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PubMed2025

CoverM: read alignment statistics for metagenomics.

SUMMARY: Genome-centric analysis of metagenomic samples is a powerful method for understanding the function of microbial communities. Calculating read coverage is a central part of analysis, enabling differential coverage binning for recovery of genomes and estimation of microbial community composition. Coverage is determined by processing read alignments to reference sequences of either contigs or genomes. Per-reference coverage is typically calculated in an ad-hoc manner, with each software package providing its own implementation and specific definition of coverage. Here we present a unified software package CoverM which calculates several coverage statistics for contigs and genomes in an ergonomic and flexible manner. It uses "Mosdepth arrays" for computational efficiency and avoids unnecessary I/O overhead by calculating coverage statistics from streamed read alignment results. AVAILABILITY AND IMPLEMENTATION: CoverM is free software available at https://github.com/wwood/coverm. CoverM is implemented in Rust, with Python (https://github.com/apcamargo/pycoverm) and Julia (https://github.com/JuliaBinaryWrappers/CoverM_jll.jl) interfaces.

Metabolomics↗
PubMed1998

rRNA-targeted fluorescent in situ hybridization analysis of bacterial community structure in river water.

An improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Flavobacterium-Cytophaga; Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes; Vibrio-Aeromonas; Pseudomonas (rRNA I): the genus Acinetobacter] were used to investigate the bacterial community structure at two sites differing in organic carbon pollution level. At the eutrophic site, 54-68% of all cells visualized by staining with DAPI (4',6-diamidino-2-phenylindole) could be detected with probe EUB338. In samples from the oligotrophic site, 39-45% of the total cells hybridized with EUB338. At the eutrophic site, approximately 50% of the total cells were identified with the five group-specific probes; the bacterial community structure was dominated by the Flavobacterium-Cytophaga group and Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes group. At the oligotrophic site, only 26-38% of the total cells were identified with the five group-specific probes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectable cells differed. No appreciable change was found in the community structure during the sampling period at either site. The improved HNPP-FISH technique should be a useful tool for the analysis of microbial community composition.

Bacteria↗
PubMed2004

Bacterial diversity in marine hydrocarbon seep sediments.

Marine seeps introduce significant amounts of hydrocarbons into oceans and create unusual habitats for microfauna and -flora. In the vicinity of chronic seeps, microbes likely exert control on carbon quality entering the marine food chain and, in turn, hydrocarbons could influence microbial community composition and diversity. To determine the effects of seep oil on marine sediment bacterial communities, we collected sediment piston cores within an active marine hydrocarbon seep zone in the Coal Oil Point Seep Field, at a depth of 22 m in the Santa Barbara Channel, California. Cores were taken adjacent to an active seep vent in a hydrocarbon volcano, on the edge of the volcano, and at the periphery of the area of active seepage. Bacterial community profiles were determined by terminal restriction fragment length polymorphisms (TRFLPs) of 16S ribosomal genes that were polymerase chain reaction (PCR)-amplified with eubacterial primers. Sediment carbon content and C/N ratio increased with oil content. Terminal restriction fragment length polymorphisms suggested that bacterial communities varied both with depth into sediments and with oil concentration. Whereas the apparent abundance of several peaks correlated positively with hydrocarbon content, overall bacterial diversity and richness decreased with increasing sediment hydrocarbon content. Sequence analysis of a clone library generated from sediments collected at the periphery of the seep suggested that oil-sensitive species belong to the gamma Proteobacteria and Holophaga groups. These sequences were closely related to sequences previously recovered from uncontaminated marine sediments. Our results suggest that seep hydrocarbons exert a strong selective pressure on bacterial communities in marine sediments. This selective pressure could, in turn, control the effects of oil on other biota in the vicinity of marine hydrocarbon seeps.

Bacteria↗
PubMed2006

Bacterial and archaeal phylotypes associated with distinct mineralogical layers of a white smoker spire from a deep-sea hydrothermal vent site (9 degrees N, East Pacific Rise).

