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1.  Cellulolytic potential under environmental changes in microbial communities from grassland litter 
In many ecosystems, global changes are likely to profoundly affect microorganisms. In Southern California, changes in precipitation and nitrogen deposition may influence the composition and functional potential of microbial communities and their resulting ability to degrade plant material. To test whether such environmental changes impact the distribution of functional groups involved in leaf litter degradation, we determined how the genomic diversity of microbial communities in a semi-arid grassland ecosystem changed under reduced precipitation or increased N deposition. We monitored communities seasonally over a period of 2 years to place environmental change responses into the context of natural variation. Fungal and bacterial communities displayed strong seasonal patterns, Fungi being mostly detected during the dry season whereas Bacteria were common during wet periods. Most putative cellulose degraders were associated with 33 bacterial genera and predicted to constitute 18% of the microbial community. Precipitation reduction reduced bacterial abundance and cellulolytic potential whereas nitrogen addition did not affect the cellulolytic potential of the microbial community. Finally, we detected a strong correlation between the frequencies of genera of putative cellulose degraders and cellulase genes. Thus, microbial taxonomic composition was predictive of cellulolytic potential. This work provides a framework for how environmental changes affect microorganisms responsible for plant litter deconstruction.
PMCID: PMC4243572  PMID: 25505459
cellulase; metagenomics; leaf litter; global change; microbial community composition
2.  Abundance of Broad Bacterial Taxa in the Sargasso Sea Explained by Environmental Conditions but Not Water Mass 
To explore the potential linkage between distribution of marine bacterioplankton groups, environmental conditions, and water mass, we investigated the factors determining the abundance of bacterial taxa across the hydrographically complex Subtropical Convergence Zone in the Sargasso Sea. Based on information from 16S rRNA gene clone libraries from various locations and two depths, abundances of the predominant taxa (eubacteria, Archaea, Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and the Roseobacter, SAR11, and SAR86 clades) were quantified by real-time PCR. In addition, the abundances of Synechococcus, Prochlorococcus, and picoalgae were determined by flow cytometry. Linear multiple-regression models determining the relative effects of eight environmental variables and of water mass explained 35 to 86% of the variation in abundance of the quantified taxa, even though only one to three variables were significantly related to any particular taxon's abundance. Most of the variation in abundance was explained by depth and chlorophyll a. The predominant phototrophs, Prochlorococcus and picoalgae, were negatively correlated with phosphate, whereas eubacteria, heterotrophic bacteria, and SAR86 were negatively correlated with nitrite. Water mass showed limited importance for explaining the abundance of the taxonomical groups (significant only for Roseobacter, explaining 14% of the variation). The results suggest the potential for predicting the abundance of broad bacterioplankton groups throughout the Sargasso Sea using only a few environmental parameters.
PMCID: PMC3993294  PMID: 24561593
3.  Microbial composition affects the functioning of estuarine sediments 
The ISME Journal  2012;7(4):868-879.
Although microorganisms largely drive many ecosystem processes, the relationship between microbial composition and their functioning remains unclear. To tease apart the effects of composition and the environment directly, microbial composition must be manipulated and maintained, ideally in a natural ecosystem. In this study, we aimed to test whether variability in microbial composition affects functional processes in a field setting, by reciprocally transplanting riverbed sediments between low- and high-salinity locations along the Nonesuch River (Maine, USA). We placed the sediments into microbial ‘cages' to prevent the migration of microorganisms, while allowing the sediments to experience the abiotic conditions of the surroundings. We performed two experiments, short- (1 week) and long-term (7 weeks) reciprocal transplants, after which we assayed a variety of functional processes in the cages. In both experiments, we examined the composition of bacteria generally (targeting the 16S rDNA gene) and sulfate-reducing bacteria (SRB) specifically (targeting the dsrAB gene) using terminal restriction fragment length polymorphism (T-RFLP). In the short-term experiment, sediment processes (CO2 production, CH4 flux, nitrification and enzyme activities) depended on both the sediment's origin (reflecting differences in microbial composition between salt and freshwater sediments) and the surrounding environment. In the long-term experiment, general bacterial composition (but not SRB composition) shifted in response to their new environment, and this composition was significantly correlated with sediment functioning. Further, sediment origin had a diminished effect, relative to the short-term experiment, on sediment processes. Overall, this study provides direct evidence that microbial composition directly affects functional processes in these sediments.
PMCID: PMC3603390  PMID: 23235294
ecosystem functioning; common garden; reciprocal transplant; estuary
4.  Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries 
Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26%) cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase (GH) families were detected. Using tetranucleotide frequency (TNF) binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.
