Julie A. Huber

Microbial diversity in the deep sea and the underexplored “rare biosphere”

The evolution of marine microbes over billions of years predicts that the composition of microbial communities should be much greater than the published estimates of a few thousand distinct kinds of microbes per liter of seawater. By adopting a massively parallel tag sequencing strategy, we show that bacterial communities of deep water masses of the North Atlantic and diffuse flow hydrothermal vents are one to two orders of magnitude more complex than previously reported for any microbial environment. A relatively small number of different populations dominate all samples, but thousands of low-abundance populations account for most of the observed phylogenetic diversity. This “rare biosphere” is very ancient and may represent a nearly inexhaustible source of genomic innovation. Members of the rare biosphere are highly divergent from each other and, at different times in earths history, may have had a profound impact on shaping planetary processes.

Accuracy and quality of massively parallel DNA pyrosequencing

BackgroundMassively parallel pyrosequencing systems have increased the efficiency of DNA sequencing, although the published per-base accuracy of a Roche GS20 is only 96%. In genome projects, highly redundant consensus assemblies can compensate for sequencing errors. In contrast, studies of microbial diversity that catalogue differences between PCR amplicons of ribosomal RNA genes (rDNA) or other conserved gene families cannot take advantage of consensus assemblies to detect and minimize incorrect base calls.ResultsWe performed an empirical study of the per-base error rate for the Roche GS20 system using sequences of the V6 hypervariable region from cloned microbial ribosomal DNA (tag sequencing). We calculated a 99.5% accuracy rate in unassembled sequences, and identified several factors that can be used to remove a small percentage of low-quality reads, improving the accuracy to 99.75% or better.ConclusionBy using objective criteria to eliminate low quality data, the quality of individual GS20 sequence reads in molecular ecological applications can surpass the accuracy of traditional capillary methods.

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.

Temporal changes in archaeal diversity and chemistry in a mid-ocean ridge subseafloor habitat.

ABSTRACT The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-μm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.

Bacterial diversity in a subseafloor habitat following a deep-sea volcanic eruption

Abstract The bacterial diversity in a diffuse flow hydrothermal vent habitat at Axial Volcano, Juan de Fuca Ridge was examined shortly after an eruptive event in 1998 and again in 1999 and 2000 using PCR-amplified 16S rRNA gene sequence analyses. While considerable overlap with deep-sea background seawater was found within the alpha- and gamma-proteobacteria, unique subseafloor phylotypes were distinguishable. These included diverse members of the epsilon-proteobacteria, high temperature groups such as Desulfurobacterium, Gram-positive bacteria, and members of novel candidate divisions WS6 and ABY1. Phylotype richness was highest in the particle-attached populations from all three sampling periods, and diversity appeared to increase over that time, particularly among the epsilon-proteobacteria. A preliminary model of the subseafloor is presented that relates microbial diversity to temperature, chemical characteristics of diffuse flow fluids and the degree of mixing with seawater.

Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.

Diverse styles of submarine venting on the ultraslow spreading Mid-Cayman Rise

Thirty years after the first discovery of high-temperature submarine venting, the vast majority of the global mid-ocean ridge remains unexplored for hydrothermal activity. Of particular interest are the world’s ultraslow spreading ridges that were the last to be demonstrated to host high-temperature venting but may host systems particularly relevant to prebiotic chemistry and the origins of life. Here we report evidence for previously unknown, diverse, and very deep hydrothermal vents along the ∼110 km long, ultraslow spreading Mid-Cayman Rise (MCR). Our data indicate that the MCR hosts at least three discrete hydrothermal sites, each representing a different type of water-rock interaction, including both mafic and ultramafic systems and, at ∼5,000 m, the deepest known hydrothermal vent. Although submarine hydrothermal circulation, in which seawater percolates through and reacts with host lithologies, occurs on all mid-ocean ridges, the diversity of vent types identified here and their relative geographic isolation make the MCR unique in the oceans. These new sites offer prospects for an expanded range of vent-fluid compositions, varieties of abiotic organic chemical synthesis and extremophile microorganisms, and unparalleled faunal biodiversity—all in close proximity.