A diffusely venting chimney spire from the East Pacific Rise (9 degrees N) was analysed by petrographic thin sectioning and 16S rRNA gene cloning and sequencing in parallel, to correlate microbial community composition with mineralogy and inferred in situ conditions within the chimney mineral matrix. Both approaches indicated a zonation of the chimney spire into distinct microhabitats for different bacteria and archaea. The thermal gradient inferred from the mineral composition and porosity of the chimney was consistent with the distribution of bacterial and archaeal phylotypes in the chimney matrix. A novel phylogenetic lineage of euryarchaeota was found that co-occurred with clones related to cultured hyperthermophilic archaea. A few phylotypes related to mesophilic bacteria were found in the hot core of the chimney, indicating that seawater influx during retrieval and cooling of these highly porous structures can entrain microorganisms into chimney layers that are not their native habitat.

Archaea↗
PubMed2006

An anaerobic methane-oxidizing community of ANME-1b archaea in hypersaline Gulf of Mexico sediments.

Sediments overlying a brine pool methane seep in the Gulf of Mexico (Green Canyon 205) were analyzed using molecular and geochemical approaches to identify geochemical controls on microbial community composition and stratification. 16S rRNA gene and rRNA clone libraries, as well as mcrA gene clone libraries, showed that the archaeal community consists predominantly of ANME-1b methane oxidizers; no archaea of other ANME subgroups were found with general and group-specific PCR primers. The ANME-1b community was found in the sulfate-methane interface, where undersaturated methane concentrations of ca. 100 to 250 microM coexist with sulfate concentrations around 10 mM. Clone libraries of dsrAB genes and bacterial 16S rRNA genes show diversified sulfate-reducing communities within and above the sulfate-methane interface. Their phylogenetic profiles and occurrence patterns are not linked to ANME-1b populations, indicating that electron donors other than methane, perhaps petroleum-derived hydrocarbons, drive sulfate reduction. The archaeal component of anaerobic oxidation of methane is comprised of an active population of mainly ANME-1b in this hypersaline sediment.

Anaerobiosis↗
PubMed1990

Molecular analysis of deep-subsurface bacteria.

Bacterial isolates from deep-sediment samples from three sites at the Savannah River site, near Aiken, S.C., were studied to determine their microbial community composition and DNA structure by using total DNA hybridization and moles percent G + C. Standard phenotypic identification underestimated the bacterial diversity at the three sites, since isolates with the same phenotype had different DNA structures in terms of moles percent G + C and DNA homology. The G + C content of deep-subsurface bacteria ranged from 20 to 77 mol%. More than 60% of the isolates tested had G + C values similar to those of Pseudomonas spp., and 12% had values similar to those of Acinetobacter spp. No isolates from deeper formations showed the same DNA composition as isolates from upper formations. Total-DNA hybridization and DNA base composition analysis provided a better resolution than phenotypic tests for the understanding of the diversity and structure of deep-subsurface bacterial communities. On the basis of the moles percent G + C values, deep-subsurface isolates tested seemed to belong to the families Pseudomonadaceae and Neisseriaceae, which might reflect a long period of adaptation to the environmental conditions of the deep subsurface.

Bacteria↗
PubMed2000

Application of neural computing methods for interpreting phospholipid fatty acid profiles of natural microbial communities.

