Extensive profiling of a complex microbial community by high-throughput sequencing

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.     

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community

Although uncultured microorganisms have important roles in ecosystems, their ecophysiology in situ remains elusive owing to the difficulty of obtaining live cells from their natural habitats. In this study, we employed a novel magnetic nanoparticle-mediated isolation (MMI) method to recover metabolically active cells of a group of previously uncultured phenol degraders, Burkholderiales spp., from coking plant wastewater biosludge; five other culturable phenol degraders—Rhodococcus sp., Chryseobacterium sp. and three different Pseudomonas spp.—were also isolated from the same biosludge using traditional methods. The kinetics of phenol degradation by MMI-recovered cells (MRCs) was similar to that of the original sludge. Stable isotope probing (SIP) and pyrosequencing of the 16S rRNA from the ‘heavy'' DNA (¹³C-DNA) fractions indicated that Burkholderiales spp. were the key phenol degraders in situ in the biosludge, consistent with the results of MRCs. Single-cell Raman micro-spectroscopy was applied to probe individual bacteria in the MRCs obtained from the SIP experiment and showed that 79% of them were fully ¹³C-labelled. Biolog assays on the MRCs revealed the impact of various carbon and nitrogen substrates on the efficiency of phenol degradation in the wastewater treatment plant biosludge. Specifically, hydroxylamine, a metabolite of ammonia oxidisation, but not nitrite, nitrate or ammonia, inhibited phenol degradation in the biosludge. Our results provided a novel insight into the occasional abrupt failure events that occur in the wastewater treatment plant. This study demonstrated that MMI is a powerful tool to recover live and functional cells in situ from a complex microbial community to enable further characterisation of their physiology.     

Proteomics of Porphyromonas gingivalis within a model oral microbial community

Background  

Porphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community.     

Demethylation of methylarsonic acid by a microbial community

Arsenic is one of the most widespread environmental carcinogens and has created devastating human health problems worldwide, yet little is known about mechanisms of biotransformation in contaminated regions. Methylarsonic acid [MAs(V)], extensively utilized as an herbicide, is largely demethylated to more toxic inorganic arsenite, which causes environmental problems. To understand the process of demethylation of methylarsenicals, soil samples commonly used on Florida golf courses were studied. Several soil extracts were found to demethylate MAs(V) to inorganic arsenite [As(III)]. From these extracts, a bacterial isolate was capable of reducing MAs(V) to MAs(III) but not of demethylating to As(III). A second bacterial isolate was capable of demethylating MAs(III) to As(III) but not of reducing MAs(V). A mixed culture could carry out the complete process of reduction and demethylation, demonstrating that demethylation of MAs(V) to As(III) is a two-step process. Analysis of the 16S ribosomal DNA sequences of the two organisms identified the MAs(V)-reducing and the MAs(III)-demethylating isolates as belong to Burkholderia and Streptomyces species respectively. This is the first report of a novel pathway of degradation of a methylarsenical herbicide by sequential reduction and demethylation in a microbial soil community, which we propose plays a significant role in the arsenic biogeocycle.     

Improving the genetic representation of rare taxa within complex microbial communities using DNA normalization methods

Complex microbial communities typically contain a large number of low abundance species, which collectively, comprise a considerable proportion of the community. This ‘rare biosphere’ has been speculated to contain keystone species and act as a repository of genomic diversity to facilitate community adaptation. Many environmental microbes are currently resistant to cultivation, and can only be accessed via culture‐independent approaches. To enhance our understanding of the role of the rare biosphere, we aimed to improve their metagenomic representation using DNA normalization methods, and assess normalization success via shotgun DNA sequencing. A synthetic metagenome was constructed from the genomic DNA of five bacterial species, pooled in a defined ratio spanning three orders of magnitude. The synthetic metagenome was fractionated and thermally renatured, allowing the most abundant sequences to hybridize. Double‐stranded DNA was removed either by hydroxyapatite chromatography, or by a duplex‐specific nuclease (DSN). The chromatographic method failed to enrich for the genomes present in low starting abundance, whereas the DSN method resulted in all genomes reaching near equimolar abundance. The representation of the rarest member was increased by approximately 450‐fold. De novo assembly of the normalized metagenome enabled up to 18.0% of genes from the rarest organism to be assembled, in contrast to the un‐normalized sample, where genes were not able to be assembled at the same sequencing depth. This study has demonstrated that the application of normalization methods to metagenomic samples is a powerful tool to enrich for sequences from rare taxa, which will shed further light on their ecological niches.     

Enrichment of microbial community generating electricity using a fuel-cell-type electrochemical cell

A fuel cell was used to enrich a microbial consortium generating electricity, using organic wastewater as the fuel. Within 30 days of enrichment the maximum current of 0.2 mA was generated with a resistance of 1 k. Current generation was coupled to a fall in chemical oxygen demand from over 1,700 mg l^–1 down to 50 mg l^–1. Denaturing gradient gel electrophoresis showed a different microbial population in the enriched electrode from that in the sludge used as the inoculum. Electron microscopic observation showed a biofilm on the electrode surface and microbial clumps. Nanobacteria-like particles were present on the biofilm surface. Metabolic inhibitors and electron acceptors inhibited the current generation. 16S ribosomal RNA gene analysis showed a diverse bacterial population in the enrichment culture. These findings demonstrate that an electricity-generating microbial consortium can be enriched using a fuel cell and that the electrochemical activity is a form of anaerobic electron transfer.     

