what we now call "metagenomics".

Longitudinal and Source-to-Tap New Orleans, LA, U.S.A. Drinking Water Microbiology.

The two municipal drinking water systems of New Orleans, LA, U.S.A. were sampled to compare the microbiology of independent systems that treat the same surface water from the Mississippi River. To better understand temporal trends and sources of microbiology delivered to taps, these treatment plants and distribution systems were subjected to source-to-tap sampling over four years. Both plants employ traditional treatment by chloramination, applied during or after settling, followed by filtration before distribution in a warm, low water age system. Longitudinal samples indicated microbiology to have stability both spatially and temporally, and between treatment plants and distribution systems. Disinfection had the greatest impact on microbial composition, which was further refined by filtration and influenced by distribution and premise plumbing. Actinobacteria spp. exhibited trends with treatment. In particular, Mycobacterium spp., very low in finished waters, occurred idiosyncratically at high levels in some tap waters, indicating distribution and/or premise plumbing as main contributors of mycobacteria. Legionella spp., another genus containing potential opportunistic pathogens, also occurred ubiquitously. Source water microbiology was most divergent from tap water, and each step of treatment brought samples more closely similar to tap waters.

Factors Influencing Bacterial Diversity and Community Composition in Municipal Drinking Waters in the Ohio River Basin, USA.

The composition and metabolic activities of microbes in drinking water distribution systems can affect water quality and distribution system integrity. In order to understand regional variations in drinking water microbiology in the upper Ohio River watershed, the chemical and microbiological constituents of 17 municipal distribution systems were assessed. While sporadic variations were observed, the microbial diversity was generally dominated by fewer than 10 taxa, and was driven by the amount of disinfectant residual in the water. Overall, Mycobacterium spp. (Actinobacteria), MLE1-12 (phylum Cyanobacteria), Methylobacterium spp., and sphingomonads were the dominant taxa. Shifts in community composition from Alphaproteobacteria and Betaproteobacteria to Firmicutes and Gammaproteobacteria were associated with higher residual chlorine. Alpha- and beta-diversity were higher in systems with higher chlorine loads, which may reflect changes in the ecological processes structuring the communities under different levels of oxidative stress. These results expand the assessment of microbial diversity in municipal distribution systems and demonstrate the value of considering ecological theory to understand the processes controlling microbial makeup. Such understanding may inform the management of municipal drinking water resources.

Microbial aerosol liberation from soiled textiles isolated during routine residuals handling in a modern health care setting.

BACKGROUND: A wide variety of specialty textiles are used in health care settings for bedding, clothing, and privacy. The ability of textiles to host or otherwise sequester microbes has been well documented; however, their reciprocal potential for liberating airborne bacteria remains poorly characterized. In response, a multi-season survey of bacterial bioaerosols was conducted in the origin and terminus of residual paths which are specifically designed to isolate soiled hospital textiles as they are moved to laundering. This survey used conventional optical particle counting which incorporated multi-channel fluorescence in conjunction with molecular phylogenetic analyses to characterize the bioaerosols liberated during soiled textile storage--immediately before and after the occupation of a modern hospital. Although outfitted with a HEPA filtration system, the number of airborne particles presenting fluorescing optical signatures consistent with airborne bacteria and fungi significantly increased in textile holding rooms soon after the hospital's commissioning, even though these isolated residual areas rarely host personnel. The bioaerosol liberated during textile storage was characterized using Illumina MiSeq sequencing of bacterial 16S ribosomal ribonucleic acid (rRNA) genes. Gene copies recovered by quantitative PCR from aerosol collected in co-located impingers were consistent with fluorescence gated optical particle counting. RESULTS: The relative abundance patterns of proximal bacterial bioaerosol were such that the air in the origin and terminus of textile storage rooms could not be differentiated once the hospital began processing soiled linens. Genes from microbes typically associating with human skin, feces, and hair--Staphylococcus, Propionibacteria, Corynebacteria, Lactobacillus, and Streptococcus spp.--dominated the aerosol abundance profiles in textile holding rooms, which were generally far less diverse than communities recovered from surfaces in patient rooms. CONCLUSIONS: These results suggest that aerosol partitioning from the routine handling of soiled textiles can contribute to airborne exposures in the health care environment.

Esophageal microbiome in eosinophilic esophagitis.

