The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained ...popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Bacteria comprise the most diverse domain of life on Earth, where they occupy nearly every possible ecological niche and play key roles in biological and chemical processes. Studying the ...composition and ecology of bacterial ecosystems and understanding their function are of prime importance. High‐throughput sequencing technologies enable nearly comprehensive descriptions of bacterial diversity through 16S ribosomal RNA gene amplicons. Analyses of these communities generally rely upon taxonomic assignments through reference data bases or clustering approaches using de facto sequence similarity thresholds to identify operational taxonomic units. However, these methods often fail to resolve ecologically meaningful differences between closely related organisms in complex microbial data sets.
In this paper, we describe oligotyping, a novel supervised computational method that allows researchers to investigate the diversity of closely related but distinct bacterial organisms in final operational taxonomic units identified in environmental data sets through 16S ribosomal RNA gene data by the canonical approaches.
Our analysis of two data sets from two different environments demonstrates the capacity of oligotyping at discriminating distinct microbial populations of ecological importance.
Oligotyping can resolve the distribution of closely related organisms across environments and unveil previously overlooked ecological patterns for microbial communities. The URL http://oligotyping.org offers an open‐source software pipeline for oligotyping.
During theDeepwater Horizonoil well blowout in the Gulf of Mexico, the application of 7 million liters of chemical dispersants aimed to stimulate microbial crude oil degradation by increasing the ...bioavailability of oil compounds. However, the effects of dispersants on oil biodegradation rates are debated. In laboratory experiments, we simulated environmental conditions comparable to the hydrocarbon-rich, 1,100 m deep plume that formed during theDeepwater Horizondischarge. The presence of dispersant significantly altered the microbial community composition through selection for potential dispersant-degradingColwellia,which also bloomed in situ in Gulf deep waters during the discharge. In contrast, oil addition to deepwater samples in the absence of dispersant stimulated growth of natural hydrocarbon-degradingMarinobacter.In these deepwater microcosm experiments, dispersants did not enhance heterotrophic microbial activity or hydrocarbon oxidation rates. An experiment with surface seawater from an anthropogenically derived oil slick corroborated the deepwater microcosm results as inhibition of hydrocarbon turnover was observed in the presence of dispersants, suggesting that the microcosm findings are broadly applicable across marine habitats. Extrapolating this comprehensive dataset to real world scenarios questions whether dispersants stimulate microbial oil degradation in deep ocean waters and instead highlights that dispersants can exert a negative effect on microbial hydrocarbon degradation rates.
Earth's subsurface environment is one of the largest, yet least studied, biomes on Earth, and many questions remain regarding what microorganisms are indigenous to the subsurface. Through the ...activity of the Census of Deep Life (CoDL) and the Deep Carbon Observatory, an open access 16S ribosomal RNA gene sequence database from diverse subsurface environments has been compiled. However, due to low quantities of biomass in the deep subsurface, the potential for incorporation of contaminants from reagents used during sample collection, processing, and/or sequencing is high. Thus, to understand the ecology of subsurface microorganisms (i.e., the distribution, richness, or survival), it is necessary to minimize, identify, and remove contaminant sequences that will skew the relative abundances of all taxa in the sample. In this meta-analysis, we identify putative contaminants associated with the CoDL dataset, recommend best practices for removing contaminants from samples, and propose a series of best practices for subsurface microbiology sampling. The most abundant putative contaminant genera observed, independent of evenness across samples, were
,
,
, and
. While the top five most frequently observed genera were
,
,
,
, and
. The majority of the most frequently observed genera (high evenness) were associated with reagent or potential human contamination. Additionally, in DNA extraction blanks, we observed potential archaeal contaminants, including methanogens, which have not been discussed in previous contamination studies. Such contaminants would directly affect the interpretation of subsurface molecular studies, as methanogenesis is an important subsurface biogeochemical process. Utilizing previously identified contaminant genera, we found that ∼27% of the total dataset were identified as contaminant sequences that likely originate from DNA extraction and DNA cleanup methods. Thus, controls must be taken at every step of the collection and processing procedure when working with low biomass environments such as, but not limited to, portions of Earth's deep subsurface. Taken together, we stress that the CoDL dataset is an incredible resource for the broader research community interested in subsurface life, and steps to remove contamination derived sequences must be taken prior to using this dataset.