Neotropical diversification: the effects of a complex history on diversity within the poison frog genus Dendrobates

Aim We study the Neotropical poison frogs of the genus Dendrobates Wagler, 1830 in order to clarify their phylogenetic relationships and biogeographical history. The genus Dendrobates is an excellent taxon for examining patterns of Neotropical diversification as the four major species groups appear to correspond roughly to distinct geographical regions: (1) trans‐Andean, (2) Andean foreland, (3) Brazilian Shield and (4) Guianan Shield/Central America. In order to test the agreement of five of the most prominent hypotheses of Amazonian diversification, phylogenetic patterns were examined for agreement with patterns predicted by these hypotheses. Location Central and South America Methods The phylogenetic relationships of the genus Dendrobates were examined from novel and existing (GenBank) sequences of four mitochondrial loci totalling c. 1400 bp from 40 specimens of 22 different species using maximum parsimony and Bayesian methods. Results were compared with traditional taxonomic arrangements by means of SH tests. Phylogenetic relationships and genetic distances were used to test the adequacy of various diversification hypotheses. Results Phylogenetic analyses support the restructuring of two species groups of Dendrobates and the creation of a new species group. Statistical tests of the traditional taxonomic arrangement indicate a significantly bad fit to the molecular data. This restructuring has important implications for the understanding of the historical biogeography of Dendrobates. Biogeographical patterns within this genus suggest that a complex interaction of biotic and abiotic factors since the Eocene have produced the diversity observed today. Main conclusions The current classification of Dendrobates into discrete species groups does not accurately reflect evolutionary history. Data presented here strongly support a monophyletic Brazilian Shield lineage whose members have previously been split among the quinquevittatus and tinctorius groups. Furthermore, previous attempts at elucidating the historical biogeography of this genus were compromised by incomplete sampling and conclusions drawn from a paraphyletic ingroup. Our findings demonstrate a role for numerous hypotheses of diversification (e.g. river, refuge, disturbance–vicariance) in the history of Dendrobates, supporting previous warnings about the dangers of over‐simplification in the study of Neotropical diversification.     

Analysis of methanotroph community composition using a pmoA-based microbial diagnostic microarray

Microbial diagnostic microarrays (MDMs) are highly parallel hybridization platforms containing multiple sets of immobilized oligonucleotide probes used for parallel detection and identification of many different microorganisms in environmental and clinical samples. Each probe is approximately specific to a given group of organisms. Here we describe the protocol used to develop and validate an MDM method for the semiquantification of a range of functional genes--in this case, particulate methane monooxygenase (pmoA)--and we give an example of its application to the study of the community structure of methanotrophs and functionally related bacteria in the environment. The development and validation of an MDM, following this protocol, takes ～6 months. The pmoA MDM described in detail comprises 199 probes and addresses ～50 different species-level clades. An experiment comprising 24 samples can be completed, from DNA extraction to data acquisition, within 3 d (12-13 h bench work).     

Pathway of indole metabolism by a denitrifying microbial community

The metabolism of indole in a mineral-salts medium inoculated with 9% anaerobically digested nitrate-reducing sewage sludge was studied. The sequential occurrence of four structurally-related compounds — oxindole, isatin, dioxindole, and anthranilic acid — was detected using high-performance liquid or thin-layer chromatography. Mass spectrometry and proton nuclear resonance were used to identify isatin and dioxindole isolated from the culture fluids. Prior exposure of the microorganisms to indole, oxindole, isatin, or anthranilic acid resulted in accelerated decomposition of these compounds in a pattern that was consistent with a proposed pathway for the metabolism of indole under denitrifying conditions.     

Degradation of chlorophenols by a defined mixed microbial community

Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp. strain B13 as a chlorocatechol-dissimilating member of the community. Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents. During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II. In the fully acclimated culture, hybrid strains such as Alcaligenes sp. strain A7-2 were detected, which are more competitive than Pseudomonas sp. strain B13 with respect to chlorophenol degradation.     

Bacteria of the genus Burkholderia as a typical component of the microbial community of sphagnum peat bogs

Bacteria of the genus Burkholderia are a typical component of the microbial complex of sphagnum peat bogs and constitute a substantial portion of the aerobic chemoorganotrophic isolates which are routinely obtained from these environments on acidic nutrient media. The ecophysiological characteristics of the 27 strains of such organisms, which were isolated from the peat of acidic sphagnum bogs of the boreal and tundra zones of Russia, Canada, and Estonia, were investigated in the present paper. The overwhelming majority of the Burkholderia strains isolated from these bogs were phylogenetically close to the species B. glathei, B. phenazinium, B. fungorum, and B. caryophylli, the typical inhabitants of soil and plant rhizosphere. The bog isolates utilized a broad range of substrates as carbon and energy sources, including organic acids, sugars, polyalcohols, and certain aromatic compounds. All the strains studied were capable of growth on nitrogen-free media. They developed in the pH ranges of 3.5 to 7.4 and from 3 to 37 degrees C, with the optima at pH 5-7 and 11-23 degrees C, respectively. They were therefore moderately acidophilic, psychroactive, dinitrogen-fixing microorganisms well adapted to the conditions of acidic northern sphagnum bogs.     