OBJECTIVE: The microbiome has been implicated in the pathogenesis of a number of allergic and inflammatory diseases. The mucosa affected by eosinophilic esophagitis (EoE) is composed of a stratified squamous epithelia and contains intraepithelial eosinophils. To date, no studies have identified the esophageal microbiome in patients with EoE or the impact of treatment on these organisms. The aim of this study was to identify the esophageal microbiome in EoE and determine whether treatments change this profile. We hypothesized that clinically relevant alterations in bacterial populations are present in different forms of esophagitis. DESIGN: In this prospective study, secretions from the esophageal mucosa were collected from children and adults with EoE, Gastroesophageal Reflux Disease (GERD) and normal mucosa using the Esophageal String Test (EST). Bacterial load was determined using quantitative PCR. Bacterial communities, determined by 16S rRNA gene amplification and 454 pyrosequencing, were compared between health and disease. RESULTS: Samples from a total of 70 children and adult subjects were examined. Bacterial load was increased in both EoE and GERD relative to normal subjects. In subjects with EoE, load was increased regardless of treatment status or degree of mucosal eosinophilia compared with normal. Haemophilus was significantly increased in untreated EoE subjects as compared with normal subjects. Streptococcus was decreased in GERD subjects on proton pump inhibition as compared with normal subjects. CONCLUSIONS: Diseases associated with mucosal eosinophilia are characterized by a different microbiome from that found in the normal mucosa. Microbiota may contribute to esophageal inflammation in EoE and GERD.

Effect of monochloramine treatment on the microbial ecology of Legionella and associated bacterial populations in a hospital hot water system.

Opportunistic pathogens, including Legionella spp. and non-tuberculous mycobacteria, can thrive in building hot water systems despite municipal and traditional on-site chlorine disinfection. Monochloramine is a relatively new approach to on-site disinfection, but the microbiological impact of on-site chloramine use has not been well studied. We hypothesized that comparison of the microbial ecology associated with monochloramine treatment versus no on-site treatment would yield highly dissimilar bacterial communities. Hot water samples were collected monthly from 7 locations for three months from two buildings in a Pennsylvania hospital complex supplied with common municipal water: (1) a hospital administrative building (no on-site treatment) and (2) an adjacent acute-care hospital treated on-site with monochloramine to control Legionella spp. Water samples were subjected to DNA extraction, rRNA PCR, and 454 pyrosequencing. Stark differences in the microbiome of the chloraminated water and the control were observed. Bacteria in the treated samples were primarily Sphingomonadales and Limnohabitans, whereas Flexibacter and Planctomycetaceae predominated in untreated control samples. Serendipitously, one sampling month coincided with dysfunction of the on-site disinfection system that resulted in a Legionella bloom detected by sequencing and culture. This study also demonstrates the potential utility of high-throughput DNA sequencing to monitor microbial ecology in water systems.

High-resolution microbial community succession of microbially induced concrete corrosion in working sanitary manholes.

Microbially-induced concrete corrosion in headspaces threatens wastewater infrastructure worldwide. Models for predicting corrosion rates in sewer pipe networks rely largely on information from culture-based investigations. In this study, the succession of microbes associated with corroding concrete was characterized over a one-year monitoring campaign using rRNA sequence-based phylogenetic methods. New concrete specimens were exposed in two highly corrosive manholes (high concentrations of hydrogen sulfide and carbon dioxide gas) on the Colorado Front Range for up to a year. Community succession on corroding surfaces was assessed using Illumina MiSeq sequencing of 16S bacterial rRNA amplicons and Sanger sequencing of 16S universal rRNA clones. Microbial communities associated with corrosion fronts presented distinct succession patterns which converged to markedly low α-diversity levels (< 10 taxa) in conjunction with decreasing pH. The microbial community succession pattern observed in this study agreed with culture-based models that implicate acidophilic sulfur-oxidizer Acidithiobacillus spp. in advanced communities, with two notable exceptions. Early communities exposed to alkaline surface pH presented relatively high α-diversity, including heterotrophic, nitrogen-fixing, and sulfur-oxidizing genera, and one community exposed to neutral surface pH presented a diverse transition community comprised of less than 20% sulfur-oxidizers.

Molecular analysis of single room humidifier bacteriology.