The microbial community compositions of surface and subsurface marine sediments and sediments lining burrows of marine polychaetes and hemichordates from the North Inlet estuary (near Georgetown, S.C. ) were analyzed by comparing ester-linked phospholipid fatty acid (PLFA) profiles with a back-propagating neural network (NN). The NNs were trained to relate PLFA inputs to sediment type outputs (e.g., surface, subsurface, and burrow lining) and worm species (e.g., Notomastus lobatus, Balanoglossus aurantiacus, and Branchyoasychus americana). Sensitivity analysis was used to determine which of the 60 PLFAs significantly contributed to training the NN. The NN architecture was optimized by changing the number of hidden neurons and calculating the cross-validation error between predicted and actual outputs of training and test data. The optimal NN architecture was found to be four hidden neurons with 60-input neurons representing the 60 PLFAs, and four output neurons coding for both sediment types and worm species. Comparison of cross-validation results using NNs and linear discriminant analysis (LDA) revealed that NNs had significantly fewer incorrect classifications (2.7%) than LDA (8.4%). For the NN cross-validation, both sediment type and worm species had 3 incorrect classifications out of 112. For the LDA cross-validation, sediment type and worm species had 7 and 12 incorrect classifications out of 112, respectively. Sensitivity analysis of the trained NNs revealed that 17 fatty acids explained 50% of variability in the data set. These PLFAs were highly different among sediments and burrow types, indicating significant differences in the microbiota.

Animals↗
PubMed2004

Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient.

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R(2) = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R(2) = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the alpha, beta, and gamma-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.

Base Sequence↗
PubMed2006

Molecular characterization of subject-specific oral microflora during initial colonization of enamel.

The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.

Actinomyces↗
PubMed2001

Impact of polychaetes (Nereis spp. and Arenicola marina) on carbon biogeochemistry in coastal marine sediments.

Known effects of bioturbation by common polychaetes (Nereis spp. and Arenicola marina) in Northern European coastal waters on sediment carbon diagenesis is summarized and assessed. The physical impact of irrigation and reworking activity of the involved polychaete species is evaluated and related to their basic biology. Based on past and present experimental work, it is concluded that effects of bioturbation on carbon diagenesis from manipulated laboratory experiments cannot be directly extrapolated to in situ conditions. The 45-260% flux (e.g., CO2 release) enhancement found in the laboratory is much higher than usually observed in the field (10-25%). Thus, the faunal induced enhancement of microbial carbon oxidation in natural sediments instead causes a reduction of the organic matter inventory rather than an increased release of CO2 across the sediment/water interface. The relative decrease in organic inventory (Gb/Gu) is inversely related to the relative increase in microbial capacity for organic matter decay (kb/ku). The equilibrium is controlled by the balance between organic input (deposition of organic matter at the sediment surface) and the intensity of bioturbation. Introduction of oxygen to subsurface sediment and removal of metabolites are considered the two most important underlying mechanisms for the stimulation of carbon oxidation by burrowing fauna. Introduction of oxygen to deep sediment layers of low microbial activity, either by downward irrigation transport of overlying oxic water or by upward reworking transport of sediment to the oxic water column will increase carbon oxidation of anaerobically refractory organic matter. It appears that the irrigation effect is larger than and to a higher degree dependent on animal density than the reworking effect. Enhancement of anaerobic carbon oxidation by removal of metabolites (reduced diffusion scale) may cause a significant increase in total sediment metabolism. This is caused by three possible mechanisms: (i) combined mineralization and biological uptake; (ii) combined mineralization and abiogenic precipitation; and (iii) alleviation of metabolite inhibition. Finally, some suggestions for future work on bioturbation effects are presented, including: (i) experimental verification of metabolite inhibition in bioturbated sediments; (ii) mapping and quantification of the role of metals as electron acceptors in bioturbated sediments; and (iii) identification of microbial community composition by the use of new molecular biological techniques. These three topics are not intended to cover all unresolved aspects of bioturbation, but should rather be considered a list of obvious gaps in our knowledge and present new and appealing approaches.

Journal Article↗
PubMed2005

Graphical representation of ribosomal RNA probe accessibility data using ARB software package.

BACKGROUND: Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments. RESULTS: The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different types of sequence associated information (SAI) can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool. CONCLUSION: Using this tool, in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of in situ hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page http://www.arb-home.de

Algorithms↗
PubMed2001

Biolog for the determination of microbial diversity in activated sludge systems.