The Deepwater Horizon (DWH) oil well blowout generated an enormous plume of dispersed hydrocarbons that substantially altered the Gulf of Mexico's deep-sea microbial community. A significant ...enrichment of distinct microbial populations was observed, yet, little is known about the abundance and richness of specific microbial ecotypes involved in gas, oil and dispersant biodegradation in the wake of oil spills. Here, we document a previously unrecognized diversity of closely related taxa affiliating with Cycloclasticus, Colwellia and Oceanospirillaceae and describe their spatio-temporal distribution in the Gulf's deepwater, in close proximity to the discharge site and at increasing distance from it, before, during and after the discharge. A highly sensitive, computational method (oligotyping) applied to a data set generated from 454-tag pyrosequencing of bacterial 16S ribosomal RNA gene V4-V6 regions, enabled the detection of population dynamics at the sub-operational taxonomic unit level (0.2% sequence similarity). The biogeochemical signature of the deep-sea samples was assessed via total cell counts, concentrations of short-chain alkanes (C1-C5), nutrients, (colored) dissolved organic and inorganic carbon, as well as methane oxidation rates. Statistical analysis elucidated environmental factors that shaped ecologically relevant dynamics of oligotypes, which likely represent distinct ecotypes. Major hydrocarbon degraders, adapted to the slow-diffusive natural hydrocarbon seepage in the Gulf of Mexico, appeared unable to cope with the conditions encountered during the DWH spill or were outcompeted. In contrast, diverse, rare taxa increased rapidly in abundance, underscoring the importance of specialized sub-populations and potential ecotypes during massive deep-sea oil discharges and perhaps other large-scale perturbations.
Modern microbial mats are potential analogues for Proterozoic ecosystems, yet only a few studies have characterized mats under low-oxygen conditions that are relevant to Proterozoic environments. ...Here, we use protein-stable isotope fingerprinting (P-SIF) to determine the protein carbon isotope (δ
C) values of autotrophic, heterotrophic, and mixotrophic organisms in a benthic microbial mat from the low-oxygen Middle Island Sinkhole, Lake Huron, USA (MIS). We also measure the δ
C values of the sugar moieties of exopolysaccharides (EPS) within the mat to explore the relationships between cyanobacterial exudates and heterotrophic anabolic carbon uptake. Our results show that Cyanobacteria (autotrophs) are
C-depleted, relative to sulfate-reducing bacteria (heterotrophs), and
C-enriched, relative to sulfur oxidizing bacteria (autotrophs or mixotrophs). We also find that the pentose moieties of EPS are systematically enriched in
C, relative to the hexose moieties of EPS. We hypothesize that these isotopic patterns reflect cyanobacterial metabolic pathways, particularly phosphoketolase, that are relatively more active in low-oxygen mat environments, rather than oxygenated mat environments. This results in isotopically more heterogeneous C sources in low-oxygen mats. While this might partially explain the isotopic variability observed in Proterozoic mat facies, further work is necessary to systematically characterize the isotopic fractionations that are associated with the synthesis of cyanobacterial exudates.
The δ
C compositions of heterotrophic microorganisms are dictated by the δ
C compositions of their organic carbon sources. In both modern and ancient photosynthetic microbial mats, photosynthetic exudates are the most likely source of organic carbon for heterotrophs. We measured the δ
C values of autotrophic, heterotrophic, and mixotrophic bacteria as well as the δ
C value of the most abundant photosynthetic exudate (exopolysaccharide) in a modern analogue for a Proterozoic environment. Given these data, future studies will be better equipped to estimate the most likely carbon source for heterotrophs in both modern environments as well as in Proterozoic environments preserved in the rock record.
