Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1؉V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7؉V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5؉V6, and V6؉V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment.Culture-independent 16S rRNA gene surveys are now routinely utilized to examine the microbial diversity in various environmental habitats. However, in surveys of highly diverse ecosystems, the size of clone libraries typically constructed (100 to 500 clones) allows for the identification only of members of the community that are present in high abundance (2,13,14,17,24,51). In addition to the failure to detect the rare members of the ecosystem, these relatively small datasets provide inaccurate estimates when used for computing species richness within an ecosystem. Regardless of the approach utilized to estimate species richness, the estimates obtained are highly dependent on sample size, and smaller datasets typically result in the underestimation of species richness (14,44,47,55).The use of a pyrosequencing-based approach (40) in 16S gene-based diversity surveys promises to overcome both of the above-mentioned problems associated with inadequate sampling. The large number of 16S...

Hydrothermal vents are a well-known source of energy that powers chemosynthesis in the deep sea. Recent work suggests that microbial chemosynthesis is also surprisingly pervasive throughout the dark oceans, serving as a significant CO 2 sink even at sites far removed from vents. Ammonia and sulfur have been identified as potential electron donors for this chemosynthesis, but they do not fully account for measured rates of dark primary production in the pelagic water column. Here we use metagenomic and metatranscriptomic analyses to show that deep-sea populations of the SUP05 group of uncultured sulfur-oxidizing Gammaproteobacteria, which are abundant in widespread and diverse marine environments, contain and highly express genes encoding group 1 Ni, Fe hydrogenase enzymes for H 2 oxidation. Reconstruction of near-complete genomes of two cooccurring SUP05 populations in hydrothermal plumes and deep waters of the Gulf of California enabled detailed population-specific metatranscriptomic analyses, revealing dynamic patterns of gene content and transcript abundance. SUP05 transcripts for genes involved in H 2 and sulfur oxidation are most abundant in hydrothermal plumes where these electron donors are enriched. In contrast, a second hydrogenase has more abundant transcripts in background deep-sea samples. Coupled with results from a bioenergetic model that suggest that H 2 oxidation can contribute significantly to the SUP05 energy budget, these findings reveal the potential importance of H 2 as a key energy source in the deep ocean. This study also highlights the genomic plasticity of SUP05, which enables this widely distributed group to optimize its energy metabolism (electron donor and acceptor) to local geochemical conditions. Guaymas | oxygen minimum zone D eep-sea hydrothermal vent ecosystems depend on microorganisms that use reduced chemicals such as sulfur, methane, ammonium, and H 2 as electron donors for chemosynthesis (1-5). Recent work suggests that microbial chemosynthesis is also far more prevalent in the broader deep oceans than previously recognized, extending throughout the water column of the dark open ocean, where it serves as a significant source of organic carbon (6, 7). The fuels for this pelagic primary production remain unknown, but recent studies show that ammonium (3) and sulfur (8, 9) are potential electron donors in the water column. H 2 , long known as an energy source for free-living bacteria in seafloor hydrothermal systems, was also recently identified as an electron donor in hydrothermal vent animal symbioses (4). Although microbial communities at seafloor hydrothermal vent sites have attracted much attention, hydrothermal vent plumes remain poorly characterized despite their importance as habitats for free-living chemolithoautotrophs (10). These plume microorganisms mediate the hydrothermal transfer of elements from the lithosphere to the oceans (11, 12) and contribute significantly to organic carbon in the deep oceans via carbon fixation (1, 13-15).We investigated hydrothermal vent ...

The soil microbiome is responsible for mediating key ecological processes; however, little is known about its sensitivity to climate change. Observed increases in global temperatures and alteration to rainfall patterns, due to anthropogenic release of greenhouse gases, will likely have a strong influence on soil microbial communities and ultimately the ecosystem services they provide. Therefore, it is vital to understand how soil microbial communities will respond to future climate change scenarios. To this end, we surveyed the abundance, diversity and structure of microbial communities over a 2-year period from a long-term in situ warming experiment that experienced a moderate natural drought. We found the warming treatment and soil water budgets strongly influence bacterial population size and diversity. In normal precipitation years, the warming treatment significantly increased microbial population size 40-150% but decreased diversity and significantly changed the composition of the community when compared with the unwarmed controls. However during drought conditions, the warming treatment significantly reduced soil moisture thereby creating unfavorable growth conditions that led to a 50-80% reduction in the microbial population size when compared with the control. Warmed plots also saw an increase in species richness, diversity and evenness; however, community composition was unaffected suggesting that few phylotypes may be active under these stressful conditions. Our results indicate that under warmed conditions, ecosystem water budget regulates the abundance and diversity of microbial populations and that rainfall timing is critical at the onset of drought for sustaining microbial populations.

Chemolithotrophy is a pervasive metabolic lifestyle for microorganisms in the dark ocean. The SAR324 group of Deltaproteobacteria is ubiquitous in the ocean and has been implicated in sulfur oxidation and carbon fixation, but also contains genomic signatures of C1 utilization and heterotrophy. Here, we reconstructed the metagenome and metatranscriptome of a population of SAR324 from a hydrothermal plume and surrounding waters in the deep Gulf of California to gain insight into the genetic capability and transcriptional dynamics of this enigmatic group. SAR324's metabolism is signified by genes that encode a novel particulate hydrocarbon monooxygenase (pHMO), degradation pathways for corresponding alcohols and short-chain fatty acids, dissimilatory sulfur oxidation, formate dehydrogenase (FDH) and a nitrite reductase (NirK). Transcripts of the pHMO, NirK, FDH and transporters for exogenous carbon and amino acid uptake were highly abundant in plume waters. Sulfur oxidation genes were also abundant in the plume metatranscriptome, indicating SAR324 may also utilize reduced sulfur species in hydrothermal fluids. These results suggest that aspects of SAR324's versatile metabolism (lithotrophy, heterotrophy and alkane oxidation) operate simultaneously, and may explain SAR324's ubiquity in the deep Gulf of California and in the global marine biosphere.

Hydrothermal plumes are an important yet understudied component of deep-sea vent microbial ecosystems. The significance of plume microbial processes can be appreciated from three perspectives: (1) mediation of plume biogeochemistry, (2) dispersal of seafloor hydrothermal vent microbes between vents sites, (3) as natural laboratories for understanding the ecology, physiology, and function of microbial groups that are distributed throughout the pelagic deep sea. Plume microbiology has been largely neglected in recent years, especially relative to the extensive research conducted on seafloor and subseafloor systems. Rapidly advancing technologies for investigating microbial communities provide new motivation and opportunities to characterize this important microbial habitat. Here we briefly highlight microbial contributions to plume and broader ocean (bio)geochemistry and review recent work to illustrate the ecological and biogeographic linkages between plumes, seafloor vent habitats, and other marine habitats such as oxygen minimum zones (OMZs), cold seeps, and oil spills. 16S rRNA gene surveys and metagenomic/-transcriptomic data from plumes point to dominant microbial populations, genes, and functions that are also operative in OMZs (SUP05, ammonia-oxidizing Archaea, and SAR324 Deltaproteobacteria) and hydrocarbon-rich environments (methanotrophs). Plume microbial communities are distinct from those on the seafloor or in the subsurface but contain some signatures of these habitats, consistent with the notion that plumes are potential vectors for dispersal of microorganisms between seafloor vent sites. Finally, we put forward three pressing questions for the future of deep-sea hydrothermal plume research and consider interactions between vents and oceans on global scales.