In this study, different carbon source profiles were generated by inoculating Biolog GN microwell plates, with different dilutions of microbial communities from a number of activated sludge systems. This led to the successful generation of patterns reflecting diversity and evenness in the different systems. The high number of substrates utilized at the lower dilutions (10(-1) and 10(-2)) indicated a high microbial diversity in the community, but not necessarily evenness of each species. Evenness of each species was reflected upon further dilution. Our results indicated differences in the microbial community composition amongst some of the activated sludge systems studied. These differences were not specifically related to phosphate removing and non phosphate removing systems.

Bacteria↗
PubMed2005

Membrane bioreactor versus conventional activated sludge system: population dynamics of nitrifiers.

Although membrane bioreactors have attracted increasing attention in recent years, little research has been undertaken on the influence of the membrane separation on the microbial community composition. This paper compares the startup behaviour and the performance of the subsequent eight months of a membrane bioreactor with a conventional activated sludge pilot plant. Both plants were operated in parallel at the same sludge age and treated the same domestic wastewater. The identification of the nitrifying community composition using fluorescent in situ hybridization revealed only minor differences between the two reactors for both ammonia-oxidizing bacteria and nitrite-oxidizing bacteria. Accordingly, both systems exhibited the same maximum nitrification rates. Confocal laser scanning microscopy showed that the aggregates formed by nitrifying bacteria were located mostly in the inner part of the flocs and were overgrown by heterotrophic bacteria. It is concluded that the membrane separation itself does affect neither the nitrifying community composition nor the nitrification performance. However, impacts on kinetic parameters are emphasized.

Ammonia↗
PubMed2000

Composition of soil microbial communities enriched on a mixture of aromatic hydrocarbons.

Soil contaminated with C5+, which contained benzene (45%, wt/wt), dicyclopentadiene (DCPD) plus cyclopentadiene (together 20%), toluene (6%), styrene (3%), xylenes (2%), naphthalene (2%), and smaller quantities of other compounds, served as the source for isolation of 55 genomically distinct bacteria (standards). Use of benzene as a substrate by these bacteria was most widespread (31 of 44 standards tested), followed by toluene (23 of 44), xylenes (14 of 44), styrene (10 of 44), and naphthalene (10 of 44). Master filters containing denatured genomic DNAs of all 55 standards were used to analyze the community compositions of C5+ enrichment cultures by reverse sample genome probing (RSGP). The communities enriched from three contaminated soils were similar to those enriched from three uncontaminated soils from the same site. The compositions of these communities were time dependent and showed a succession of Pseudomonas and Rhodococcus spp. before convergence on a composition dominated by Alcaligenes spp. The dominant community members detected by RSGP were capable of benzene degradation at all stages of succession. The enrichments effectively degraded all C5+ components except DCPD. Overall, degradation of individual C5+ hydrocarbons followed first-order kinetics, with the highest rates of removal for benzene.

Alcaligenes↗
PubMed2006

H2S degradation is reflected by both the activity and composition of the microbial community in a compost biofilter.

In this study, 16S rRNA- and rDNA-based denaturing gradient gel electrophoresis (DGGE) were used to study the temporal and spatial evolution of the microbial communities in a compost biofilter removing H(2)S and in a control biofilter without H(2)S loading. During the first 81 days of the experiment, the H(2)S removal efficiencies always exceeded 93% at loading rates between 4.1 and 30 g m(-3) h(-1). Afterwards, the H(2)S removal efficiency decreased to values between 44 and 71%. RNA-based DGGE analysis showed that H(2)S loading to the biofilter increased the stability of the active microbial community but decreased the activity-based diversity and evenness. The most intense band in both the RNA- and DNA-based DGGE patterns of the H(2)S-degrading biofilter represented the sulfur oxidizing bacterium Thiobacillus thioparus. This suggested that T. thioparus constituted a major part of the bacterial community and was an important primary degrader in the H(2)S-degrading biofilter. The decreasing H(2)S removal efficiencies near the end of the experiment were not accompanied by a substantial change of the DGGE patterns. Therefore, the decreased H(2)S removal was probably not caused by a failing microbiology but rather by a decrease of the mass transfer of substrates after agglutination of the compost particles.

