Luis M. Rodriguez-R

Microbiome of the upper troposphere: Species composition and prevalence, effects of tropical storms, and atmospheric implications

The composition and prevalence of microorganisms in the middle-to-upper troposphere (8–15 km altitude) and their role in aerosol-cloud-precipitation interactions represent important, unresolved questions for biological and atmospheric science. In particular, airborne microorganisms above the oceans remain essentially uncharacterized, as most work to date is restricted to samples taken near the Earth’s surface. Here we report on the microbiome of low- and high-altitude air masses sampled onboard the National Aeronautics and Space Administration DC-8 platform during the 2010 Genesis and Rapid Intensification Processes campaign in the Caribbean Sea. The samples were collected in cloudy and cloud-free air masses before, during, and after two major tropical hurricanes, Earl and Karl. Quantitative PCR and microscopy revealed that viable bacterial cells represented on average around 20% of the total particles in the 0.25- to 1-μm diameter range and were at least an order of magnitude more abundant than fungal cells, suggesting that bacteria represent an important and underestimated fraction of micrometer-sized atmospheric aerosols. The samples from the two hurricanes were characterized by significantly different bacterial communities, revealing that hurricanes aerosolize a large amount of new cells. Nonetheless, 17 bacterial taxa, including taxa that are known to use C1–C4 carbon compounds present in the atmosphere, were found in all samples, indicating that these organisms possess traits that allow survival in the troposphere. The findings presented here suggest that the microbiome is a dynamic and underappreciated aspect of the upper troposphere with potentially important impacts on the hydrological cycle, clouds, and climate.

Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics

This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ.

Nonpareil: a redundancy-based approach to assess the level of coverage in metagenomic datasets

MOTIVATION Determining the fraction of the diversity within a microbial community sampled and the amount of sequencing required to cover the total diversity represent challenging issues for metagenomics studies. Owing to these limitations, central ecological questions with respect to the global distribution of microbes and the functional diversity of their communities cannot be robustly assessed. RESULTS We introduce Nonpareil, a method to estimate and project coverage in metagenomes. Nonpareil does not rely on high-quality assemblies, operational taxonomic unit calling or comprehensive reference databases; thus, it is broadly applicable to metagenomic studies. Application of Nonpareil on available metagenomic datasets provided estimates on the relative complexity of soil, freshwater and human microbiome communities, and suggested that ∼200 Gb of sequencing data are required for 95% abundance-weighted average coverage of the soil communities analyzed. AVAILABILITY AND IMPLEMENTATION Nonpareil is available at https://github.com/lmrodriguezr/nonpareil/ under the Artistic License 2.0.

Detecting Nitrous Oxide Reductase (nosZ) Genes in Soil Metagenomes: Method Development and Implications for the Nitrogen Cycle

ABSTRACT Microbial activities in soils, such as (incomplete) denitrification, represent major sources of nitrous oxide (N2O), a potent greenhouse gas. The key enzyme for mitigating N2O emissions is NosZ, which catalyzes N2O reduction to N2. We recently described “atypical” functional NosZ proteins encoded by both denitrifiers and nondenitrifiers, which were missed in previous environmental surveys (R. A. Sanford et al., Proc. Natl. Acad. Sci. U. S. A. 109:19709–19714, 2012, doi:10.1073/pnas.1211238109). Here, we analyzed the abundance and diversity of both nosZ types in whole-genome shotgun metagenomes from sandy and silty loam agricultural soils that typify the U.S. Midwest corn belt. First, different search algorithms and parameters for detecting nosZ metagenomic reads were evaluated based on in silico-generated (mock) metagenomes. Using the derived cutoffs, 71 distinct alleles (95% amino acid identity level) encoding typical or atypical NosZ proteins were detected in both soil types. Remarkably, more than 70% of the total nosZ reads in both soils were classified as atypical, emphasizing that prior surveys underestimated nosZ abundance. Approximately 15% of the total nosZ reads were taxonomically related to Anaeromyxobacter, which was the most abundant genus encoding atypical NosZ-type proteins in both soil types. Further analyses revealed that atypical nosZ genes outnumbered typical nosZ genes in most publicly available soil metagenomes, underscoring their potential role in mediating N2O consumption in soils. Therefore, this study provides a bioinformatics strategy to reliably detect target genes in complex short-read metagenomes and suggests that the analysis of both typical and atypical nosZ sequences is required to understand and predict N2O flux in soils. IMPORTANCE Nitrous oxide (N2O) is a potent greenhouse gas with ozone layer destruction potential. Microbial activities control both the production and the consumption of N2O, i.e., its conversion to innocuous dinitrogen gas (N2). Until recently, consumption of N2O was attributed to bacteria encoding “typical” nitrous oxide reductase (NosZ). However, recent phylogenetic and physiological studies have shown that previously uncharacterized, functional, “atypical” NosZ proteins are encoded in genomes of diverse bacterial groups. The present study revealed that atypical nosZ genes outnumbered their typical counterparts, highlighting their potential role in N2O consumption in soils and possibly other environments. These findings advance our understanding of the diversity of microbes and functional genes involved in the nitrogen cycle and provide the means (e.g., gene sequences) to study N2O fluxes to the atmosphere and associated climate change. Nitrous oxide (N2O) is a potent greenhouse gas with ozone layer destruction potential. Microbial activities control both the production and the consumption of N2O, i.e., its conversion to innocuous dinitrogen gas (N2). Until recently, consumption of N2O was attributed to bacteria encoding “typical” nitrous oxide reductase (NosZ). However, recent phylogenetic and physiological studies have shown that previously uncharacterized, functional, “atypical” NosZ proteins are encoded in genomes of diverse bacterial groups. The present study revealed that atypical nosZ genes outnumbered their typical counterparts, highlighting their potential role in N2O consumption in soils and possibly other environments. These findings advance our understanding of the diversity of microbes and functional genes involved in the nitrogen cycle and provide the means (e.g., gene sequences) to study N2O fluxes to the atmosphere and associated climate change.