Anoxygenic cyanobacteria that use sulfide as the electron donor for photosynthesis are a potentially influential but poorly constrained force on Earth's biogeochemistry. Their versatile metabolism ...may have boosted primary production and nitrogen cycling in euxinic coastal margins in the Proterozoic. In addition, they represent a biological mechanism for limiting the accumulation of atmospheric oxygen, especially before the Great Oxidation Event and in the low-oxygen conditions of the Proterozoic. In this study, we describe the draft genome sequence of
sp. PCC 9228, formerly
'Solar Lake', a mat-forming diazotrophic cyanobacterium that can switch between oxygenic photosynthesis and sulfide-based anoxygenic photosynthesis (AP).
possesses three variants of
, which encodes protein D1, a core component of the photosystem II reaction center. Phylogenetic analyses indicate that one variant is closely affiliated with cyanobacterial
genes that code for a D1 protein used for oxygen-sensitive processes. Another version is phylogenetically similar to cyanobacterial
genes that encode D1 proteins used under microaerobic conditions, and the third variant may be cued to high light and/or elevated oxygen concentrations.
has the canonical gene for sulfide quinone reductase (SQR) used in cyanobacterial AP and a putative transcriptional regulatory gene in the same operon. Another operon with a second, distinct
and regulatory gene is present, and is phylogenetically related to
genes used for high sulfide concentrations. The genome has a comprehensive
gene suite for nitrogen fixation, supporting previous observations of nitrogenase activity.
possesses a bidirectional hydrogenase rather than the uptake hydrogenase typically used by cyanobacteria in diazotrophy. Overall, the genome sequence of
sp. PCC 9228 highlights potential cyanobacterial strategies to cope with fluctuating redox gradients and nitrogen availability that occur in benthic mats over a diel cycle. Such dynamic geochemical conditions likely also challenged Proterozoic cyanobacteria, modulating oxygen production. The genetic repertoire that underpins flexible oxygenic/anoxygenic photosynthesis in cyanobacteria provides a foundation to explore the regulation, evolutionary context, and biogeochemical implications of these co-occurring metabolisms in Earth history.
Seasonal changes in light and physicochemical conditions have strong impacts on cyanobacteria, but how they affect community structure, metabolism, and biogeochemistry of cyanobacterial mats remains ...unclear. Light may be particularly influential for cyanobacterial mats exposed to sulphide by altering the balance of oxygenic photosynthesis and sulphide‐driven anoxygenic photosynthesis. We studied temporal shifts in irradiance, water chemistry, and community structure and function of microbial mats in the Middle Island Sinkhole (MIS), where anoxic and sulphate‐rich groundwater provides habitat for cyanobacteria that conduct both oxygenic and anoxygenic photosynthesis. Seasonal changes in light and groundwater chemistry were accompanied by shifts in bacterial community composition, with a succession of dominant cyanobacteria from Phormidium to Planktothrix, and an increase in diatoms, sulphur‐oxidizing bacteria, and sulphate‐reducing bacteria from summer to autumn. Differential abundance of cyanobacterial light‐harvesting proteins likely reflects a physiological response of cyanobacteria to light level. Beggiatoa sulphur oxidation proteins were more abundant in autumn. Correlated abundances of taxa through time suggest interactions between sulphur oxidizers and sulphate reducers, sulphate reducers and heterotrophs, and cyanobacteria and heterotrophs. These results support the conclusion that seasonal change, including light availability, has a strong influence on community composition and biogeochemical cycling of sulphur and O2 in cyanobacterial mats.
We studied temporal shifts in irradiance, water chemistry, and community structure and function of microbial mats in Middle Island Sinkhole (MIS), an O2‐poor benthic sinkhole in Lake Huron, Michigan where anoxic and sulphate‐rich groundwater provides habitat for cyanobacteria that conduct both oxygenic and anoxygenic photosynthesis. We found that higher availability of sulphate‐rich groundwater, together with higher light intensity, coincided with dominance of the metabolically flexible cyanobacterium Phormidium during the summer. Diverse sulphur cycling bacteria were more successful in other seasons when the mat experienced lower light and sulphate availability. These results provide insights into how seasonal environmental dynamics can shape the community structure and metabolisms of microbial mats, ultimately controlling biogeochemical cycling in these ecological hotspots.