Bacteria of the genus Burkholderia as a typical component of the microbial community of Sphagnum peat bogs

Bacteria of the genus Burkholderia are a typical component of the microbial complex of Sphagnum peat bogs and constitute a substantial portion of the aerobic chemoorganotrophic isolates which are routinely obtained from these environments on an acidic nutrient media. The ecophysiological characteristics of the 27 strains of such organisms, which were isolated from the peat of acidic Sphagnum bogs of the boreal and tundra zones of Russia, Canada, and Estonia, were investigated in the present study. Most of the Burkholderia strains isolated from these bogs were phylogenetically close to the species B. glathei, B. phenazinium, B. fungorum, and B. caryophylli, the typical inhabitants of soil and plant rhizosphere. The bog isolates utilized a broad range of substrates as carbon and energy sources, including organic acids, sugars, polyalcohols, and certain aromatic compounds. All the strains studied were capable of growth on nitrogen-free media. They developed in the pH range of 3.5 to 7.4 and from 3 to 37°C, with the optima at pH 5–7 and 11–23°C, respectively. They were therefore moderately acidophilic, psychroactive, dinitrogen-fixing microorganisms well adapted to the conditions of acidic northern Sphagnum bogs.     

Comparison of CE-SSCP and DGGE for monitoring a complex microbial community remediating mine drainage

Capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) is a promising high-throughput tool for profiling complex bioremediation communities, but has not been well characterized with respect to other methods such as denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to compare CE-SSCP with DGGE with respect to: 1) overall representation of the community in terms of the dominant species identified and corresponding Shannon diversity indices; 2) reproducibility and resolution; and 3) artifacts, using a complex sulfate-reducing community remediating mine drainage as a model system. Some of the dominant microorganisms were detected by both methods, but there were also differences in the reported community compositions, and more phylogenetic groups were detected by CE-SSCP. CE-SSCP Shannon diversity indices were slightly higher than those determined from DGGE data, and differed in terms of the time point at which the community was reported to have the highest diversity. Both methods had high reproducibility, but CE-SSCP resolution was higher in terms of the total number of peaks resolved, reduced co-migration of distinct DNA sequences, and length and legibility of the DNA sequencing data of clones used to identify peaks. Ten double bands in the same lane representing the same species were found by DGGE, whereas only one such artifact was observed by CE-SSCP. Finally, less overall sample preparation and analysis time was required for CE-SSCP than for DGGE. The results suggest that CE-SSCP offers several advantages over DGGE, especially for high-throughput monitoring.     

Disentangling the complex microbial community of coral reefs using standardized Autonomous Reef Monitoring Structures (ARMS)

Autonomous Reef Monitoring Structures (ARMS) have been applied worldwide to describe eukaryotic cryptic reef fauna. Conversely, bacterial communities, which are critical components of coral reef ecosystem functioning, remain largely overlooked. Here we deployed 56 ARMS across the 2,000‐km spread of the Red Sea to assay biodiversity, composition and inferred underlying functions of coral reef‐associated bacterial communities via 16S rRNA gene sequencing. We found that bacterial community structure and diversity aligned with environmental differences. Indeed, sea surface temperature and macroalgae cover were key in explaining bacterial relative abundance. Importantly, taxonomic and functional alpha diversity decreased under more extreme environmental conditions (e.g., higher temperatures) in the southern Red Sea. This may imply a link between bacterial community diversity and functional capabilities, with implications for conservation management. Our study demonstrates the utility of ARMS to investigate the response of coral reef‐associated bacterial communities to environmental change.     

Denitrification by the sessile microbial community of a polluted river

Denitrification by the sessile microbial community of the River Tamagawa was studied in laboratory experiments. Inorganic nitrogen loss was observed when river water was incubated with sessile microbial community of the river in a continuously circulating system. It was confirmed by the ¹⁵N tracer technique that both sessile microbial communities of unpolluted and polluted areas had denitrifying activity, even though they were incubated in oxygenated river water. The denitrification rate of the sessile microbial community taken from a polluted area, measured by the ¹⁵N tracer technique, was 8–16 mg N/m²/day in October and December, 1977, and it was enhanced 10-fold by raising the water temperature from 14 to 30° C. Denitrification in the river was also suggested by determining the N₂: Ar ratio of gases evolved from the river bed.     

Degradation of chlorophenols by a defined mixed microbial community.

Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp. strain B13 as a chlorocatechol-dissimilating member of the community. Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents. During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II. In the fully acclimated culture, hybrid strains such as Alcaligenes sp. strain A7-2 were detected, which are more competitive than Pseudomonas sp. strain B13 with respect to chlorophenol degradation.