Portable, single-room humidifiers are commonly used in homes for comfort and health benefits, but also create habitats for microbiology. Currently there is no information on home humidifier microbiology aside from anecdotal evidence of infection with opportunistic pathogens and irritation from endotoxin exposure. To obtain a broader perspective on humidifier microbiology, DNAs were isolated from tap source waters, tank waters, and biofilm samples associated with 26 humidifiers of ultrasonic and boiling modes of operation in the Front Range of Colorado. Humidifiers sampled included units operated by individuals in their homes, display models continuously operated by a retail store, and new humidifiers operated in a controlled laboratory study. The V1V2 region of the rRNA gene was amplified and sequenced to determine the taxonomic composition of humidifier samples. Communities encountered were generally low in richness and diversity and were dominated by Sphingomonadales, Rhizobiales, and Burkholderiales of the Proteobacteria, and MLE1-12, a presumably non-photosynthetic representative of the cyanobacterial phylum. Very few sequences of potential health concern were detected. The bacteriology encountered in source waters sampled here was similar to that encountered in previous studies of municipal drinking waters. Source water bacteriology was found to have the greatest effect on tank water and biofilm bacteriology, an effect confirmed by a controlled study comparing ultrasonic and boiler humidifiers fed with tap vs. treated (deionized, reverse osmosis, 0.2 µm filtered) water over a period of two months.

Specific microbiome changes in a mouse model of parenteral nutrition associated liver injury and intestinal inflammation.

BACKGROUND: Parenteral nutrition (PN) has been a life-saving treatment in infants intolerant of enteral feedings. However, PN is associated with liver injury (PN Associated Liver Injury: PNALI) in a significant number of PN-dependent infants. We have previously reported a novel PNALI mouse model in which PN infusion combined with intestinal injury results in liver injury. In this model, lipopolysaccharide activation of toll-like receptor 4 signaling, soy oil-derived plant sterols, and pro-inflammatory activation of Kupffer cells (KCs) played key roles. The objective of this study was to explore changes in the intestinal microbiome associated with PNALI. METHODOLOGY AND PRINCIPAL FINDINGS: Microbiome analysis in the PNALI mouse identified specific alterations within colonic microbiota associated with PNALI and further association of these communities with the lipid composition of the PN solution. Intestinal inflammation or soy oil-based PN infusion alone (in the absence of enteral feeds) caused shifts within the gut microbiota. However, the combination resulted in accumulation of a specific taxon, Erysipelotrichaceae (23.8% vs. 1.7% in saline infused controls), in PNALI mice. Moreover, PNALI was markedly attenuated by enteral antibiotic treatment, which also was associated with significant reduction of Erysipelotrichaceae (0.6%) and a Gram-negative constituent, the S24-7 lineage of Bacteroidetes (53.5% in PNALI vs. 0.8%). Importantly, removal of soy oil based-lipid emulsion from the PN solution resulted in significant reduction of Erysipelotrichaceae as well as attenuation of PNALI. Finally, addition of soy-derived plant sterol (stigmasterol) to fish oil-based PN restored Erysipelotrichaceae abundance and PNALI. CONCLUSIONS: Soy oil-derived plant sterols and the associated specific bacterial groups in the colonic microbiota are associated with PNALI. Products from these bacteria may directly trigger activation of KCs and promote PNALI. Furthermore, the results indicate that lipid modification of PN solutions may alter specific intestinal bacterial species associated with PNALI, and thus suggest strategies for management of PNALI.

Carbon dioxide and hydrogen sulfide associations with regional bacterial diversity patterns in microbially induced concrete corrosion.

The microbial communities associated with deteriorating concrete corrosion fronts were characterized in 35 samples taken from wastewater collection and treatment systems in ten utilities. Bacterial communities were described using Illumina MiSeq sequencing of the V1V2 region of the small subunit ribosomal ribonucleic acid (SSU-rRNA) gene recovered from fresh corrosion products. Headspace gas concentrations (hydrogen sulfide, carbon dioxide, and methane), pore water pH, moisture content, and select mineralogy were tested for correlation to community outcomes and corrosion extent using pairwise linear regressions and canonical correspondence analysis. Corroding concrete was most commonly characterized by moisture contents greater than 10%, pore water pH below one, and limited richness (<10 taxa). Bacterial community composition was not correlated to geographic location when considered independently from other environmental factors. Corrosion was most severe in sites with high levels of hydrogen sulfide (>100 ppm) and carbon dioxide (>1%) gases, conditions which also were associated with low diversity biofilms dominated by members of the acidophilic sulfur-oxidizer genus Acidithiobacillus.

Molecular analysis of point-of-use municipal drinking water microbiology.