Human-associated microbial communities include fungi, but we understand little about which fungal species are present, their relative and absolute abundances, and how antimicrobial therapy impacts ...fungal communities. The disease cystic fibrosis (CF) often involves chronic airway colonization by bacteria and fungi, and these infections cause irreversible lung damage. Fungi are detected more frequently in CF sputum samples upon initiation of antimicrobial therapy, and several studies have implicated the detection of fungi in sputum with worse outcomes. Thus, a more complete understanding of fungi in CF is required.
We characterized the fungi and bacteria in expectorated sputa from six CF subjects. Samples were collected upon admission for systemic antibacterial therapy and upon the completion of treatment and analyzed using a pyrosequencing-based analysis of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA sequences. A mixture of Candida species and Malassezia dominated the mycobiome in all samples (74%-99% of fungal reads). There was not a striking trend correlating fungal and bacterial richness, and richness showed a decline after antibiotic therapy particularly for the bacteria. The fungal communities within a sputum sample resembled other samples from that subject despite the aggressive antibacterial therapy. Quantitative PCR analysis of fungal 18S rDNA sequences to assess fungal burden showed variation in fungal density in sputum before and after antibacterial therapy but no consistent directional trend. Analysis of Candida ITS1 sequences amplified from sputum or pure culture-derived genomic DNA from individual Candida species found little (<0.5%) or no variation in ITS1 sequences within or between strains, thereby validating this locus for the purpose of Candida species identification. We also report the enhancement of the publically available Visualization and Analysis of Microbial Population Structures (VAMPS) tool for the analysis of fungal communities in clinical samples.
Fungi are present in CF respiratory sputum. In CF, the use of intravenous antibiotic therapy often does not profoundly impact bacterial community structure, and we observed a similar stability in fungal species composition. Further studies are required to predict the effects of antibacterials on fungal burden in CF and fungal community stability in non-CF populations.
The sedimentary pyrite sulfur isotope (δ34S) record is an archive of ancient microbial sulfur cycling and environmental conditions. Interpretations of pyrite δ34S signatures in sediments deposited in ...microbial mat ecosystems are based on studies of modern microbial mat porewater sulfide δ34S geochemistry. Pyrite δ34S values often capture δ34S signatures of porewater sulfide at the location of pyrite formation. However, microbial mats are dynamic environments in which biogeochemical cycling shifts vertically on diurnal cycles. Therefore, there is a need to study how the location of pyrite formation impacts pyrite δ34S patterns in these dynamic systems. Here, we present diurnal porewater sulfide δ34S trends and δ34S values of pyrite and iron monosulfides from Middle Island Sinkhole, Lake Huron. The sediment–water interface of this sinkhole hosts a low‐oxygen cyanobacterial mat ecosystem, which serves as a useful location to explore preservation of sedimentary pyrite δ34S signatures in early Earth environments. Porewater sulfide δ34S values vary by up to ~25‰ throughout the day due to light‐driven changes in surface microbial community activity that propagate downwards, affecting porewater geochemistry as deep as 7.5 cm in the sediment. Progressive consumption of the sulfate reservoir drives δ34S variability, instead of variations in average cell‐specific sulfate reduction rates and/or sulfide oxidation at different depths in the sediment. The δ34S values of pyrite are similar to porewater sulfide δ34S values near the mat surface. We suggest that oxidative sulfur cycling and other microbial activity promote pyrite formation in and immediately adjacent to the microbial mat and that iron geochemistry limits further pyrite formation with depth in the sediment. These results imply that primary δ34S signatures of pyrite deposited in organic‐rich, iron‐poor microbial mat environments capture information about microbial sulfur cycling and environmental conditions at the mat surface and are only minimally affected by deeper sedimentary processes during early diagenesis.
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