PMCID: PMC3779933  PMID: 24069019
functional metagenomics; carbon cycling; trait-based modeling; gene annotation; microbial communities; decomposers; metagenomics; enzyme activity
5.  Fundamentals of Microbial Community Resistance and Resilience 
Microbial communities are at the heart of all ecosystems, and yet microbial community behavior in disturbed environments remains difficult to measure and predict. Understanding the drivers of microbial community stability, including resistance (insensitivity to disturbance) and resilience (the rate of recovery after disturbance) is important for predicting community response to disturbance. Here, we provide an overview of the concepts of stability that are relevant for microbial communities. First, we highlight insights from ecology that are useful for defining and measuring stability. To determine whether general disturbance responses exist for microbial communities, we next examine representative studies from the literature that investigated community responses to press (long-term) and pulse (short-term) disturbances in a variety of habitats. Then we discuss the biological features of individual microorganisms, of microbial populations, and of microbial communities that may govern overall community stability. We conclude with thoughts about the unique insights that systems perspectives – informed by meta-omics data – may provide about microbial community stability.
PMCID: PMC3525951  PMID: 23267351
microbial ecology; disturbance; stability; sensitivity; structure-function; perturbation; community structure; time series
6.  The Effect of Nitrogen Enrichment on C1-Cycling Microorganisms and Methane Flux in Salt Marsh Sediments 
Methane (CH4) flux from ecosystems is driven by C1-cycling microorganisms – the methanogens and the methylotrophs. Little is understood about what regulates these communities, complicating predictions about how global change drivers such as nitrogen enrichment will affect methane cycling. Using a nitrogen addition gradient experiment in three Southern California salt marshes, we show that sediment CH4 flux increased linearly with increasing nitrogen addition (1.23 μg CH4 m−2 day−1 for each g N m−2 year−1 applied) after 7 months of fertilization. To test the reason behind this increased CH4 flux, we conducted a microcosm experiment altering both nitrogen and carbon availability under aerobic and anaerobic conditions. Methanogenesis appeared to be both nitrogen and carbon (acetate) limited. N and C each increased methanogenesis by 18%, and together by 44%. In contrast, methanotrophy was stimulated by carbon (methane) addition (830%), but was unchanged by nitrogen addition. Sequence analysis of the sediment methylotroph community with the methanol dehydrogenase gene (mxaF) revealed three distinct clades that fall outside of known lineages. However, in agreement with the microcosm results, methylotroph abundance (assayed by qPCR) and composition (assayed by terminal restriction fragment length polymorphism analysis) did not vary across the experimental nitrogen gradient in the field. Together, these results suggest that nitrogen enrichment to salt marsh sediments increases methane flux by stimulating the methanogen community.
PMCID: PMC3307020  PMID: 22470369
methylotrophy; methanogenesis; methanotrophy; methane flux; nitrogen gradient; nutrient limitation; mxaF; acetate
7.  Patterns of fungal diversity and composition along a salinity gradient 
The ISME journal  2010;5(3):379-388.
Estuarine salinity gradients are known to influence plant, bacterial and archaeal community structure. We sequenced 18S rRNA genes to investigate patterns in sediment fungal diversity (richness and evenness of taxa) and composition (taxonomic and phylogenetic) along an estuarine salinity gradient. We sampled three marshes—a salt, brackish and freshwater marsh—in Rhode Island. To compare the relative effect of the salinity gradient with that of plants, we sampled fungi in plots with Spartina patens and in plots from which plants were removed 2 years prior to sampling. The fungal sediment community was unique compared with previously sampled fungal communities; we detected more Ascomycota (78%), fewer Basidiomycota (6%) and more fungi from basal lineages (16%) (Chytridiomycota, Glomeromycota and four additional groups) than typically found in soil. Across marshes, fungal composition changed substantially, whereas fungal diversity differed only at the finest level of genetic resolution, and was highest in the intermediate, brackish marsh. In contrast, the presence of plants had a highly significant effect on fungal diversity at all levels of genetic resolution, but less of an effect on fungal composition. These results suggest that salinity (or other covarying parameters) selects for a distinctive fungal composition, and plants provide additional niches upon which taxa within these communities can specialize and coexist. Given the number of sequences from basal fungal lineages, the study also suggests that further sampling of estuarine sediments may help in understanding early fungal evolution.
PMCID: PMC3105720  PMID: 20882058
community composition; estuary; fungi; ribosomal RNA; salinity
8.  The Abundance of Pink-Pigmented Facultative Methylotrophs in the Root Zone of Plant Species in Invaded Coastal Sage Scrub Habitat 
PLoS ONE  2012;7(2):e31026.