Little is known about the nature of the microbiology in tap waters delivered to consumers via public drinking water distribution systems (DWDSs). In order to establish a broader understanding of the microbial complexity of public drinking waters we sampled tap water from seventeen different cities between the headwaters of the Arkansas River and the mouth of the Mississippi River and determined the bacterial compositions by pyrosequencing small subunit rRNA genes. Nearly 98% of sequences observed among all systems fell into only 5 phyla: Proteobacteria (35%), Cyanobacteria (29%, including chloroplasts), Actinobacteria (24%, of which 85% were Mycobacterium spp.), Firmicutes (6%), and Bacteroidetes (3.4%). The genus Mycobacterium was the most abundant taxon in the dataset, detected in 56 of 63 samples (16 of 17 cities). Among the more rare phylotypes, considerable variation was observed between systems, and was sometimes associated with the type of source water, the type of disinfectant, or the concentration of the environmental pollutant nitrate. Abundant taxa (excepting Cyanobacteria and chloroplasts) were generally similar from system to system, however, regardless of source water type or local land use. The observed similarity among the abundant taxa between systems may be a consequence of the selective influence of chlorine-based disinfection and the common local environments of DWDS and premise plumbing pipes.

Explicet: graphical user interface software for metadata-driven management, analysis and visualization of microbiome data.

Studies of the human microbiome, and microbial community ecology in general, have blossomed of late and are now a burgeoning source of exciting research findings. Along with the advent of next-generation sequencing platforms, which have dramatically increased the scope of microbiome-related projects, several high-performance sequence analysis pipelines (e.g. QIIME, MOTHUR, VAMPS) are now available to investigators for microbiome analysis. The subject of our manuscript, the graphical user interface-based Explicet software package, fills a previously unmet need for a robust, yet intuitive means of integrating the outputs of the software pipelines with user-specified metadata and then visualizing the combined data.

Culture-independent analysis of aerosol microbiology in a metropolitan subway system.

The goal of this study was to determine the composition and diversity of microorganisms associated with bioaerosols in a heavily trafficked metropolitan subway environment. We collected bioaerosols by fluid impingement on several New York City subway platforms and associated sites in three sampling sessions over a 1.5-year period. The types and quantities of aerosolized microorganisms were determined by culture-independent phylogenetic analysis of small-subunit rRNA gene sequences by using both Sanger (universal) and pyrosequencing (bacterial) technologies. Overall, the subway bacterial composition was relatively simple; only 26 taxonomic families made up ~75% of the sequences determined. The microbiology was more or less similar throughout the system and with time and was most similar to outdoor air, consistent with highly efficient air mixing in the system. Identifiable bacterial sequences indicated that the subway aerosol assemblage was composed of a mixture of genera and species characteristic of soil, environmental water, and human skin commensal bacteria. Eukaryotic diversity was mainly fungal, dominated by organisms of types associated with wood rot. Human skin bacterial species (at 99% rRNA sequence identity) included the Staphylococcus spp. Staphylococcus epidermidis (the most abundant and prevalent commensal of the human integument), S. hominis, S. cohnii, S. caprae, and S. haemolyticus, all well-documented human commensal bacteria. We encountered no organisms of public health concern. This study is the most extensive culture-independent survey of subway microbiota so far and puts in place pre-event information required for any bioterrorism surveillance activities or monitoring of the microbiological impact of recent subway flooding events.

Tetracycline resistance and Class 1 integron genes associated with indoor and outdoor aerosols.

Genes encoding tetracycline resistance and the integrase of Class 1 integrons were enumerated using quantitative PCR from aerosols collected from indoor and outdoor environments. Concentrated animal feeding operations (CAFOs) and human-occupied indoor environments (two clinics and a homeless shelter) were found to be a source of airborne tet(X) and tet(W) genes. The CAFOs had 10- to 100-times higher concentrations of airborne 16S rRNA, tet(X), and tet(W) genes than other environments sampled, and increased concentrations of aerosolized bacteria correlated with increased concentrations of airborne resistance genes. The two CAFOs studied had statistically similar concentrations of resistance genes in their aerosol samples, even though antibiotic use was markedly different between the two operations. Additionally, tet(W) genes were recovered in outdoor air within 2 km of livestock operations, which suggests that antibiotic resistance genes may be transported via aerosols on local scales. The integrase gene (intI1) from Class 1 integrons, which has been associated with multidrug resistance, was detected in CAFOs but not in human-occupied indoor environments, suggesting that CAFO aerosols could serve as a reservoir of multidrug resistance. In conclusion, our results show that CAFOs and clinics are sources of aerosolized antibiotic resistance genes that can potentially be transported via air movement.

Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat.

The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119,000 nearly full-length sequences and 28,000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.

Novel device to sample the esophageal microbiome--the esophageal string test.