Shifts in bacterial communities of two caribbean reef-building coral species affected by white plague disease

Coral reefs are deteriorating at an alarming rate mainly as a consequence of the emergence of coral diseases. The white plague disease (WPD) is the most prevalent coral disease in the southwestern Caribbean, affecting dozens of coral species. However, the identification of a single causal agent has proved problematic. This suggests more complex etiological scenarios involving alterations in the dynamic interaction between environmental factors, the coral immune system and the symbiotic microbial communities. Here we compare the microbiome of healthy and WPD-affected corals from the two reef-building species Diploria strigosa and Siderastrea siderea collected at the Tayrona National Park in the Caribbean of Colombia. Microbiomes were analyzed by combining culture-dependent methods and pyrosequencing of 16S ribosomal DNA (rDNA) V5-V6 hypervariable regions. A total of 20 410 classifiable 16S rDNA sequences reads were obtained including all samples. No significant differences in operational taxonomic unit diversity were found between healthy and affected tissues; however, a significant increase of Alphaproteobacteria and a concomitant decrease in the Beta- and Gammaproteobacteria was observed in WPD-affected corals of both species. Significant shifts were also observed in the orders Rhizobiales, Caulobacteriales, Burkholderiales, Rhodobacterales, Aleteromonadales and Xanthomonadales, although they were not consistent between the two coral species. These shifts in the microbiome structure of WPD-affected corals suggest a loss of community-mediated growth control mechanisms on bacterial populations specific for each holobiont system.

Microbial community successional patterns in beach sands impacted by the Deepwater Horizon oil spill

Although petroleum hydrocarbons discharged from the Deepwater Horizon (DWH) blowout were shown to have a pronounced impact on indigenous microbial communities in the Gulf of Mexico, effects on nearshore or coastal ecosystems remain understudied. This study investigated the successional patterns of functional and taxonomic diversity for over 1 year after the DWH oil was deposited on Pensacola Beach sands (FL, USA), using metagenomic and 16S rRNA gene amplicon techniques. Gamma- and Alphaproteobacteria were enriched in oiled sediments, in corroboration of previous studies. In contrast to previous studies, we observed an increase in the functional diversity of the community in response to oil contamination and a functional transition from generalist populations within 4 months after oil came ashore to specialists a year later, when oil was undetectable. At the latter time point, a typical beach community had reestablished that showed little to no evidence of oil hydrocarbon degradation potential, was enriched in archaeal taxa known to be sensitive to xenobiotics, but differed significantly from the community before the oil spill. Further, a clear succession pattern was observed, where early responders to oil contamination, likely degrading aliphatic hydrocarbons, were replaced after 3 months by populations capable of aromatic hydrocarbon decomposition. Collectively, our results advance the understanding of how natural benthic microbial communities respond to crude oil perturbation, supporting the specialization-disturbance hypothesis; that is, the expectation that disturbance favors generalists, while providing (microbial) indicator species and genes for the chemical evolution of oil hydrocarbons during degradation and weathering.