Pink-pigmented facultative methylotrophic bacteria (PPFMs) are associated with the roots, leaves and seeds of most terrestrial plants and utilize volatile C1 compounds such as methanol generated by growing plants during cell division. PPFMs have been well studied in agricultural systems due to their importance in crop seed germination, yield, pathogen resistance and drought stress tolerance. In contrast, little is known about the PPFM abundance and diversity in natural ecosystems, let alone their interactions with non-crop species. Here we surveyed PPFM abundance in the root zone soil of 5 native and 5 invasive plant species along ten invasion gradients in Southern California coastal sage scrub habitat. PPFMs were present in every soil sample and ranged in abundance from 102 to 105 CFU/g dry soil. This abundance varied significantly among plant species. PPFM abundance was 50% higher in the root zones of annual or biennial species (many invasives) than perennial species (all natives). Further, PPFM abundance appears to be influenced by the plant community beyond the root zone; pure stands of either native or invasive species had 50% more PPFMs than mixed species stands. In sum, PPFM abundance in the root zone of coastal sage scrub plants is influenced by both the immediate and surrounding plant communities. The results also suggest that PPFMs are a good target for future work on plant-microorganism feedbacks in natural ecosystems.
PMCID: PMC3286463  PMID: 22383990
9.  Functional Metagenomics Reveals Previously Unrecognized Diversity of Antibiotic Resistance Genes in Gulls 
Wildlife may facilitate the spread of antibiotic resistance (AR) between human-dominated habitats and the surrounding environment. Here, we use functional metagenomics to survey the diversity and genomic context of AR genes in gulls. Using this approach, we found a variety of AR genes not previously detected in gulls and wildlife, including class A and C β-lactamases as well as six tetracycline resistance gene types. An analysis of the flanking sequences indicates that most of these genes are present in Enterobacteriaceae and various Gram-positive bacteria. In addition to finding known gene types, we detected 31 previously undescribed AR genes. These undescribed genes include one most similar to an uncharacterized gene in Verrucomicrobium and another to a putative DNA repair protein in Lactobacillus. Overall, the study more than doubled the number of clinically relevant AR gene types known to be carried by gulls or by wildlife in general. Together with the propensity of gulls to visit human-dominated habitats, this high diversity of AR gene types suggests that gulls could facilitate the spread of AR.
PMCID: PMC3275322  PMID: 22347872
herring gulls; antibiotic resistance; Verrucomicrobia; Larus argentatus; metagenomics
10.  Global Patterns of Bacterial Beta-Diversity in Seafloor and Seawater Ecosystems 
PLoS ONE  2011;6(9):e24570.
Marine microbial communities have been essential contributors to global biomass, nutrient cycling, and biodiversity since the early history of Earth, but so far their community distribution patterns remain unknown in most marine ecosystems.
Methodology/Principal Findings
The synthesis of 9.6 million bacterial V6-rRNA amplicons for 509 samples that span the global ocean's surface to the deep-sea floor shows that pelagic and benthic communities greatly differ, at all taxonomic levels, and share <10% bacterial types defined at 3% sequence similarity level. Surface and deep water, coastal and open ocean, and anoxic and oxic ecosystems host distinct communities that reflect productivity, land influences and other environmental constraints such as oxygen availability. The high variability of bacterial community composition specific to vent and coastal ecosystems reflects the heterogeneity and dynamic nature of these habitats. Both pelagic and benthic bacterial community distributions correlate with surface water productivity, reflecting the coupling between both realms by particle export. Also, differences in physical mixing may play a fundamental role in the distribution patterns of marine bacteria, as benthic communities showed a higher dissimilarity with increasing distance than pelagic communities.
This first synthesis of global bacterial distribution across different ecosystems of the World's oceans shows remarkable horizontal and vertical large-scale patterns in bacterial communities. This opens interesting perspectives for the definition of biogeographical biomes for bacteria of ocean waters and the seabed.
PMCID: PMC3169623  PMID: 21931760
11.  The minimum information about a genome sequence (MIGS) specification 
Nature biotechnology  2008;26(5):541-547.
With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the ‘transparency’ of the information contained in existing genomic databases.
PMCID: PMC2409278  PMID: 18464787
12.  Selection and Characterization of Cyanophage Resistance in Marine Synechococcus Strains▿  
Applied and Environmental Microbiology  2007;73(17):5516-5522.
Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.
PMCID: PMC2042077  PMID: 17630310

Results 1-12 (12)