Host-specificity among abundant and rare taxa in the sponge microbiome.

Microbial communities have a key role in the physiology of the sponge host, and it is therefore essential to understand the stability and specificity of sponge–symbiont associations. Host-specific bacterial associations spanning large geographic distance are widely acknowledged in sponges. However, the full spectrum of specificity remains unclear. In particular, it is not known whether closely related sponges host similar or very different microbiota over wide bathymetric and geographic gradients, and whether specific associations extend to the rare members of the sponge microbiome. Using the ultra-deep Illumina sequencing technology, we conducted a comparison of sponge bacterial communities in seven closely related Hexadella species with a well-resolved host phylogeny, as well as of a distantly related sponge Mycale. These samples spanned unprecedentedly large bathymetric (15–960 m) gradients and varying European locations. In addition, this study included a bacterial community analysis of the local background seawater for both Mycale and the widespread deep-sea taxa Hexadella cf. dedritifera. We observed a striking diversity of microbes associated with the sponges, spanning 47 bacterial phyla. The data did not reveal any Hexadella microbiota co-speciation pattern, but confirmed sponge-specific and species-specific host–bacteria associations, even within extremely low abundant taxa. Oligotyping analysis also revealed differential enrichment preferences of closely related Nitrospira members in closely related sponges species. Overall, these results demonstrate highly diverse, remarkably specific and stable sponge–bacteria associations that extend to members of the rare biosphere at a very fine phylogenetic scale, over significant geographic and bathymetric gradients.

Isolated communities of Epsilonproteobacteria in hydrothermal vent fluids of the Mariana Arc seamounts.

Low-temperature hydrothermal vent fluids represent access points to diverse microbial communities living in oceanic crust. This study examined the distribution, relative abundance, and diversity of Epsilonproteobacteria in 14 low-temperature vent fluids from five volcanically active seamounts of the Mariana Arc using a 454 tag sequencing approach. Most vent fluids were enriched in cell concentrations compared with background seawater, and quantitative PCR results indicated that all fluids were dominated by bacteria. Operational taxonomic unit-based statistical tools applied to 454 data show that all vents from the northern end of the Mariana Arc grouped together, to the exclusion of southern arc seamounts, which were as distinct from one another as they were from northern seamounts. Statistical analysis also showed a significant relationship between seamount and individual vent groupings, suggesting that community membership may be linked to geographical isolation and not geochemical parameters. However, while there may be large-scale geographic differences, distance is not the distinguishing factor in the microbial community composition. At the local scale, most vents host a distinct population of Epsilonproteobacteria, regardless of seamount location. This suggests that there may be barriers to exchange and dispersal for these vent endemic microorganisms at hydrothermal seamounts of the Mariana Arc.

Mixing, Reaction and Microbial Activity in the Sub‐Seafloor Revealed by Temporal and Spatial Variation in Diffuse Flow Vents at Axial Volcano

To begin to understand the relationship between microbial communities and the geochemical environment, we have conducted systematic sampling and in situ analysis of a range of seafloor vents on or near the January 1998 lava flow at the summit of Axial Volcano on the Juan de Fuca ridge. The systematics of the chemical composition indicate that low-temperature diffuse fluids (3°C-78°C) at Axial Volcano have a high-temperature (>350°C) reaction-zone component overprinted by lower-temperature reactions. The low-temperature reactions include production of methane, ammonia and particulate elemental sulfur, oxidation of hydrogen sulfide, nitrate reduction, stripping of metals from seawater, and reactions that dissolve iron and produce alkalinity. High concentrations of CO 2 from magmatic degassing maintain acidic pH conditions and may be important in promoting low-temperature hydrolysis reactions. H 2 S oxidation is the dominant chemical energy source for microbial metabolism at Axial Volcano, and the energy available from either methanogenesis or iron oxidation is ∼100 times less. Chemical evidence, genetic signatures of thermophilic, non-seawater organisms, presence of culturable thermophiles, and cell counts elevated above backgound seawater in low-temperature fluids indicate microbial activity below the seafloor. Metabolic activity of organisms identified in venting fluids matches the chemical processes occurring in low-temperature sub-seafloor reservoirs.