Bioreactors↗
PubMed2006

Alteration and resilience of the soil microbial community following compost amendment: effects of compost level and compost-borne microbial community.

Compost amendment has been reported to impact soil microbial activities or community composition. However, little information is available on (i) to what extent compost amendment concurrently affects the activity, size and composition of soil microbial community, (ii) the relative effect of the addition of a material rich in organic matter versus addition of compost-borne microorganisms in explaining the effects of amendment and (iii) the resilience of community characteristics. We compared five treatments in microcosms: (i) control soil (S), (ii) soil + low level of compost (Sc), (iii) soil + high level of compost (SC), (iv) sterilized soil + high level of compost [(S)C] and (v) soil + high level of sterilized compost [S(C)]. The actual C mineralization rate, substrate-induced respiration, size of microbial community (biomass and heterotrophic cells number), and structure of total microbial (phospholipid fatty acids) and bacterial (automated ribosomal intergenic spacer analysis, A-RISA) communities were surveyed during 6 months after amendment. Our results show that (i) compost amendment affected the activity, size and composition of the soil microbial community, (ii) the effect of compost amendment was mainly due to the physicochemical characteristics of compost matrix rather than to compost-borne microorganisms and (iii) no resilience of microbial characteristics was observed 6-12 months after amendment with a high amount of compost.

Bacteria↗
PubMed2006

Site-specific variation in Antarctic marine biofilms established on artificial surfaces.

The community structure and composition of marine microbial biofilms established on glass surfaces was investigated across three differentially contaminated Antarctic sites within McMurdo Sound. Diverse microbial communities were revealed at all sites using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques. Sequencing of excised DGGE bands demonstrated close affiliation with known psychrophiles or undescribed bacteria also recovered from the Antarctic environment. The majority of bacterial sequences were affiliated to the Gammaproteobacteria, Cytophaga/Flavobacteria of Bacteroidetes (CFB), Verrucomicrobia and Planctomycetales. Principal components analysis of quantitative FISH data revealed distinct differences in community composition between sites. Each of the sites were dominated by different bacterial groups: Alphaproteobacteria, Gammaproteobacteria and CFB at the least impacted site, Cape Armitage; green sulfur and sulfate reducing bacteria near the semi-impacted Scott Base and Planctomycetales and sulfate reducing bacteria near the highly impacted McMurdo Station. The highest abundance of archaea was detected near Scott Base (2.5% of total bacteria). Multivariate analyses (non-metric multidimensional scaling and analysis of similarities) of DGGE patterns revealed greater variability in community composition between sites than within sites. This is the first investigation of Antarctic biofilm structure and FISH results suggest that anthropogenic impacts may influence the complex composition of microbial communities.

Actinobacteria↗
PubMed1999

Bacterial function and community structure in reactors treating biopolymers and surfactants at mesophilic and thermophilic temperatures.

Microbial communities capable of degrading biopolymers and surfactants typically found in graywater were selected in continuous-flow bioreactors operated at 30, 44, 53, or 62 degrees C. The effect of temperature upon microbial activity and community composition was determined. Microbial respiration of the organic components of the medium (including linear alkylbenzene sulfonate) was detected in samples from each reactor. The microbial community in each reactor was adapted to the operating temperature. Nucleic acid-based analyses of community composition showed that distinct consortia were present at each temperature. Community complexity was inversely related to temperature. The specific maintenance rate was twofold higher at 62 degrees C than at the lower temperatures. Under starvation conditions, microbes in the 62 degrees C system lost membrane integrity 30- to 100-fold faster than microbes at lower temperatures.

Journal Article↗