A growing number of studies implicate the microbiome in the pathogenesis of intestinal inflammation. Previous work has shown that adults with esophagitis related to gastroesophageal reflux disease have altered esophageal microbiota compared to those who do not have esophagitis. In these studies, sampling of the esophageal microbiome was accomplished by isolating DNA from esophageal biopsies obtained at the time of upper endoscopy. The aim of the current study was to identify the esophageal microbiome in pediatric individuals with normal esophageal mucosa using a minimally invasive, capsule-based string technology, the Enterotest™. We used the proximal segment of the Enterotest string to sample the esophagus, and term this the "Esophageal String Test" (EST). We hypothesized that the less invasive EST would capture mucosal adherent bacteria present in the esophagus in a similar fashion as mucosal biopsy. EST samples and mucosal biopsies were collected from children with no esophageal inflammation (n = 15) and their microbiome composition determined by 16S rRNA gene sequencing. Microbiota from esophageal biopsies and ESTs produced nearly identical profiles of bacterial genera and were different from the bacterial contents of samples collected from the nasal and oral cavity. We conclude that the minimally invasive EST can serve as a useful device for study of the esophageal microbiome.

Inflammatory bowel diseases phenotype, C. difficile and NOD2 genotype are associated with shifts in human ileum associated microbial composition.

We tested the hypothesis that Crohn's disease (CD)-related genetic polymorphisms involved in host innate immunity are associated with shifts in human ileum-associated microbial composition in a cross-sectional analysis of human ileal samples. Sanger sequencing of the bacterial 16S ribosomal RNA (rRNA) gene and 454 sequencing of 16S rRNA gene hypervariable regions (V1-V3 and V3-V5), were conducted on macroscopically disease-unaffected ileal biopsies collected from 52 ileal CD, 58 ulcerative colitis and 60 control patients without inflammatory bowel diseases (IBD) undergoing initial surgical resection. These subjects also were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R) and the ATG16L1 risk allele (T300A). The samples were linked to clinical metadata, including body mass index, smoking status and Clostridia difficile infection. The sequences were classified into seven phyla/subphyla categories using the Naïve Bayesian Classifier of the Ribosome Database Project. Centered log ratio transformation of six predominant categories was included as the dependent variable in the permutation based MANCOVA for the overall composition with stepwise variable selection. Polymerase chain reaction (PCR) assays were conducted to measure the relative frequencies of the Clostridium coccoides - Eubacterium rectales group and the Faecalibacterium prausnitzii spp. Empiric logit transformations of the relative frequencies of these two microbial groups were included in permutation-based ANCOVA. Regardless of sequencing method, IBD phenotype, Clostridia difficile and NOD2 genotype were selected as associated (FDR ≤ 0.05) with shifts in overall microbial composition. IBD phenotype and NOD2 genotype were also selected as associated with shifts in the relative frequency of the C. coccoides--E. rectales group. IBD phenotype, smoking and IBD medications were selected as associated with shifts in the relative frequency of F. prausnitzii spp. These results indicate that the effects of genetic and environmental factors on IBD are mediated at least in part by the enteric microbiota.

Altered vaginal microbiota are associated with perinatal mother-to-child transmission of HIV in African women from Burkina Faso.

BACKGROUND: Mother-to-child transmission (MTCT) of HIV remains a significant problem in resource-limited settings, despite the advent of antiretroviral therapies. Because perturbations in vaginal microbial communities are associated with sexual transmission of HIV, we determined whether perinatal MTCT is associated with the vaginal microbiotas of HIV-infected mothers. METHODS: We conducted a retrospective analysis of cervicovaginal microbiotas by pyrosequencing of bacterial 16S rRNA genes (median 350 sequences per sample) from 10 transmitters and 54 nontransmitters during a perinatal MTCT prevention clinical trial of azidothymidine and the microbicide benzalkonium chloride. Logistic regression was performed adjusting for multiple covariates, including CD4(+) T-cell numbers and treatment group, to correlate abundances of microbial taxa with perinatal MTCT. RESULTS: The vaginal microbiotas of these subjects were dominated by several lactobacilli species, although a subset of subjects was colonized by diverse anaerobic species. MTCT of HIV was associated with significantly greater relative abundances of several groups of microorganisms. Most notably, among the abundant bacterial species, Gardnerella vaginalis was significantly enriched in cases of antepartum transmission, compared with nontransmission (odds ratio 1.7; P = 0.004). Neither azidothymidine nor benzalkonium chloride treatment was associated with shifts in microbial distributions compared with the placebo control group. CONCLUSIONS: These data suggest that alterations in vaginal microbial communities are associated with an increased risk for perinatal MTCT, consistent with results with horizontal transmission of HIV. Therefore, determining the mucosal features associated with alterations in vaginal microbial communities may guide efforts to modulate the risk for HIV MTCT.

Phototrophic phylotypes dominate mesothermal microbial mats associated with hot springs in Yellowstone National Park.

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.