Soil microbial community responses to a decade of warming as revealed by comparative metagenomics.

ABSTRACT Soil microbial communities are extremely complex, being composed of thousands of low-abundance species (<0.1% of total). How such complex communities respond to natural or human-induced fluctuations, including major perturbations such as global climate change, remains poorly understood, severely limiting our predictive ability for soil ecosystem functioning and resilience. In this study, we compared 12 whole-community shotgun metagenomic data sets from a grassland soil in the Midwestern United States, half representing soil that had undergone infrared warming by 2°C for 10 years, which simulated the effects of climate change, and the other half representing the adjacent soil that received no warming and thus, served as controls. Our analyses revealed that the heated communities showed significant shifts in composition and predicted metabolism, and these shifts were community wide as opposed to being attributable to a few taxa. Key metabolic pathways related to carbon turnover, such as cellulose degradation (∼13%) and CO2 production (∼10%), and to nitrogen cycling, including denitrification (∼12%), were enriched under warming, which was consistent with independent physicochemical measurements. These community shifts were interlinked, in part, with higher primary productivity of the aboveground plant communities stimulated by warming, revealing that most of the additional, plant-derived soil carbon was likely respired by microbial activity. Warming also enriched for a higher abundance of sporulation genes and genomes with higher G+C content. Collectively, our results indicate that microbial communities of temperate grassland soils play important roles in mediating feedback responses to climate change and advance the understanding of the molecular mechanisms of community adaptation to environmental perturbations.

MyTaxa: an advanced taxonomic classifier for genomic and metagenomic sequences

Determining the taxonomic affiliation of sequences assembled from metagenomes remains a major bottleneck that affects research across the fields of environmental, clinical and evolutionary microbiology. Here, we introduce MyTaxa, a homology-based bioinformatics framework to classify metagenomic and genomic sequences with unprecedented accuracy. The distinguishing aspect of MyTaxa is that it employs all genes present in an unknown sequence as classifiers, weighting each gene based on its (predetermined) classifying power at a given taxonomic level and frequency of horizontal gene transfer. MyTaxa also implements a novel classification scheme based on the genome-aggregate average amino acid identity concept to determine the degree of novelty of sequences representing uncharacterized taxa, i.e. whether they represent novel species, genera or phyla. Application of MyTaxa on in silico generated (mock) and real metagenomes of varied read length (100–2000 bp) revealed that it correctly classified at least 5% more sequences than any other tool. The analysis also showed that ∼10% of the assembled sequences from human gut metagenomes represent novel species with no sequenced representatives, several of which were highly abundant in situ such as members of the Prevotella genus. Thus, MyTaxa can find several important applications in microbial identification and diversity studies.

Genomes-based phylogeny of the genus Xanthomonas

BackgroundThe genus Xanthomonas comprises several plant pathogenic bacteria affecting a wide range of hosts. Despite the economic, industrial and biological importance of Xanthomonas, the classification and phylogenetic relationships within the genus are still under active debate. Some of the relationships between pathovars and species have not been thoroughly clarified, with old pathovars becoming new species. A change in the genus name has been recently suggested for Xanthomonas albilineans, an early branching species currently located in this genus, but a thorough phylogenomic reconstruction would aid in solving these and other discrepancies in this genus.ResultsHere we report the results of the genome-wide analysis of DNA sequences from 989 orthologous groups from 17 Xanthomonas spp. genomes available to date, representing all major lineages within the genus. The phylogenetic and computational analyses used in this study have been automated in a Perl package designated Unus, which provides a framework for phylogenomic analyses which can be applied to other datasets at the genomic level. Unus can also be easily incorporated into other phylogenomic pipelines.ConclusionsOur phylogeny agrees with previous phylogenetic topologies on the genus, but revealed that the genomes of Xanthomonas citri and Xanthomonas fuscans belong to the same species, and that of Xanthomonas albilineans is basal to the joint clade of Xanthomonas and Xylella fastidiosa. Genome reduction was identified in the species Xanthomonas vasicola in addition to the previously identified reduction in Xanthomonas albilineans. Lateral gene transfer was also observed in two gene clusters.

SAR11 bacteria linked to ocean anoxia and nitrogen loss

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world’s largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth’s most abundant organismal group.